Advances in Cancer Research Volume 76

Advances in

CANCER RESEARCH Volume 76

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Advances in

CANCER RESEARCH Volume 76

Edited by

George F. Vande Woude ABL-Basic Research Program National Cancer Institute Frederick Cancer Research and Development Center Frederick, Maryland

George Klein Microbiology and Tumor Biology Center Karolinska lnstitutet Stockholm, Sweden

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Contents

Contributors to Volume 76 ix

Fibronectin and Its Integrin Receptors in Cancer Erkki Ruoslahti I. Introduction

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11. Reduced Adhesiveness Is Needed for Detachment and Migration

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111. Anchorage Dependence and Anoikis 5 IV. Cell Migration and Invasion 10 References 16

Myb and Oncogenesis Brigitte Canter a n d Joseph S. Lipsick I. Introduction 21 11. The M y b Genes 22 111. Structural and Functional Features of the Myb Proteins IV. Regulation of v-Myb and c-Myb 41 V. Transcriptional Regulation by v-Myb and c-Myb 46 VI. The Myb-Chromatin Connection 50 References 52

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c-Src, Receptor Tyrosine Kinases, and Human Cancer Jacqueline S. Biscardi, David A. Tice, a n d Sarah 1. Parsons I. Introduction

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11. Receptor Tyrosine Kinases and Human Cancers

63 111. c-Src and c-Src Family Members in Human Cancers 78 IV. Mechanisms of c-Src Action 89 V. Potential Therapeutic Applications of c-Src/HERl Interactions References 103

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Contents

Epidemiology of Kaposi’s Sarcoma-Associated Herpesvirushluman Herpesvlrus 8 Thomas F. Schulz I. 11. 111. IV. V.

Introduction 121 KSHV Phylogeny and Molecular Epidemiology 122 Geographic Distribution 124 KSHV Prevalence in Risk Groups for HIV-1 Transmission Transmission of KSHV 141 VI. Association of KSHV with Disease 145 VII. Conclusion 153 References 154

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Consensus on Synergism between Cigarette Smoke and Other Environmental Carcinogens in the Causation of Lung Cancer Amold E. Reif a n d Timothy Heeren 1. Introduction 161 11. Testing the Significance of a Finding of Synergism 165 III. Carcinogenic Synergism and Public Health 172 IV. Previous Findings on Synergism Involving Cigarette Smoke 176 V. Multistep Carcinogenesis 177 VI. Varying the Time Frame of Data Collection 180 VII. Conclusion 182 References 182

Carcinogenesis and Natural Selection: A New Perspective to the Genetics and Epigenetics of Colorectal Cancer JadeBreivik a n d Gustav Gaudemack I. Introduction 187 11. Evolution and Cancer 188 111. The Microsatellite Instability Pathway 192 IV. The Chromosomal Instability Pathway 196 V. MIN versus CIN 199 VI. DNA Methylation and the Epigenetics of Cancer 200 W. Location-Related Carcinogenic Environments 206 VIII. Conclusion and Perspectives 208 References 209

Antitumor lmmunity at Work in a Melanoma Patient Pierre G . Coulie, Hideyuki Ikeda, Jean-Francois Baurain, a n d Rita Chiari I. htroduction 214

Contents 11. Melanoma Patient LB33 and Melanoma Cell Lines 216 111. Autologous CTLs against MEL.A Cells 218 IV.Identificationof Antigens Recognized by CTLs on MEL.A Cells 223 V. The MEL.B Cells 227 VI. A New Class of Antitumor CTL 232 VII. Conclusions 238 References 239

Index 243

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Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Jean-Franqois Baurain, Catholic University of Louvain, Cellular Genetics Unit, B-1200 Brussels, Belgium (213) Jacqueline S. Biscardi, Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 (61) Jarle Breivik, Section for Immunotherapy, The Norwegian Radium Hospital, N-0310 Oslo, Norway (187) Rita Chiari, Catholic University of Louvain, Cellular Genetics Unit, B-1200 Brussels, Belgium (213) Pierre G. Coulie, Catholic University of Louvain, Cellular Genetics Unit, B1200 Brussels, Belgium (213) Brigitte Ganter, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 (21) Gustav Gaudernack, Section for Immunotherapy, The Norwegian Radium Hospital, N-0310 Oslo, Norway (187) Timothy Heeren, Department of Epidemiology and Biostatistics, Boston University School of Public Health, Boston, Massachusetts 021 18 (161) Hideyuki Ikeda, Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, 160 Tokyo, Japan (213) Joseph S . Lipsick, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 (21) Sarah J. Parsons, Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 (61) Arnold E. Reif, Mallory Institute of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118 (161) Erkki Ruoslahti, Cancer Research Center, The Burnham Institute, La Jolla, California 92037 (1)

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Contributors

Thomas F. Schulz, Molecular Virology Group, Department of Medical Microbiology,The University of Liverpool, Liverpool L69 3GA, United Kingdom (121) David A. Tice, Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 (61)

Fibronectin and Its Integrin Receptors in Cancer Erkkl Ruoslahtl Cancer Research Center The Burnham Institute LA Jolla, California 92037

I. Introduction 11. Reduced Adhesiveness Is Needed for Detachment and Migration 111. Anchorage Dependence and Anoikis IV. Cell Migration and Invasion A. Tumor Cells in Circulation-Possible Antimetastatic Effect of Fibronectin in Plasma B. Site-SpecificMetastasis-Vascular Specificities References

The adhesive extracellular matrix protein fibronectin and its integrin receptors play important roles at several stages of tumor development. Tumor cells are generally less adhesive than normal cells and deposit less extracellular matrix. The loosened matrix adhesion that results may contribute to the ability of tumor cells to leave their original position in the tissue. Normal cells, when detached, stop growing and undergo anoikis (apoptosiscaused by loss of adhesion). Integrin-activated pathways mediated by focal adhesion kinase (FAK) and the adapter protein Shc seem to be particularly important in anchorage dependence; many oncoproteins are capable of shunting these pathways. Malignant cells circumvent anchorage dependence with the help of oncoproteins. Once invading tumor cells have gained access to the circulation, adhesion to the endothelia and other tissue components facilitates the establishment of tumor colonies at distant sites. Specific tissue affinitiesmay underlie the tendency of some tumors to metastasize preferentially to certain tissues. Interfering with tumor cell attachment with integrin-binding peptides has been shown to he an effective antimetastatic strategy in animal experiments. Tumor angiogenesisis yet another aspect of malignancy wherein extracellular matrices and integrins are important. Angiogenic endothelial cells in tumor vessels depend on the av family of integrins for survival. Inhibiting angiogenesis with compounds that block the activity of av integrins, and targeting drugs into tumors through these integrins, show promise as new anticancer strategies. 0 1999 Academic Press.

I. INTRODUCTION Cancer is a disease of tissue architecture. In forming tissues and organs during development, cells specialize and migrate to their appropriate places Advances in CANCER RWURCH

0065-230W99 $30.00

Copyright B 1999 by Academic Press. All rights of reproduction in any form reserved.

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in an orderly way. After this process is completed, the body plan is strictly maintained, except in cancer. Cancerous cells acquire the ability to breach tissue barriers, trespassing into adjacent tissues and distant sites in the body. This process, metastasis, is the most devastating aspect of cancer. Metastatic cancer has usually reached so many places that cure by surgery becomes impossible. For that reason, invasion into normal tissue and metastasis are the hallmarks of malignancy. A benign tumor that is not removed can get very large; the cells that make up such a tumor obviously overproliferate, but unlike malignant cancer cells, they do not invade or metastasize. Adhesive interactions are thought to play a major role in the construction of the body plan during development. These interactions comprise an area code system that guides cells to their appropriate locations in the body and anchors them there. Adhesion is also important in the maintenance of the body plan. Thus, it is not surprising that cell adhesion molecules-integrins and their extracellular matrix ligands in particular-are important in cancer. Integrins are the main class of cell adhesion receptors for extracellular matrices and can also serve as cell-cell adhesion receptors (Hynes, 1992; Springer, 1994; Ruoslahti, 1996). Integrins are membrane proteins that consist of an 01 and a p subunit, each with a molecular mass in the 100- to 200kDa range. Both subunits span the cell membrane, with most of the polypeptide outside the cell. The cytoplasmic domains of the integrin subunits, with one exception, are short, 30- to 50-amino acid peptides. There are 15 known 01 subunits and 8 p subunits, which combine into some 25 different integrins. The P l , p2, and a v subunits can pair with a particularly large number of possible companion subunits, each defining its subfamily among integrins. The extracellular ligand-binding specificity of an integrin is generated jointly by the a and p subunits of the integrin. Integrins display specificity on several levels. First, they are expressed in a cell-type- and stage-specific manner. Thus, one group of integrins is associated with migration and proliferation in various types of cells. These “emergency integrins” include a 5 p 1 , avp3, and avp6 (Sheppard, 1996). These integrins may be particularly important in cancer. Many other integrins are selectively expressed in a certain cell type or a few cell types. Examples of cell-type-specific integrins include aIIbp3 in platelets and a 6 p 4 in epithelial cells. Another level of integrin specificity is manifested in their ligand binding. Many of the integrins bind the RGD cell attachment sequence, but they recognize that sequence differentially in the context of various extracellular matrix proteins, such that some bind primarily to fibronectin and others to vitronectin (Ruoslahti, 1996). At yet another level of specificity, individual integrins mediate distinct signals into the interior of the cell (Schwartz et al., 1995; Clark and Brugge, 1995; Juliano and Haskill, 1993). Integrins have been shown to be signaling molecules capable of generating both common signals and signals that are specific for individual integrins. It

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has also been found that information can flow in the reverse direction through integrins; their ligand-binding ability is regulated from inside the cell (Chan and Hemler, 1993; Zhang et al., 1996; Kolanus et al., 1996; Hughes et al., 1997). In this article, I review the current understanding of the role of integrins and their ligands in the development and dissemination of cancer. Also discussed are a number of emerging integrin-based cancer treatments.

11. REDUCED ADHESIVENESS IS NEEDED

FOR DETACHMENT AND MIGRATION To be able to emigrate to another tissue, cancer cells have to detach from their original location, invade a blood or lymphatic vessel, travel in the circulation to a distant site, and establish a new cellular colony (Fig. 1).At every one of these steps, they must escape a number of controls that keep normal cells in place. The first step in cancer invasion is likely to be loosening of the adhesive restraint between cells. Both cell-extracellular matrix adhesion and cell-cell adhesion contribute to keeping cells in place. Indeed, adhesion molecules that mediate these interactions are frequently missing or compromised in cancer cells. The first adhesion anomaly discovered was the loss of fibronectin matrix in malignantly transformed cells in the early 1970s (Ruoslahti, 1988). Fibronectin is the prototype cell attachment protein found in the matrix surrounding normal cells. The fibronectin matrix mediates cell adhesion and anchorage through a number of fibronectin-binding integrins, including a5pl. The fibronectin matrix appears to be an important constraint on cells, because its restoration by various means suppresses cell migration and tumorigenicity. Fibronectin does not form matrix spontaneously-the cell uses its a5p1 integrin to capture secreted fibronectin and convert it into the fibrils that are then deposited into the matrix. Forced expression of the a5pl integrin in tumor cells reduces their motility and tumorigenicity (Giancotti and Ruoslahti, 1990). Conversely, a decrease in a5pl expression increases the tumorigenicity of CHO cells (Schreiner et al., 1991). Some of the a5pl integrin effect may be related to a signaling role of the integrin (Varner et al., 1995; Varner and Cheresh, 1996). However, the increase in fibronectin matrix assembly that accompanies an increase in a5pl expression or activity (Giancotti and Ruoslahti, 1990; Wu et al., 1996) appears to be responsible for most of the effect. Thus, expression of fibronectin from a transfected cDNA can convert tumorigenic cells into nontumorigenic ones (Wu et al., 1996), and the

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Fig. 1 Critical stages in tumor metastasis.

fibrillar form of fibronectin, superfibronectin, has a tumor-suppressive effect in vitro and in vivo. I discuss superfibronectin later on in this review. Malignant transformation by oncogenes generally reduces cellular adhesiveness, and this may be one critical element in tumorigenesis. At least two oncogenes, Src and Ras, can impair integrin activity (Akamatsu et al., 1996; Hughes et al., 1997). The Src oncogene can directly phosphorylate the p l integrin subunit, causing loss of p l integrin ligand-binding affinity (Akamatsu et a/., 1996). Src also phosphorylates a number of signaling molecules associated with integrins; the effect appears to be to shunt the integrin sig-

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naling pathways that control anchorage dependence (below). The influence of Ras on integrins was discovered in experiments designed to reveal cDNAs capable of reducing integrin affinity for their ligands (Hughes et af., 1997). Importantly, by impairing the a5pl integrin, both Src and Ras reduce fibronectin matrix deposition. In addition, oncogenic Src reduces fibronectin synthesis by the cells transformed with it (Adams et al., 1977). All these changes contribute to reduced adhesiveness and the increased propensity to migrate characteristic of transformed cells. Among the cell-cell adhesion molecules, E-cadherin behaves as a tumor suppressor. It is often lost on malignant transformation, either because of disabling mutations in its gene or in genes for the cytoplasmic proteins, catenins, that activate E-cadherin (Birchmeier, 1995). Alternatively, the expression of the E-cadherin gene can become down-regulated. Moreover, by manipulating this molecule in cultured cancer cells, one can change the cells’ ability to invade tissues and form tumors. Blocking the function of E-cadherin can turn a cultured lineage of cells from noninvasive to invasive. Conversely, restoring E-cadherin to cancer cells that lack it can negate their ability to form tumors when they are injected into mice. The a5pl integrin and N-cadherin coordinately regulate cell proliferation and migration (Huttenlocher et al., 1998). Moreover, integrin-linked kinase (ILK), a kinase that binds to the cytoplasmic domains of integrin p subunits and regulates integrin activity, also influences the activity of the LEF-l/TNF transcription factor, which is associated with p-catenin (Novak et al., 1998). Thus, both cell-matrix and cell-cell adhesion molecules individually and concordantly develop abnormalities that permit cancer cells to leave their original tissue site.

111. ANCHORAGE DEPENDENCE AND ANOIKIS The loss of tissue attachment discussed above creates a dilemma for a cancer cell about to invade; cells need attachment to be able to grow and even to survive. This leads to one of the most fundamental requirements in cancer progression, that cancer cells must become independent of anchorage. Fibroblasts dissociated from their extracellular matrix become arrested in their growth (Folkman, 1978). This phenomenon has been recognized for many years and is known as anchorage dependence on growth. The arrested growth of detached fibroblasts is reversible; on reattaching they regain their ability to proliferate. More recent is the realization that anchorage dependence is an integrin-mediated event; the cell attachment must be through integrins-other cell surface molecules will not suffice (Meredith et al., 1993; Frisch and Francis, 1994; Re et al., 1994; Boudreau et al., 1995). Moreover,

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endothelial and epithelial cells, when displaced from the extracellular matrix, undergo apoptosis. The likely biological purpose of this response, termed anoikis (Frisch and Francis, 1994), is that it would cause detached cells to die before they could reattach at new locations and disturb normal tissue architecture. Evidence now indicates that to satisfy the anchorage dependence requirement it is not enough for cells to attach to a matrix through any integrin, they have to attach through the correct integrin. The a5pl integrin may be particularly important in providing proper attachment. Thus, experiments show that cells engineered to express the a5Pl integrin as their fibronectin receptor survive for an extended period when cultured on fibronectin in the absence of serum, whereas cells with an alternative fibronectin receptor, olvpl, undergo apoptosis under the same conditions (Zhang et aL, 1995; O’Brien et al., 1996). No other integrin among several tested was able to substitute for d p l , even when the test cells were cultured on matrix proteins that serve as ligands for those integrins (Zhang et al., 1995; Z. Zhang and E. Ruoslahti, unpublished). The reason for the survival of the a5plexpressing cells appears to be that they express increased amounts of the antiapoptotic protein Bcl-2 (Zhang et al., 1995). Other integrins may be needed under other conditions. Thus, angiogenic endothelial cells depend on the avP3 integrin and epithelial cells on laminin-binding integrins in vivo (Brooks, 1995).The antiapoptotic effect of av@3in endothelial cells may be routed through NF-KB(Scatena et al., 1998). Figure 2 depicts some of these pathways. The integrin-selective survival signals are likely to provide a further safeguard for the maintenance of the integrity of normal tissues, because cells of one type would not be able to survive a change of location by attaching to a new place with a different matrix. All these results show that a molecular explanation for anchorage dependence is beginning to take shape, although much still remains to be learned. Neoplastic cells develop changes that enable them to circumvent the integrin signaling requirement and become independent of their anchorage. It is important to note that I am using here the terms anchorage dependence and anchorage independence as manifested in suspension culture. The classical definition of these terms derives from behavior of cells in semisolid media. Although that method of determining anchorage dependence correlates with in vivo tumorigenicity (Kahn and Shin, 1979), there are exceptions. For example, transforming growth factor @ (TGFP)can support the growth of anchorage-dependent, nontumorigenic cells in semisolid media, apparently because TGFP increases the expression of fibronectin and the integrins that bind to fibronectin (MassaguP, 1987).The ability of cells to survive and grow in suspension without any support is less likely to be altered by TGFP. Cancer cells get around the requirement of integrin-mediated attachment by shunting the integrin signaling pathways that control anchorage depen-

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f

Ras

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n

I

f

-\

- extracellular xp

matrix

Fig. 2 Integrin pathways preventing anoikis and some of their oncoprotein substitutes. See text for references.

dence. They can also protect themselves against anoikis by up-regulating an antiapoptotic protein, such as Bcl-2 (Zhang et al., 1995). Oncogenes often have as one of their activities the ability to confer a cell with anchorage independence. In some cases, this may be the only activity of an oncogene. One can distinguish between an oncogenic activity that affects both growth and anchorage from an effect that is directed only at anchorage by determining whether the transformed cells require serum for growth in culture. When the oncoprotein activity affects only anchorage dependence, the cells are able to survive and grow in suspension, but they require serum (or growth factors). The anchorage-related signalling pathways overtaken by oncoproteins originate at subcellular structures known as focal adhesions. Focal adhesions are specialized cell-substrate contacts, in which integrin clusters link actin filaments to the substrate or to extracellular matrix. As a result of the integrin and cytoskeletal clustering, various signaling molecules become concentrated in focal adhesions at the cytoplasmic surface of the cell membrane. Thus, focal adhesions appear to contain the highest concentration of proteins phosphorylated at tyrosine residues, a hallmark of signaling molecules (Burridge and Chrzanowska-Wodnicka, 1996). The high reactant concentration in focal adhesions and the proximities of multiple components necessary to complete signal transduction through a pathway facilitate efficient signaling. One important proximity is that of the cell membrane, because many signaling molecules require membrane association to be active

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(e.g., Ras) or have their enzymatic substrates in the membrane leg., phosphoinositol-3' (PI-3') kinase]. The formation of focal adhesion on integrin-mediated cell attachment results in the activation of a number of protein tyrosine kinases. These kinases include focal adhesion kinase (FAK),a cytoplasmic protein tyrosine kinase that binds directly to integrin cytoplasmic domains and plays a central role in integrin signaling (Parsons, 1996). Activated FAK connects to the Ras/ Faf/MAP kinase mitogenic pathway through the adapter protein Grb-2 (Schlaepferet al., 1994) and FAK activation also leads to an activation of PI3' kinase (Chen and Guan, 1994). The activation of FAK on the binding of integrins to their extracellular matrix ligands and the ensuing cell spreading may be the critical requirement that makes a normal cell anchorage dependent; without FAK activation, the cell will not grow and may undergo apoptosis. The evidence that suggests this role for FAK is that FAK confers anchorage independence to epithelial [Madin-Darby canine kidney (MDCK)]cells without affecting their other growth properties (Frisch et al., 1996), and that reducing FAK expression with antisense cDNA can induce anoikis in previously anchorage-independent celis (Hungerford et al., 1996). However, there is some controversy in the literature regarding the importance of FAK in anoikis. An alternative pathway involving the adapter protein Shc has been proposed as the conduit of this effect (Wary et al., 1996). Interestingly, this pathway is activated only by certain integrins, not all of them. FAK and Shc, and as yet unidentified pathways, all of which converge on Grb2, cooperate in mediating the activation of the extracellular signal-regulated kinase/mitogen-activated protein (ERWMAP) kinase pathway through Ras and Raf-1 (Schlaepfer et al., 1994). The activation of the phosphatidyl inositol-3' kinase (PI3-K) seems to be more important in anoikis than the MAP kinase pathway (Marte et al., 1997; Frisch and Ruoslahti, 1997), but it may be that this pathway may also respond to cell attachment through FAK and other signaling molecules. FAK is associated with the oncoproteins Src, p13OCUs,a recently characterized docking protein, and certain cytoskeleton-associated proteins such as paxillin and cortactin, all of which become activated by phoshorylation on cell attachment and spreading (Parsons, 1996; Polte and Hanks, 1997).One important activity of various oncoproteins appears to be that they cause downstream activation of the integrin-FAK pathway so that integrin-mediated adhesion is no longer needed for cell survival and growth. This makes the cells anchorage independent. Some tumor cells express elevated levels of FAK (Brunton et al., 1997), and this may be at least partly responsible for loss of anchorage dependence. The oncogenic variants of Src cause increased phosphorylation of several focal adhesion components, including FAK and pl3OCas (Kanner et aL, 1990). The phosphorylation of these proteins is independent of cell adhesion in Src-transformed cells (Schlaepfer and Hunter,

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1996; Vuori et al., 1996), and, therefore, presumably generates a “false” anchorage signal. The oncogenic adapter protein v-Crk may function similarly, because it causes increased phosphorylation of p13OcuSand changes the subcellular localization of p13OcUs (Matsuda et al., 1990; Nievers et al., 1997). Ilk is a recently discovered serinehhreonine kinase that may play a role in integrin regulation of malignancy (Hannigan et al., 1996). Ilk binds to the cytoplasmic domain of the PI integrin subunit and the binding suppresses kinase activity of Ilk. When Ilk is overexpressed and active, it promotes malignant transformation and anchorage independence. The ability to circumvent the anchorage requirement may be as important an activity of oncoproteins as is their ability to stimulate cell growth. The FAK-Ras-MAPK and Shc-Ras-MAPK pathways are examples of how oncogene activation can substitute for cell attachment. How the signals that couple loss of cell attachment to inhibited growth and apoptosis are carried into the nucleus is not entirely clear. It may be simply the lack of the positive signals discussed above, but there is also evidence that an “empty” (not extracellular ligand-bound) integrin can generate a negative signal (Varner et al., 1995). In their natural environment tissue cells are in contact with extracellular matrix, rather than a cell culture substrate. Malignantly transformed cells generally elaborate less extracellular matrix around themselves than do normal cells, but they may need some matrix to be able to grow. It seems that a dense matrix and a total loss of matrix will both prevent a cell from growing and migrating, and that there is an optimal amount of matrix for these functions. Thus, forced expression of the a5Pl integrin causes abundant fibronectin matrix production and suppresses tumorigenicity (Giancotti and Ruoslahti, 1990),whereas preventing cultured tumor cells from making any fibronectin matrix inhibits their growth (Saulineret al., 1996) and migration (Bourdoulous et al., 1998). The link between the matrix and the regulation of growth and survival is likely to be the actin cytoskeleton. Actin stress fiber organization in spread cells depends on fibronectin matrix (Bourdoulous et al., 1998). On the other hand, a close correlation exists between actin stress fiber formation and fibronectin matrix assembly. Thus, disrupting the cytoskeleton with cytochalasin D inhibits fibronectin matrix assembly, whereas lysophosphatidic acid (LPA), which induces stress fiber formation, enhances it (Zhang et al., 1994). LPA probably acts through the small GTPase Rho, because inhibiting Rho with C3 transferase inhibits the LPA-induced matrix assembly (Zhong et al., 1998). Rho is a member of the Ras superfamily of GTPases that controls the cytoskeletal rearrangements accompanying cell spreading. Integrins provide the connection that makes the extracellular matrix and the cytoskeleton interdependent. Interaction of integrins with the actin cy-

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toskeleton i s also essential for matrix assembly (Hynes, 1992) and truncation of the PI cytoplasmic domain blocks fibronectin matrix assembly by preventing the interaction of the ligand-occupied integrin with the cytoskeleton (Wu e t a / . , 1996). One indication of the importance of the extracellular matrix and cytoskeleton in cell growth, migration, and malignancy is the evidence that farnesyl transferase inhibitors may suppress malignancy through Rho, as well as Kas (Lebowitz et a/., 1997). Like Ras, Rho requires farnesylation to be active, and it has now been found that Rho may be an important target for the drugs that were designed as inhibitors of Ras farnesylation. In the nucleus, the growth inhibitory signals from the loss of cell attachment appear to be mediated by changes in nuclear cyclidcdk complexes. The two main changes are reduced expression of cyclin D at the protein level, and a loss of cyclin E/Cdk2 activity that is secondary to an up-regulation of the p21Cip and p27Kip inhibitors of cyclidcdk kinases (Assoian and Marcantonio, 1996; Fang et al., 1996; Shulze et al., 1996; Zhu et al., 1996).Removal of extracellular matrix has similar effects (Bourdoulous et al., 1998). Importantly, the cyclin E/Cdk2 complex stays active in anchorage-independent cells that are not attached. One mechanism whereby this is accomplished by a malignant cell is increased expression of cyclin E, which neutralizes the inhibitors (Fang et al., 1996), but other mechanisms, such as displacement of the Cdk inhibitor from the nucleus into the cytoplasm (Orend et al., 1998), are likely to play a role as well. The end result is that malignant cells can proliferate regardless of attachment to a substrate. In contrast, removal of all fibronectin matrix, despite the similarity of some of its effects to those from loss of substrate attachment, does not seem to be as easily circumvented by tumor cells as is anchorage dependence (Sauliner et al., 1996; Bourdoulous et al., 1998). Perhaps, the matrix also controls pathways other than the induction of cyclin inhibitors. This dual role of cell attachment and matrix formation may explain why fibronectin and its receptors do act as classical tumor suppressors (Taverna et al., 1998); downregulation of their functions enhances the malignant behavior of cells, but their complete absence is a disadvantage to a tumor cell.

IV. CELL MIGRATION A N D INVASION Anchorage dependence is only one of the constraints that a cancer cell must overcome to move away from its original site. Epithelial cells are separated from the underlying tissue by a basement membrane, a thin layer of specialized extracellular matrix. Basement membranes form a barrier that most types of normal cells cannot breach; the main exception is leukocytes,

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whose ability to penetrate basement membranes and other tissue barriers makes it possible for them to reach sites of inflammation. Tumor cells are also adept at penetrating basement membranes and invading tissues. Moreover, highly metastatic cells generally invade more effectively than do those from nonmetastatic tumors in assays that model the invasive process in vitro, and their invasive properties can be suppressed with integrin-blocking peptides (Gehlsen et al., 1988). Apparently, integrin-mediated cell adhesion provides the traction for tumor cell migration (Albelda et al., 1990).Various proteases that tumor cells make or activate are important for tissue penetration by tumor cells (Stetler-Stevensonet al., 1993; Zou et al., 1994). Integrins control the expression of some of these proteases (Werb et al., 1989; Seftor et al., 1992).They also regulate the cell surface localization of another protease important in tumor invasion, urokinase, by directing its receptor to focal adhesions (Seftor et al., 1992; Pollanen et al., 1987). Proteases on cancer cell surfaces may have roles other than helping the cell take down matrix barriers; some cell surface proteases process growth factors (Blobel, 1997). Another activity that may be important in invasion relates to deposition of fibrin around tumor nodules (Brown et al., 1988).Fibrin, together with the fibronectin that binds to it, forms a provisional wound matrix (Gailit and Clark, 1994).Like wound matrix, the new matrix around the tumor nodules may provide a favorable substrate for cells to migrate into, facilitating invasion. The d p l , avf33, and other a v integrins are among the integrins that play a prominent role in cell migration associated with tissue remodeling and cancer. Thus, for example, skin epithelial cells express the a v integrin subunit at the leading edge of the epidermis migrating into a wound. The a v is paired with the f35 and f36subunits in these cells (Sheppard, 1996; Zambruno et al., 1995; Gailit et al., 1994). The asp1 integrin is also expressed in migrating, but not resting, keratinocytes (Zambruno et al., 1995). Melanomas appear to depend on the avp3 integrin. This integrin has been shown to be important for the migration, survival, and tumorigenicity of melanoma cells (Montgomery et al., 1994). These melanoma cells lose their ability to form tumors if they are selected not to express the avp3 integrin, and they regain that ability when avf33 expression is restored by cDNA transfection. These changes are thought to depend on a role of avp3 in maintaining the survival of the melanoma cells; they die by apoptosis if their avp3 integrin is not engaged in substrate attachment. That the a v p 3 integrin is important for progression of melanomas in vivo is further suggested by a correlation that has been found between avp3 expression and invasiveness of the tumors; those melanomas that have progressed to the vertical growth mode (penetrating deeper into the dermis) express avp3 (Albelda et al., 1990; Natali et al., 1997). Although integrins mediate cell migration, their role in tumor invasion is

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unlikely to depend solely on this activity. Rather, their signaling functions are probably important. In this regard, the avp3 and a5pl integrins have special properties. As discussed above, cell attachment through these integrins activates the intracellular signaling molecule Shc (Wary et af., 1996). The connection to Shc is shared by the collagen-binding integrin u l p l . Shc activates the RadMAP kinase growth-regulatory pathway, and the ability of the a5Pl and a v integrins to activate that pathway through Shc may explain why these integrins are associated with tissue regeneration and tumor invasion. Despite their similarities, the avla5 class of integrins also possesses individually distinct signaling pathways. Thus, avp3 is associated with certain growth factor receptors and can enhance their function, whereas asp1 can control apoptosis through the antiapoptosis protein Bcl-2. avp3 is associated in cells with the intracellular pathways that mediate platelet-derived growth factor (PDGF),insulin, and insulin-like growth factor signaling pathways (Bartfeld etaf., 1993; Schneller etaf., 1997). Highly activated forms of the insulin receptor and PDGFP receptor coimmunoprecipitate with the avp3 integrin, and the mitogenic and chemotactic activities of these growth factors are enhanced in cells bound to a subratrate through this integrin (Vuori and Ruoslahti, 1994; Schneller et al., 1997; Jones et al., 1996). Thus, the avp3-growth factor receptor association may play a role in promoting tumor cell survival, growth, and invasion. As discussed below, the uvp3 integrin is also important in tumor angiogenesis. Having acquired invasive properties and having left its original position, a tumor cell (or a group of tumor cells) is poised to gain access to the circulation. This process involves penetrating the endothelial basement membrane that surrounds small blood vessels, and then the endothelial cell layer. The penetration of blood vessels is likely to be aided by poor integrity of small blood vessels near the advancing tumor cells: tumors cause angiogenesis, and the newly formed vessels have discontinuities (Folkman, 1995).

Tumor Cells in Circulation-Possible Effect of Fibronectin in Plasma

A.

Antimetastatic

Cancer usually metastasizes through the blood circulation or the lymphatics. Experimental metastasis studies suggest that very few cells survive the stay in the circulation. When cultured tumor cells are injected intravenously into mice, only a small minority of them successfully establish a colony in tissues, usually in the lungs, of the animal (Stetler-Stevensonet al., 1993). In one study, patients with intraperitoneal tumors were provided relief from the accumulation of ascites by providing a shunt that allowed the ascitic fluid to drain back into the circulation. Along with the ascites, vast

Fibronectin and Its Integrin Receptors

13

numbers of tumor cells were introduced into the circulation in this procedure. Surprisingly, those patients who survived for extended periods of time did not have extensive new metastases, as one might have expected (Tarin et al., 1984).Thus, it appears that the blood is an unfavorable environment for tumor cells. The vulnerability of malignant cells in the circulation is likely to have many underlying reasons, most of them unknown, but some of them are adhesion related. There may be natural defense mechanisms to eliminate tissue cells, such as tumor cells, that have entered the circulation. Plasma fibronectin may be one of the factors facilitating the elimination of circulating tumor cells. Saba and Cho (1977) injected a purified plasma fraction rich in fibronectin together with tumor cells and found less hematogenous metastasis in the fibronectintreated mice. Superfibronectin (sFN), a recently discovered polymeric form of fibronectin (Morla et al., 1994), inhibits cell migration in cell culture and has striking antimetastatic activities in vivo (Pasqualini et al., 1996). Treatment of tumor cells with sFN in vitro renders the cells nontumorigenic on subsequent injection into mice. More importantly, systemic treatment of mice with sFN strongly inhibits spontaneous metastasis from subcutaneously implanted tumors. A wide variety of tumor types respond to sFN in this manner, including human breast, colon, and ovarian carcinomas, melanoma, and 0steosarcoma implanted into nude mice. The mode of action of sFN requires further study, but when used to treat cells in suspension, it makes them incapable of adhering to any extracellular matrix substrate (Pasqualini et al., 1996). It may be that sFN coats tumor cells that are in transit in vivo, preventing them from attaching to the sites where metastases would otherwise form, and that the tumor cells incapacitated in this manner would then be susceptible to elimination by natural defense mechanisms. One of these mechanisms may be uptake of the sFN-coated tumor cells by the reticuloendothelial system, as has been postulated for fibronectin (Saba and Cho, 1977). sFN has been shown to gain access to the blood after an intrapecitoneal injection (Pasqualini et al., 1996). The fact that sFN does not affect the size of the primary tumor, only suppresses metastasis, also supports the assumption that it may be exerting its effects on circulating tumor cells. Peptides containing the RCD cell attachment sequence also have antimetastatic effects. These peptides mimic the ligands of a number of integrins, and they prevent the binding of integrins to their natural ligands (Ruoslahti, 1996). When RGD peptides are coinjected with tumor cells, fewer colonies will appear in the lungs than if the peptide is omitted, or if an inactive control peptide is injected (Humphries et al., 1986; Hardan et al., 1993; Pasqualini and Ruoslahti, 1996). One reason for this activity of the RGD peptides may be that they may prevent circulating tumor cells from binding

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platelets (Nierodzik et al., 1992).Platelets can provide growth factors for the tumor cells and may heip them become lodged in small blood vessels, a location favorable for extravasation of the tumor cells (Stetler-Stevenson et al., 1993). However, the RGD peptide effect on experimental metastasis is also seen in mice whose platelets have been depleted (Humphries et al., 1988), indicating that other RGD-dependent adhesion or signaling events are important as well. As discussed later, binding of tumor cells to the endotheliurn of blood vessels is a likely target of the peptide inhibition. Blocking integrin activity may also reduce malignancy through signaling events (Weaver et al., 1997). Current techniques make it possible to detect and quantitate tumor cells in the circulation of patients. Polymerase chain reaction (PCR) detection of mRNAs for epithelial proteins that are not present in normal circulating cells has been particularly useful in this regard (Gross et al., 1995; Jaakkola et al., 199.5; Hoon et al., 1995).As techniques evolve, it should be possible to learn more about the factors that influence the spread of cancers through the bloodstream.

B. Site-Specific Metastasis-Vascular Specificities Various metastatic cancers spread preferentially to certain tissues. Circulatory patterns explain much of this selectivity, because circulating tumor cells usually get trapped in the first vascular bed they encounter, the lungs or the liver. Accordingly, the lungs are the most common site of metastasis for some tumors, and the liver for others. The simplest mechanism for circulating tumor cells to attach to the endothelium is to become physically trapped in small blood vessels, the diameter of which is smaller than that of the tumor cell or a cluster of tumor cells. Tumor cells can produce factors that make platelets aggregate around them. The platelet connection may be the reason why antiplatelet drugs have anticancer effects in some experimental systems (Hardingham et al., 1993). Physical trapping of cancer cells in the blood vessels a t the site of metastasis does not explain all site-specific metastasis; some types of cancer show a striking preference for organs other than those that receive the venous blood. For example, prostate cancer metastasized almost exclusively into the bones. More than 100 years ago, Paget formulated an explanation for the site-specific metastasis of cancers. He postulated that the metastasis preferences would be influenced both by the frequency at which tumor cells are delivered to a given site (“seed”) and by the suitability of the tissue environment for the tumor (“soil”).Specific affinity between the adhesion molecules on tumor cells and those on the endothelium of blood vessels in the preferred tissues would cause more seeding of tumor cells in the preferred tissues than

Fibronectin and Its lntegrin Receptors

15

would be the case otherwise. Given the ability of adhesion receptors to elicit cellular signals, cell adhesion could be contributing to a favorable “soil” as well. It has been shown that the metastatic spread of tumor cells can be directed to a predetermined site by an adhesion molecule. Using transgenic mice expressing the leukocyte adhesion molecule E-selectin in their vasculature has shown an altered pattern of metastatic distribution of cells that express the selectin ligand Le” (Biancone et al., 1996).The selectin system is one that leukocytes use to find inflammatory sites, at which they exit the circulation to enter tissues. Not surprisingly, lymphomas show preferential tissue homing that depends on adhesion molecules that are expressed specifically in certain lymphoid organs and that mediate leukocyte homing into these tissues (Weissman, 1994).Among the fibronectin receptors, the a4pl integrin has been shown to redirect metastasis into the bone marrow. Intravenously injected Chinese hamster ovary cells formed colonies in the lungs of nude mice, whereas forced expression of a4pl in these same cells caused them to metastasize to bones in addition to the lungs (Matsuura et al., 1996).Antibody inhibition experiments showed that the ligand for a4pl in the bone marrow is VCAM-1, rather than fibronectin. Other studies have shown that tumor cells bind preferentially to endothelial cells from their preferred tissue site of metastasis (Auerbach et al., 1991),suggesting that endothelial specificities are likely to direct natural tumor homing as well. A peptidase and an ion channellike receptor have been identified as tumor-homing receptors in the lungs (Johnson et al., 1993;Elble et al., 1997),but little else is known about such receptors. We have developed a new method for studying specific features of the vasculature in different tissues. The method is in vivo screening of peptide libraries for peptides that direct the phage to home to a selected tissue. We looked for and found phage that bound preferentially to blood vessels in mouse brain and kidney (Pasqualini and Ruoslahti, 1996).Additionally, we have identified specific homing peptides for a number of other organs and tissues (Rajotte et al., 1998).The extensive diversity of endothelia revealed by these studies has led us to postulate that most, perhaps all, tissues display individual markers in their vasculature. The nature of these vascular “addresses” is obviously of great interest, and studies are underway to use the homing peptides to isolate the relevant target molecules. Endothelial cell vessels that are undergoing angiogenesis differ from endothelial cells in resting blood vessels in that they express a number of proteins that are not expressed at detectable levels in established blood vessels (Brooks, 1994;Martiny-Baron and Marmy, 1995).We have also used the phage technique to find peptides capable of homing into tumor blood vessels and have identified several peptide motifs that selectively direct phage into tumors (Arap et al., 1998;W. Arap, R. Pasqualini, and E. Ruoslahti, un-

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published results). One such peptide binds selectively to the QV integrins avp3 and avp5 (Koivunen et al., 1995; Pasqualini et al., 1997; Arap et al., 1998). The avp3 integrin (and avp5) is a known marker of angiogenic vessels (Varner and Cheresh, 1996; Brooks, 1994). Originally discovered as an RGD-directed receptor for vitronectin (Pytela et al., 1985), avP3 is now known to bind to a number of extracellular matrix proteins including, under some circumstances, fibronectin (Ruoslahti, 1996). The avP3 integrin is not only a marker of angiogenic vessels, it is functionally important in the angiogenic process; growing endothelial cells require avp3 for survival. Antibodies and soluble peptides capable of inhibiting the binding of avp3 to its extracellular matrix ligands cause endothelial cell apoptosis and destruction of the neovasculature (Brooks, 1995; Chen et al., 1997). We have used peptides that bind to a v integrins to show that phages carrying these peptides home into tumors in a highly selective manner (Arap et al., 1998). More recently, tumor imaging was accomplished by directing liposomes to tumor blood vessels with the help of anti-avp3 (Sipkins et al., 1998).These results indicate that the QV integrins are present on the luminal surface of the tumor vessels and that they, therefore, can be used to target drugs, cells, liposomes, and other therapeutic devices into tumors. In summary, studies on integrin-mediated cell attachment to extracellular matrices have greatly increased our understanding of central cell biological phenomena, such as anchorage dependence, and have generated a number of possible approaches to new therapies of cancer.

ACKNOWLEDGMENTS The author’s work is supported by the following grants from the National Institutes of Health, Department of Health and Human Services: CA28896, CA62042, CA67224, and Cancer Center Support Grant CA30199.

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Myb and Oncogenesis Brigitte Canter and loseph S. Upsick Department of Pathology Stanford University School o f Medicine Stanford, California 94305

I. Introduction 11. The Myb Genes A. Discovery of v-Myb and c-Myb B. Oncogenic Activation of c-Myb C. Expression and Function of c-Myb during Development and Differentiation

D. c-My6 in Cell Growth and Cell Death E. Regulation of c-Myb Expression F. Myb-Related Genes 111. Structural and Functional Features of the Myb Proteins A. The Myb Repeat B. The Myb DNA-Binding Domain C. Transcriptional Activation Domain and Heptad Leucine Repeat D. Regulation by the Carboxyl Terminus IV. Regulation of v-Myb and c-Myb A. Sites of Phosphorylation B. Interactions with Other Proteins V. Transcriptional Regulation by v-Myb and c-Myb A. Genes Activated by v-Myb and c-Myb B. Transcriptional Repression by Myb Proteins VI. The Myb-Chromatin Connection References

I. INTRODUCTION This review will focus on v-My6 and its normal cellular counterpart c-My6 with an emphasis on the biological and biochemical functions of their protein products. Both the v-Myb and c-Myb proteins are nuclear, bind directly to DNA, have short half-lives, and can regulate gene expression. As various eukaryotic genome projects proceed, new members of the My6 gene family are discovered at an increasingly rapid rate, particularly in plants. In addition, genetic and biochemical analyses of differentiation, transcriptional regulation, chromosome function, and the cell cycle have identified additional My6-related genes. However, outside of the signature 50-amino acid Myb repeats that constitute its DNA-binding domain, the c-Myb protein has Advances in CANCER RESEARCH 0065-23OW99$30.00

Copyright 8 1999 by Academic Press. All rights of reproduction in any form reserved.

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Brigitte Canter and Joseph S. Lipsick

significant homology only with two other proteins of vertebrates, A-Myb and B-Myb. A single closely related protein is present in the sea urchin and in the fruit fly. In this review we draw upon our current knowledge of Mybrelated genes and proteins insofar as they inform us about v-Myb and c-Myb. Other reviews about v-Myb and c-Myb are also available (Graf, 1992; Introna et ai., 1994; Ness, 1996; Shen-Ong, 1990; Thompson and Ramsay, 1995; Wolff, 1996). More information about the evolution of the Myb repeats and gene family can be found elsewhere (Lipsick, 1996), as can more detailed reviews of the vertebrate B-Myb gene (Saville and Watson, 1998) and the Myb-related genes of plants (Martin and Paz-Ares, 1997).

11. THE My6

GENES

A. Discovery of v-My6 and c-My6 The first member of the Myb gene family to be identified was the v-Myb oncogene of the avian myeloblastosis virus (AMV), which causes rapidly fatal monoblastic leukemia in chickens (Baluda and Reddy, 1994). This acutely transforming retrovirus is unusual in that it causes only leukemias in vivo and transforms only cells of the monocyte/macrophage lineage in culture. Indeed, the first evidence for oncogene specificity came from the observation that AMV could transform macrophages but not fibroblasts in culture, whereas Rous sarcoma virus did the converse, although both viruses could replicate in both cell types (Baluda, 1963; Durban and Boettiger, 1981). As is the case for other acutely transforming retroviruses, AMV arose via two recombinations between a replication-competent retrovirus and sequences of cellular origin (Klempnauer et al., 1982; Rushlow et al., 1982a). In particular, the enu gene of the myeloblastosis-associated virus type 1 (MAV-1) has been replaced by the v-Myb oncogene in AMV (Perbal et al., 1985). The 5' transduction event appears likely to have occurred at the DNA level because AMV has retained a portion of a c-Myb intron. The remainder of vMyb is an intronless cDNA copy of seven internal exons of c-Myb, the last of which is incomplete. As a result of this transduction process, v-Myb is expressed as a spliced subgenomic viral mRNA. Conditional alleles have been used to demonstrate that v-Myb is required for both the initiation and the maintenance of the transformed phenotype (Engelke et al., 1997; Moscovici and Moscovici, 1983). The first six amino acids of AMV v-Myb are encoded by the gag sequences upstream of the viral splice donor site. The last eleven amino acids of v-Myb are encoded by the 3' end of the env gene. However, neither the gag- nor the env-encoded amino acids are required for oncogenic transformation (Tbanez

Myb and Oncogenesis

23

and Lipsick, 1988; Lipsick and Ibanez, 1987). The MAVIAMV viruses have an unusual U3 region in their long terminal repeats (LTRs) and the MAV virus has a propensity to cause nephroblastomas and osteopetrosis in chickens, unlike other Rous-related helper viruses that generally cause longlatency lymphomas (Rushlow et al., 1982b). However, these unusual LTR sequences are not absolutely required for transformation by v-Myb in cell culture, even though there appears to be strong selection for them when AMV is passaged in chickens (Engelke and Lipsick, 1994). The 48-kDa v-Myb protein is truncated relative to the normal 75-kDa cMyb protein at both its amino and carboxyl termini (Figs. 1 and 2 ) (Gerondakis and Bishop, 1986; Rosson and Reddy, 1986). In addition, v-Myb contains 10 amino acid substitutions relative to the homologous region of c-Myb (a previously described eleventh substitution was not identified in c-Myb cDNA sequences). A second acutely transforming avian leukemia virus, E26, causes a rapidly fatal erythroblastosis in vivo and transforms multipotent hematopoietic progenitors in culture (Graf et al., 1992; Moscovici et al., 1983).E26 encodes a tripartite 135-kDa Gag-Myb-Ets fusion protein (Leprince et al., 1983; Nunn et al., 1983). c-Myb and the c-Ets protooncogene are present on different chicken chromosomes and it remains unclear whether E26 arose by sequential viral transduction of two different cellular genes or by transduction of a preexisting chromosomal translocation (Symonds et al., 1986). The Myb segment encoded by the E26 virus has even larger truncations than v-Myb of AMV and also contains one amino acid substitution that is different than any of those present in v-Myb of AMV (Fig. 2) (Nunn et al., 1984). The shorter Myb fragment present in E26 is only weakly transforming in culture without Ets and can be complemented by a variety of other oncogenes, including tyrosine kinases that result in autocrine growth factor production (Metz et al., 1991). In contrast, the Myb and Ets open reading frames cannot complement one another well in trans and there is a strong selection for the production of Myb-Ets fusion proteins in vivo from viruses that encode these proteins separately (Metz and Graf, 1991).

B. Oncogenic Activation of c-My6 Truncation of the c-Myb protein is required for the efficient transformation of myelomonocytic cells in culture (Gonda et al., 1989; Grasser et al., 1991). In general, amino-terminal truncations are more effective than carboxy-terminal truncations in this regard (Dini and Lipsick, 1993). Interestingly, although the amino acid substitutions in v-Myb are not required for oncogenic transformation, they strongly influence the phenotype of the transformed cells (Dini et al., 1995; Introna et al., 1990; Stober-Grasser and Lipsick, 1988). Cells transformed by AMV resemble monoblasts that are

-regulation

-

acidic

n

C-Myb

DNA binding

activation

Myb repeats I R l I R Z I R 3 1

I I

n

1

I

acidic

n I

llUl

I

negative regulation

~

n H .I

1 01

4

exon9A L

L

n n R 1

~ 1

I

m

u

)

1

E

m

D

A-Myb B-Myb

Urch Myb Dros Myb

Fig. 1 Topography of Myb proteins. The chicken c-Myb, A-Myb, and B-Myb proteins, the sea urchin Myb protein, and the Drosophilu Myb protein were aligned using the MACAW program (Schuler et ul., 1991). Exon YA was not included in the c-Myb sequence. Boxes indicate regions of statistically significant similarity. Shading indicates the degree of similarity within each box based on mean scores. v-Myb indicates the portions of c-Myb that are present in the oncoprotein encoded by AMV. R1, R2, and R3 indicate the three Myb repeats. HLR indicates a heptad leucine repeat in c-Myb that has also been referred to as the “leucine zipper.” See the text for a more detailed discussion.

Myb and Oncogenesis

25

committed to differentiate into macrophages. Indeed, either phorbol esters or liganded retinoic acid receptor-a can promote the differentiation of AMVtransformed monoblasts despite the continued presence of v-Myb (Pessano et al., 1979; Smarda et al., 1995; Symonds et al., 1984). Amino acid substitutions within both the DNA-binding domain and the transcriptional activation domain of AMV v-Myb are required for the monoblast phenotype (Dini et al., 1995; Introna et al., 1990). Cells transformed by a variant of vMyb that lacks all of these substitutions have a myelomonoblastic phenotype and bear cell surface markers of both the granulocytic and monocyte/macrophage lineage. In addition, the reversion of single amino acid substitutions within AMV v-Myb can result in the transformation of cells with a promyelocytic phenotype when grown in the presence of chicken myeloid growth factor (cMGF), a chicken cytokine related to mammalian granulocyte colony stimulating factor (G-CSF)(Introna et d., 1990). It has been shown that constitutive expression of full-length c-Myb can also cause transformation of bipotential myelomonocytic cells in culture (Ferrao et al., 1995; Fu and Lipsick, 1997). However, the cells grow more slowly and differentiate more frequently than do cells transformed by truncated c-Myb proteins. On the other hand, protein truncation is not required for the transformation of chick neural retinal cells by c-Myb (Garrido et al., 1992). Although endogenous c-Myb is not expressed in hematopoietic cells transformed by v-Myb or truncated forms of c-Myb, additional experiments have shown that the presence of c-Myb is compatible with transformation by v-Myb and that v-Myb does not repress the endogenous c-Myb gene (Lipsick, 1987; Smarda and Lipsick, 1994). These results suggest that v-Myb transforms hematopoietic cells that have turned off c-Myb expression as part of their normal differentiation process, but that the absence of c-Myb expression is not required for transformation. The c-Myb protooncogene has been further implicated in cancer as a result of retroviral insertional mutagenesis in two well-characterized experimental systems. In the presence of an inflammatory response, the replicationcompetent, oncogene-deficient Moloney murine leukemia virus (MuLV) causes myelomonocytic tumors rather than the more typical thymic lymphomas (Wolff et al., 1988).In virtually all of these myelomonocytic tumors (formerly known as plasmacytoid lymphosarcomas), there is an insertion of the retrovirus within the endogenous c-My6 locus (Shen-Ong et al., 1984, 1986). Most of these insertions result in amino-terminal or, less frequently, in carboxy-terminal truncations of the c-Myb protein that are very similar to those found in the AMV and E26 oncoproteins. However, some tumors contain insertions that are predicted to result in the deletion of only 38 residues from the carboxyl terminus of c-Myb (Fig. 2) (Nazarov and Wolff, 1995). In a second experimental system, the injection of a replication-competent,

26

Brigitte Canter and Joseph S. Lipsick

oncogene-deficient avian leukosis virus into day 1 2 chicken embryos causes nonbursal B cell lymphomas with a remarkably short latency (Kanter et al., 1988; Pizer and Humphries, 1989). In these tumors there is invariably a retroviral insertion in the endogenous c-My6 locus that results in a much smaller amino-terminal truncation than that found in AMV, E26, or the MuLV-induced tumors (Fig. 2). Interestingly, when a recombinant retrovirus expressing a cDNA with this 20-residue amino-terminal truncation was injected into chickens, sarcomas and carcinomas as well as lymphomas were induced (Jiang et al., 1997).Detailed analyses of tumor progression strongly suggest that the insertional activation of c-Myb alone is not sufficient for tumor formation in either the chicken or mouse model system (Belli et al., 1995; Pizer et al., 1992). Other experiments have also implicated c-My6 in oncogenesis. An avian retrovirus containing a carboxy-terminal truncation of the c-Myb protein was reported to cause muscular fibrosarcomas in chickens (Press et al., 1994).The resulting tumors were monoclonal and of rather long latency, unlike most tumors caused by acutely transforming retroviruses. Retroviral insertion into c-My6 was also found in a Marek’s disease chicken T lymphoma cell line (Le Rouzic and Perbal, 1996). This is of historical interest because

Fig. 2 Alignment of Myb protein sequences. Myb protein sequences were aligned using the CIIJSTALW implementation of the Parsimony After Progressive Alignment (PAPA) method (Feng and Doolittle, 1990; Thompson et al., 1994),with some additional adjustments based on blocks identified with the MACAW local alignment program (Schuler et ul., 1991). Shading was then performed using the BOXSHADE program. Black shading indicates identical residues at a given position; gray shading indicates similarity. Hu, Human; Ch, chicken; Xe, Xenopus; urchin, sea urchin; Dros, Drosophilu. Exon 9A-encoded sequences were included for human and chicken c-Myb, but not Xenopus c-Myb. The XX near the carboxyl terminus of Xenopus B-Myb indicates an additional 62 residues that were unaligned and were not included in the numbering scheme in order to conserve space. CKII, Casein kinase I1 phosphorylation site; FL, amino-terminal truncation of the murine FL variant of c-Myb; ALV, amino-terminal truncation caused by avian leukosis virus insertion in B cell lymphomas; R1, R2, and R3 indicate the Myb repeats; AMV, amino- and carboxy-terminal truncations of v-Myb of AMY E26, amino- and carboxy-terminal truncations of v-Myb of E26 leukemia virus; cys, an essential cysteine required for transcriptional activation and oncogenic transformation by AMV v-Myb that has been proposed to be subject to redox regulation; GSKIII, a peptide phosphorylated in v-Myb in vivo and by glycogen synthase kinase Ill in vitro; shaded hexagons, two heptad hydrophobic repeats, the darker of which has been referred to as the “leucine zipper”; exon 9A, residues encoded by an alternatively spliced exon in c-Myb; 1120 and 1151, carboxy-terminal truncations in mutants of AMV v-Myb; asterisks, highly conserved putative phosphorylation sites for proline-directed kinases; PS and BN, deletions in c-Myb that together activate transcription and oncogenic transformation; EVES, sequence that mediates interaction with the DNA-binding domain in yeast two-hybrid assays and that also contains a serine that is phosphorylated in c-Myb in vivo and by MAPK in vitro; F-MuLV, carboxy-terminal truncations caused by Friend murine leukemia virus in some myelomonocytic tumors; Dm myb’, position of a glycine that is mutated to a serine in a temperature-sensitive mutant of Drosophila Myb.

27

Myb and Oncogenesis

..

CKlI Hu-c-Myb Ch-c-Myb Xe-0-Myb Ru-A-Myb Ch-A-Myb Xe-A-Myb Eu-B-Myb Ch-B-Myb Xe-B-Myb Urchin-Myb Droa-Myb

Eu-c-Myb Ch-c-Myb Xe-c-Myb Bu-A-Myb Ch-A-Myb Xe-A-Myb Hu-B-Yyb Ch-B-Myb Xe-B-Myb Urchin-Myb D ro a -Myb

Eu-c-Myb Ch-c- Myb Xe-c-Myb Hu-A-Myb Ch-A-Myb Xe-A-Myb Ru-B-Myb Ch-B-Myb Xo-B-Myb Urchin-Myb Dr o s-M yb

Hu-c-Myb Ch-c-Myb Xe-c-Myb Hu-A-Myb Ch-A-Myb Xe-A-Myb Hu-B-Myb Ch-B-Myh Xe-B-Myb Urchin-Myb Dros-Myb

Hu-c-Myb Ch-c-Myb Xe-c-Myb Hu-A-Myb Ch-A-Myb Xe-A-Myb Hu-B Myb C h-B- Myb Xa-B My b Urchin-Myb Dr o s-Myb

-

-

196 196 193 191 191 190 187 187 187 194 240

Fig. 2

FL ALV V

V

acidic

Brigitte Canter and JosephS. Lipsick

28

acidic

Siu-c-Myb Ch-c-Myb Xe-c-Myb Bu-A- Myb Ch-A-Myb Xe-A-Myb Bu-B-Myb Ch-E-Myb Xe -B- My b Urchin-Myb D r 0 B -M yb

Ku-c-Uyb Ch-c-Myb Xe-c-Myb Eu-A-Myb Ch A-Mvb XeIA-M%b Xu-B-Myb Ch-B-Myb Xa-B-Myb Urchin-Myb Droa-Myb

287 251 248 240 251 297 "leucine zipper" '2I' w-)

Bu-c-Myb Ch-c-Myb Xe-c-Myb Eu-A-Myb Ch-A-Myb Xe-A-Myb Bu-B-Uyb Ch-B-Myb Xe-B-Myb Urchin-Myb Drc8-Myb

374 375 362 340 345 338 292 284 273 282 329

Ku-c-Myb Ch-c-Myb Xe-c-Myb Bu-A-Mpb Ch-A-Myb Xe-A-Myb Bu-B-Myb Ch-B-Myb Xe-8-Myb Urchin-Myb Drc8-Myb

428 429 386 393 398 390 345 338 316 330 364

Ku-c-Myb Ch-c-Myb Xe-c-Myb HU-A-Myb Ch-A-Myb Xe-A-Myb EU-E-Myb Ch-B-Uyb Xa-E-Myb Urchin-Myb DroB-Myb

474 415

m'

w-)

+...................... "..........._.I ".I..

#

1151 V?..""

.......".............

.....................................................................

exon 9A..... .....-... ............... ............................................................. --..............-.............................. "

"

"

GR-ALQrQ-----QR1GNgTKPAGSPSPRVNK~-GT-AVQLQ----- EGGAS~LCRPPGLPISNLSKT~&--

I

~-LMRIQ-----ENLGAMECQFNVSLV Wt-LMRIQ-----ENrR1UrCQINVSVn

~-WI--------E-BISPDCALNSCLV ~QLQASEQQQVLPPRQPS~LVPSVTZYR ~~~-QT--------PSKPTPSLPNVTBYR TB-MV------TDKPQ&SN--VTBIR

~%-~T--------EMNTKQs----IDIR

-----________---------------

* *

*

... ....................".".......-............................ I

386

441

446 429 404 385 362 373 374

Fig. 2 (continued)

.*

SSLD-PPKV-L~PARES---SPP~-SPKS-LSASQGS----

29

Myb and Oncogenesis

* Hu-c-Myb Ch-c-Myb Xe-c-Myb Hu-A-Myb Ch-A-Myb Xa A-Myb Hu-H-Myb Ch-B-Myb Xe-8-Myb Urchan-Myb Dros Mvb

524 524 387 492 497 474 157

Hu-c-Myb Ch-c-Myb Xr-c-Myb Hu-A-Myb Ch-A-Myb Xe-A-Myb Hu-B-Myb Ch-B-Myb Xa-B-Myb Urchin-Myb Dros-Myb

583 583

iiu-c-Myb Ch-c-Myb Xe-c-Myb Hu-A- M y b Ch-A-Myb Xe-A-Myb Eu-B-Myb Ch-B-Myb Xa-8-Myb Urchin-Myb Dros-Myb

639 639 503 604 609 585 563 546 522 537 501

Bu-c-Myb C h-c -Myb Xa-c-Myb Hu-A-Myb Ch-A-Myb Xe-A-Myb Hu-B -Myb C h-B- M y b Xe-B-Myb Urchin-Myb Dros-Myb

718 718 583 705

440

416 433 394

447 551

556 532 514 197 473 483 443

*

710 680 652 638 632 642 596

Fig. 2 (continued)

*

AMV rPS

*

30

Brigitte Ganter and JosephS. Lipsick

AMV arose in a Marek’s disease virus-infected chicken (Baluda and Reddy, 1994). Expression of a v-My6 transgene under control of the T cell-specific CD2 promoter caused late-onset T cell lymphomas in laboratory mice (Badiani et al., 1996). These experiments together imply that additional genetic alterations are required for oncogenic transformation by activated forms of c-My6. The human c-My6 gene is located at 6q24 (Harper et al., 1983). Thus far, no consistent chromosomal translocations involving c-My6 have been identified in human leukemias or lymphomas. However, the level of c-My6 expression is quite high in most leukemias and lymphomas with an immature phenotype (Westin et al., 1982). In addition, the levels of c-My6 expression have been correlated with prognosis in human breast and colon carcinomas (Greco et al., 1994; Guerin et al., 1990; Torelli et al., 1987). Interestingly, the c-My6 gene is amplified in a number of human colon carcinoma, pancreatic carcinoma, but not leukemia cell lines (Alitalo et al., 1984; Henderson and Wolman, 1988; Wallrapp et al., 1997). In particular, it was recently found that the c-My6 gene is amplified in 10% of primary pancreatic carcinomas and that high levels of c-My6 expression are also observed in the majority of all pancreatic carcinomas (Wallrapp et al., 1997). It has also been suggested that mutations within the first intron of c-My6 may correlate with increased expression in human colon cancers by altering gene expression (Thompson et al., 1997).

C. Expression and Function of c-MyG during Development and Differentiation The normal c-My6 protooncogene is expressed at high levels in immature hematopoietic cells of all lineages, and its expression decreases as the cells differentiate (Chen, 1980; Duprey and Boettiger, 1985; Gonda and Metcalf, 1984; Kirsch et al., 1986; Ramsay et al., 1986; Westin et al., 1982). Constitutive expression of c-My6 can block the differentiation of various established erythroid and myeloid cell lines, suggesting that c-Myb may control hematopoietic differentiation (Clarke et al., 1988; Selvakumaran et al., 1992; Smarda and Lipsick, 1994; Todokoro et al., 1988). Consistent with these observations, laboratory mice with a homozygous mutation in c-My6 die in utero due to a failure of fetal liver hematopoiesis (Mucenski et al., 1991). In contrast, the earlier yolk sac hematopoiesis appears to be normal in these mice, as does the development of megakaryocytes within the fetal liver. Additional studies have suggested that c-Myb function is required for T lymphocyte development. In particular, a transgene that produces a fusion of the c-Myb DNA-binding domain and the Drosophila Engrailed repressor

Myb and Oncogenesis

31

domain can prevent normal thymocyte maturation (Badiani et al., 1994). Further studies have shown that this transgene causes apoptosis (Taylor et al., 1996). The expression of c-My6 has also been detected in immature epithelial cells in a variety of tissues, including the colon, respiratory tract, skin, and retina (Queva et al., 1992; Sitzmann et al., 1995).The role of c-Myb in the normal development of these tissues remains unclear at present. Transgenic mice bearing a c-Myb gene driven by the ubiquitously expressed p-actin promoter displayed no thymic abnormalities, but did develop degenerative abnormalities in skeletal and cardiac muscles (Furuta et al., 1993). In Xenopus, a c-Myb homolog is expressed throughout development and continues in adult tissues, with the highest levels in the intestine, heart, liver, lung, and ovary (Amaravadi and King, 1994).

D. e M y 6 in Cell Growth and Cell Death In addition to its role in regulating differentiation, c-Myb has also been implicated in control of the cell cycle. When resting lymphocytes are stimulated to divide, c-Myb mRNA and protein expression begin in the late GI phase of the cell cycle and continue into the S phase (Lipsick and Boyle, 1987; Torelli et al., 1985). Interestingly, cell lines that represent B lymphocytes at different stages of maturation appear to regulate c-My6 expression differently with respect to the cell cycle (Catron et al., 1992). In addition, the constitutive expression of c-Myb can rescue v-Myb-transformed cells from phorbol ester-induced differentiation and cell cycle arrest in both GI and G, (Smarda and Lipsick, 1994). Surprisingly, the DNA-binding domain of cMyb alone is sufficient for such rescue (Engelke et al., 1995a). Interestingly, experiments with a temperature-sensitive mutant of the E26 virus suggest that cells must traverse S phase for the Gag-Myb-Ets fusion protein to alter cell morphology after a shift to the permissive temperature (Beug et al., 1987). It has also been reported that c-Myb can permit fibroblasts to progress through the cell cycle in the absence of exogenous IGF-1, apparently by inducing endogenous IGF-1 (Reiss et al., 1991; Travali et al., 1991). Although c-Myb expression has been reported in normal fibroblasts, others have failed to reproduce this result (Catron et al., 1992; Thompson et al., 1986). Experiments with antisense oligonucleotides have suggested that c-Myb expression is required for the progression of hematopoietic cells into the S phase of the cell cycle (Gewirtz et al., 1989). However, other investigators have raised serious doubts about the specificity of this technique (Burgess et al., 1995). c-Myb has also been proposed to prevent apoptosis by directly regulating

32

Brigitte Canter and loseph S. Lipsick

the expression of the Bcl-2 gene (Frampton et al., 1996; Salomoni et al., 1997; Taylor et al., 1996). However, other investigators have found that Bcl2 expression persists after functional inactivation of a hormone-inducible form of the c-Myb protein (Hogg et al., 1997). Still other investigators have suggested that c-My6 promotes rather than inhibits apoptosis in neuronal cells (Estus et al., 1994).It therefore remains unclear whether a primary role of c-My6 is to prevent or accelerate apoptosis, or rather whether c-Myb regulates apoptosis indirectly simply by maintaining cells in the cycle in a fashion similar to the action of many extracellular growth factors.

E. Regulation of c-My6 Expression The regulation of c-My6 expression has remained rather enigmatic (Boise et al., 1992). In addition to regulation at the level of transcriptional initiation, evidence has been presented for regulation at the level of transcriptional elongation within the first intron, as had previously been proposed for the cMyc gene (Bender et al., 1987; Watson, 1988). However, in the case of cMyc the conclusions reached by analyzing nuclear run-on experiments have generally not been supported by more detailed analyses of mRNAs in whole cells (Krumm et al., 1992; Strobl and Eick, 1992). Rather than being regulated by attenuation at some distance from the promoter as initially thought, c-Myc now appears to be regulated by the release of RNA polymerase engaged near the start site in a fashion reminiscent of a proposed mechanism of action of the lac repressor of Escherichia coli (Lee and Goldfarb, 1991). Similar experiments have not yet been reported for c-Myb. The c-My6 promoter has no classical TATA motif and transcription appears to initiate at multiple sites within the promoter (Bender and Kuehl, 1986; Dvorak et al., 1989; Watson et al., 1987).An additional promoter has also been identified within the first intron of c-Myb (Jacobs et al., 1994).Although numerous proteins have been reported to bind to the c-My6 promoter and first intron, it remains unclear which, if any, of these proteins and binding sites is relevant in vivo (Calabretta and Nicolaides, 1992; McCann etal., 1995; Phan et al., 1996; Reddy and Reddy, 1989; Sullivan et al., 1997; Toth et al., 1995). For example, the c-Myb protein has been reported to either repress or activate its own promoter in transient assays (Guerra et al., 1995; Nicolaides et al., 1991). In this regard, an 8-kb fragment of chicken genomic DNA extending upstream from the first intron was unable to recapitulate the regulation of the endogenous c-Myb gene in a number of different cell lines, suggesting the presence of more widely dispersed regulatory elements (Y. Vaishnav and J. s. Lipsick, unpublished). The advent of homologous recombination in murine embryonic stem cells and various “knock-in” strategies should eventually help to answer these questions. A

Myb and Oncogenesis

33

region of particular interest is a 124-nucleotide motif within the c-My6 promoter that is 92% identical in chicken and mouse (Urbanek et al., 1988). Also, a consensus E2F-binding site is present within the c-My6 promoter and, by analogy with B-My6, may be required for cell cycle regulation (DeGregori et al., 1995; Mudryj et al., 1990).

F. My6-Related Genes In mammals, birds, and amphibians, two My6-related genes in addition to c-My6 (A-My6 and B-My6) have been identified (Nomura et al., 1988). The c-My6 and A-My6 genes appear to have arisen from a recent gene duplication event following an earlier duplication of a B-My6-like gene that is more closely related to the sole My6-related genes of sea urchin and Drosophilu (Fig. 3). Like c-My6, the expression of A-My6 appears to be rather tissue specific. The A-My6 gene is expressed in mammals at high levels in the developing central nervous system, in germinal center B lymphocytes, in mammary gland ductal epithelium, and in testis (Golay et al., 1998; Mettus et al., 1994; Trauth et al., 1994). A-Myb has also been reported to cooperate with c-Myc in driving smooth muscle cells into S phase (Marhamati et al., 1997). In Xenopus, A-My6 expression is high in mitotic spermatogonial cells, but ceases on meiosis (Sleeman, 1993). Laboratory mice with a homozygous mutation in the A-My6 gene are viable but display a failure of spermatogenesis and of mammary gland development in response to pregnancy (Toscani et al., 1997). The role of A-My6 in malignancies has not been extensively investigated, but mice that widely expressed A-My6 transgenes developed follicular hyperplasia of the spleen and lymph nodes due to a proliferation of B cells with a germinal center phenotype (DeRocco et al., 1997). The B-My6 gene is expressed throughout mouse development, unlike either A-My6 or c-My6 (Sitzmann et al., 1996).The expression of B-My6 appears to correlate with cell division during embryogenesis. When quiescent cultured fibroblasts enter the cell cycle, B-My6 mRNA is induced in late G, and early S phase (Lam et ul., 1992). The B-My6 promoter contains a binding site for the E2F transcription factor that negatively regulates gene expression during Go and early G, (Lam and Watson, 1993; Zwicker et al., 1996). Either p107 or p130 but not Rb is required for this negative regulation (Hurford et al., 1997). Other genes that are similarly regulated include cdc2, cyclin A, tbymidylate synthetase, ribonucleotide reductase, and E2 F1 . Interestingly,constitutive expression of B-My6 has been reported to bypass p53-induced G, arrest, even though p21 induction occurs (Lin et al., 1994). These results suggest that the regulation of B-My6 expression may be a critical checkpoint in G,. In addition, the peak expression of B-Myb in S phase

Brigitte Canter and loseph S. Lipsick

34

A-Myb

Fig. 3 Phyiogenetic tree of Myb protein sequences. Myb protein sequences were aligned using CLUSTALW and a boot-strapped tree was generated (Thompson et al., 1994). Hum, Human; Chi, chicken; Xen, Xenopus; urch, sea urchin; Dros, Drosophila; Dicty, Dictyostelium; Asper, Aspergillus nidulans; Scerv, Saccharomyces cerevisiae.

and its short half-life raise the possibility that the essential function of B-Myb may be transcriptional repression or even nontranscriptional. Experiments with antisense nucleic acids are consistent with the idea that B-Myb expression is essential for cell cycle progression (Arsura et ul., 1992; Sala and Calabretta, 1992). However, these studies are subject to the same caveats noted above for similar studies of c-Myb. No mutations of the B-Myb gene have yet been reported in vertebrates. There is a single Myb-related gene in Drosophilu that is more closely related to B-Myb than to A- or c-Myb (Figs. 2 and 3). In particular, the Drosophilu Myb protein lacks the central acidic region and the heptad leucine repeatIFAETL regions. Drosophilu Myb expression is seen throughout embryonic development and generally correlates with cell division, similar to B-Myb expression in vertebrates (Katzen et ul., 1985). However, Drosophilu Myb does not appear to be expressed in the larval tissues that undergo endoreduplication in which repeated S phases without M phases are

Myb and Oncogenesis

35

used to create polyploid nuclei. Temperature-sensitivemutants of Drosophila My6 result in lethality at several different points during development (Katzen and Bishop, 1996). Drosophila My6 may be required in the GJM transition because cell cycles that fail during late wing development at the nonpermissive temperature can be restored by ectopic expression of cdc2 or string (cdc25) (Katzen et ul., 1998). There appears to be only one sea urchin My6-related gene that is closely related to B-My6 (Figs. 2 and 3). It was discovered as a transcriptional repressor of a specialized actin gene during development (Coffman et al., 1997). Two My6-related genes have appeared in the Caenorhabditis elegans genome project database thus far. One, contained in cosmid D1081, is a homolog of Schizosaccharomycespombe cdc5, a gene that is required in the G, phase of the cell cycle (Ohi et al., 1994, 1998). Cdc5 homologs are also present in vertebrates, Drosophila, and green plants (Bernstein and Coughlin, 1997; Hirayama and Shinozaki, 1996; Ohi et al., 1998). The other My6related gene in C. eleguns, contained in cosmid F32H2, appears to be more closely related to c-Myb than to Cdc5. However, its DNA-binding domain is more evolutionarily distant from c-Myb than that of a Myb identified in the cellular slime mold Dictyostelium discoideum (Stober-Grasser et al., 1992; S. McCann and J. S. Lipsick, unpublished). These results suggest that another gene more closely related to c-My6 is likely to be present in the nematode C. elegans. Our analysis of the sequences and functional domains of the Myb proteins of animals suggests a model for Myb evolution. A single Myb gene most similar to Drosophila My6, sea urchin My6, and vertebrate B-My6 underwent a gene duplication event during the genesis of vertebrates. One copy of this gene was selected for retention of its function and is the modern-day B-Myb that is required in all cell types. The second copy of this gene drifted and acquired a central transcriptional activation domain that was then selected for a more specialized function in specific tissue(s). This second gene then underwent another duplication, giving rise to modern-day AMy6 and c-Myb. These two genes were then selected for a similar function in different specialized tissue(s) and have therefore both retained their central transcriptional activation domains. Such a model in which a gene has undergone two rounds of duplication and divergence during vertebrate evolution is consistent with observations in other regulatory gene families (Sidow, 1996).It will therefore be of interest to determine the repertoire of My6 genes in the “intermediate” species that have not yet been examined, including tunicates, Amphioxus, jawless fish, shark, and bony fish. Preliminary data suggest that tunicates have a single My6 gene whereas bony fish have three My6-related genes (E. Chen, S . McCann, and J. S. Lipsick, unpublished).

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Brigitte Canter and loseph S. Lipsick

Ill. STRUCTURAL AND FUNCTIONAL FEATURES OF THE MYB PROTEINS The c-Myb protein is present in the cell nucleus and contains three main functional domains-an amino-terminal DNA-binding domain, a central transcriptional activation domain, and a carboxy-terminal regulatory domain (Fig. 1)(Sakura et al., 1989).

A. The

Myb Repeat

The family of Myb proteins is defined by the presence of a highly conserved Myb domain (repeat) of approximately 50 amino acids (Klempnauer and Sippel, 1987). Each repeat contains three conserved tryptophans, spaced 18 or 19 amino acids apart, that form a hydrophobic core and that play a critical role in sequence-specific DNA binding (Anton and Frampton, 1988; Kanei-Ishii et al., 1990; Saikumar et af., 1990).Each repeat folds into three well-defined helices that form a helix-turn-helix-related structural motif as was shown by nuclear magnetic resonance (NMR)spectroscopy for single cMyb repeats and for the c-Myb repeats R2R3 complexed with DNA (Frarnpton et af., 1991; Gabrielsen et a!., 1991; Ogata et al., 1992, 1994-1996). However, the b-Myb R2R3 are somewhat different (McIntosh et al., 1998).

B. The M y b DNA-Binding Domain The c-Myb DNA-binding domain (DBD) consists of three of tandem imperfect Myb repeats as described above ( R l , R2, and R3) (Gonda et al., 1985; Klempnauer and Sippel, 1987).The vertebrate A-Myb and B-Myb, the Drosophila Myb, and the Dictyostelium Myb proteins have conserved all three of these Myb repeats (Lipsick, 1996). During the genesis of v-Myb from c-Myb, deletion of part of the first repeat ( R l )has occurred (Gerondakis and Bishop, 1986; Rosson and Reddy, 1986) (Fig. 2). Such deletions correlate well with efficient oncogenic transformation of myelomonocytic cells (Dini and Lipsick, 1993). However, despite the lack of R1, v-Myb localizes to the cell nucleus and binds to DNA in a fashion similar to that of c-Myb. The minimal region of c-Myb that is both necessary and sufficient for sequencespecific DNA binding has been narrowed down to repeats R2 and R3 (Gabrielsen et af., 1991; Garcia et af., 1991; Howe et al., 1990; Oehler et al., 1990). Unlike many other sequence-specific DNA-binding proteins, the Myb proteins bind to DNA as monomers. Although repeat R1 was not shown to be involved in sequence-specificDNA recognition, it has been shown to in-

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crease the affinity for DNA and the stability of the Myb-DNA complex (Dini and Lipsick, 1993; Ebneth etal., 1994b; Tanikawa etal., 1993).DNA binding has also been suggested to be regulated by the redox state of a highly conserved Cys at position 130 within repeat R2 (Guehmann et al., 1992; Myrset et al., 1993).In other experiments substitution of the homologous Cys by a Ser in v-Myb did not diminish DNA binding in vitro, but did abolish oncogenic transformation and reduce transcriptional activation in animal cells and in yeast (Chen and Lipsick, 1993; Grasser et al., 1992). Selection of a pool of chicken genomic DNA fragments with bacterially produced v-Myb led to the initial identification of the recognition sequence PyAAC(T/G)G(Myb recognition element, or MRE) (Biedenkappet al., 1988). Gratifyingly,three such sites were found 5’ of the transcriptional start site within the promoter of the E26-inducible mim-1 gene (Ness et al., 1989). Similar consensus sequenceswere later found by random oligonucleotideselection with either v-Myb or c-Myb proteins (Howe and Watson, 1991; Weston, 1992). Structural data imply that R3 and R2 cooperatively recognize the AAC core and the last G residue, respectively (Ogata et al., 1994). The third helix of each repeat is the recognition helix that makes specific DNA contacts within the major groove, although the precise mode of recognition by repeats R2 and R3 differs. The linker between R2 and R3 appears to be critical for DNA binding as well (Hegvold and Gabrielsen, 1996). Both v-Myb and c-Myb show greatly reduced affinity for DNA when the recognition sequence is methylated, as often occurs in inactive promoter regions (Klempnauer, 1993). Circular permutation assays have suggested that the Myb proteins bend DNA on binding (Saikumar et al., 1994). However, it remains controversial whether this technique is a valid measure of DNA bending (Hagerman, 1996; Sitlani and Crothers, 1998). A more extensive analysis of the MRE has revealed that the downstream flanking sequence is important for binding as well as for trans activation by the Myb proteins (B. Ganter, S . Chao, and J. S. Lipsick, unpublished). The v-Myb protein, but not the c-Myb protein, requires a stretch of six Ts (motif #2) downstream of the PyAACT/GG site (motif #1) for efficient in vitro binding to the strong mim-1 A site. The presence of repeat R1 within the cMyb DBD allows c-Myb to bind efficiently to the MRE without a downstream motif #2. This observation may explain why c-Myb can bind to a bigger pool of target sites compared to v-Myb. Although the T-stretch improves binding on either strand, it is required on a specific strand for transcriptional activation by both v-Myb and c-Myb. These results suggest that the local promoter structure is very important in this process. A stretch of adjacent T-A base pairs can adopt a Z-DNA-like confrontation that can result in a local bend at the junction of B- to Z-DNA (Wu and Crothers, 1984). Therefore, we suggest that the Myb proteins may require a specific local DNA conformation to induce transcriptional activation. Amino terminal to the three Myb repeats are 30 residues that include a ca-

38

Brigitte Canter and loseph S . Lipsick

sein kinase I1 (CKII) phosphorylation site and a run of acidic amino acids (10 of 15 are Glu or Asp) (Fig. 2). The presence of acidic residues but not the CKII sites is conserved among all known animal Myb proteins. Truncation of the first 30 residues of c-Myb removes both the CKII site and the acidic residues and results in increased DNA binding and transcriptional activation, but not in transformation of myelomonocytic cells in culture (Dini and Lipsick, 1993). However, retroviral insertional mutagenesis in nonbursal B cell lymphomas in chickens removes the first 20 residues of c-Myb, and a retrovirus producing this protein is oncogenic in animals (Jiang et al., 1997).Together these results suggest that the amino-terminal acidic residues and R1 regulate both DNA binding and cell proliferation, but that individual mutations may be cell-type specific in their transforming abilities. In this regard, many published experiments with variants of murine c-Myb called FL lack the first 1 7 residues of c-Myb, raising questions about whether this protein and various mutations derived from it are influenced by this potentially activating N-terminal mutation (Gonda et af., 1989). One of two published chicken c-My6 cDNA clones predicts an additional 60-codon open reading frame upstream of the initiation codon found in all other c-My6 clones from different species (Rosson and Reddy, 1986). Some investigators have reported that this additional open reading frame is specific for the thymus and results from intermolecular recombination with an unlinked gene that encodes an RNA splicing factor (Vellard et al., 1992). However, most other investigators have failed to detect either the hybrid mRNA or the predicted thymus-specific protein product. Therefore, an alternative hypothesis that must be considered is that this upstream open reading frame resulted from the ligation of two different cDNA fragments during the generation of a particular cDNA clone.

C. Transcriptional Activation Domain and Heptad Leucine Repeat v-Myb and c-Myb can activate transcription of model reporter genes that contain multiple Myb-binding sites upstream of a minimal promoter (Ibanez and Lipsick, 1990; Klempnauer et al., 1989; Nishina et af., 1989; Weston and Bishop, 1989). This transcriptional activation can be detected both in animal cells and in budding yeast (Chen and Lipsick, 1993; Punyammalee et al., 1991; Seneca et af., 1993). The transcriptional activation (TA) domain of the c-Myb protein has been mapped near the center of the protein. The limits of the TA domain are not well-defined and appear to depend on the cell line and reporter construct used (Chen et al., 1995; Ibanez and Lipsick, 1990; Kalkbrenner et al., 1990; Sakura et af., 1989). Fusion proteins that contain a heterologous DNA-binding domain and various fragments of vMyb were used to identify a small central acidic domain as the region re-

Myb and Oncogenesis

39

sponsible for transcriptional activation (Weston and Bishop, 1989). However, other studies have shown that this small central domain is not sufficient for transcriptional activation in the context of the native v-Myb protein (Ibanez and Lipsick, 1990). Rather, v-Myb contains several redundant regions that in various combinations are sufficient for transcriptional activation (Chen et al., 1995; Fu and Lipsick, 1996). Furthermore, none of the acidic residues within this central domain are necessary for transcriptional activation either in animal cells or in budding yeast. However, all of these activation regions together are required for oncogenic transformation by vMyb. The central acidic domain is highly conserved among the c-Myb and A-Myb proteins, but to a lesser degree if at all in the B-Myb, urchin Myb, or Drosophila Myb proteins (Figs. 1and 2). This observation is consistent with the failure to demonstrate transcriptional activation by the latter three proteins except under special conditions or in particular cell lines (Foos et al., 1992; Hou et al., 1997; Lane et al., 1997; Mizuguchi et al., 1990; Tashiro et al., 1995; Watson et al., 1993; Ziebold et al., 1997; (J. Manak and J. S. Lipsick, unpublished). In this regard, studies in yeast have shown that -1% of random bacterial open reading frames score positively as eukaryotic transcriptional activation domains, even though that is not their real function (Ma and Ptashne, 1987).The identification of an endogenous gene that is directly activated by B-Myb or Drosophila Myb therefore remains an important unanswered question. Carboxy-terminal to the central acidic domain of c-Myb is a region retained in AMV v-Myb that contains a heptad leucine repeat (HLR) that has been referred to as the “leucine zipper” (Kanei-Ishii et al., 1992). This term was originally coined to describe the dimerization domains of B-ZIP transcription factors such as C/EBP, GCN4, Fos, and Jun, which adopt a specific interdigitating, parallel coiled-coil structure (Landschulz et al., 1988; O’Shea et al., 1989). However, no similar structure has yet been demonstrated for c-Myb (Ebneth et al., 1994a). This heptad leucine repeat of cMyb has been proposed to function as a negative regulator because substitution of specific leucines with proline residues activates the protein, possibly by inhibiting dimerization (Nomura et al., 1993). In contrast, mutational analyses of v-Myb have shown that this region is essential for both transcriptional activation and oncogenic transformation by v-Myb (mutant 1120 versus 1151in Fig. 2), although the leucine residues are not required for these functions (Fu and Lipsick, 1996; Ibanez and Lipsick, 1988,1990). Although substitution of these leucines with alanine did not abolish transformation in culture, leukemogenicity in chickens was abolished, presumably due to temperature sensitivity of the mutant proteins (Bartunek et al., 1997).These results are consistent with the observation that v-Myb of E26, which lacks this region, transforms only weakly in the absence of fusion with the Ets protein (Metz and Graf, 1991).Interestingly, the leucine, isoleucine, and methionine residues of this leucine zipper are not all conserved in Xenopus c-Myb or the

40

Brigitte Canter and JosephS.Lipsick

closely related A-Myb proteins of mammals, birds, and amphibians (Fig. 2). However, other amino acids in this region that are required for transformation and transcriptional activation by v-Myb are highly conserved among the c-Myb, A-Myb, and to a lesser degree the B-Myb proteins (EFAETLQLID). Of additional interest, the alternatively spliced exon 9A of c-Myb inserts approximately 120 amino acids (E9A) just carboxy terminal to these conserved sequences prior to the final leucine of the zipper (Rosson et al., 1987; Shen-Ong, 1989). This larger protein, including E9A-encoded residues, represents 20% or less of the total c-Myb protein in cell types examined thus far. In contrast, sequence motifs encoded by this alternatively spliced exon of c-Myb are present in the major forms of A-Myb, B-Myb, urchin Myb, and Drosophila Myb proteins. B-My6 has been reported to display similar alternative splicing of this exon, albeit to a far lesser degree than c-Myb (Kamano et al., 1995). Exon 9A-encoded residues are not present in either the AMV or E26 v-Myb proteins. However, other studies have shown that the presence of exon 9A is compatible with oncogenic transformation (Woo et al., 1998). Furthermore, exon 9A appears to increase the transcriptional activation by c-Myb proteins that retain either their normal amino or carboxyl termini.

D. Regulation by the Carboxyl Terminus The carboxyl terminus of c-Myb that is deleted in v-Myb has been highly conserved during evolution and appears to function as a regulator of the remainder of the protein (Figs. 1 and 2). Truncation of this region occurs in the v-Myb oncoproteins of AMV and E26, and also occurs as a result of retroviral insertional mutagenesis in some murine myeloid leukemias. A recent publication suggests that carboxy-terminal truncation may activate cMyb by increasing protein stability (Bies and Wolff, 1997). However, a variety of other studies have not revealed significant steady-state differences in the abundance of strongly and weakly transforming mutants of c-Myb (Dini and Lipsick, 1993; Grasser et al., 1991; Hu et al., 1991). Many experiments have suggested that the carboxyl terminus of c-Myb is a negative regulator (NR) of transcriptional activation. First, truncation of this domain results in increased transcriptional activation of model reporter genes bearing Myb-binding sites (Hu et al., 1991; Sakura et al., 1989). Second, replacement of the c-Myb DNA-binding domain with that of the yeast GAL4 protein results in a complete lack of transcriptional activation (Dubendorff et al., 1992; Kalkbrenner et al., 1990). However, a carboxyterminal truncation similar to that of v-Myb strongly activates such GAL4-Myb fusion proteins. The deletion of two nonadjacent regions within the carboxyl terminus (PS and BN in Fig. 2) was required for the activation of GAL4-Myb fusion proteins in lieu of a complete truncation (Duben-

Myb and Oncogenesis

41

dorff et al., 1992). The same double deletion can also significantly activate the oncogenic potential of c-Myb (D. M. Wang and J. S. Lipsick, unpublished). Interestingly, the most conserved region of the carboxyl terminus lies between these two negative regulatory regions, suggesting that it has a different function. However, it is also possible that this region is important in negative regulation but that its structure is perturbed by these adjacent deletions. An analysis of LexA-Myb fusion proteins further demonstrated that the carboxyl terminus of c-Myb inhibited transcriptional activation but neither nuclear transport nor DNA binding. In addition, it was shown that the carboxyl terminus could specificallyinhibit the transcriptional activation domain of c-Myb or v-Myb in trans. These results led to the hypothesis that the c-Myb protein is regulated by intramolecular interactions. Additional experiments have suggested that the carboxyl terminus can also inhibit DNA binding by c-Myb (Ramsay et al., 1991). This was proposed to occur by homodimerization via the heptad leucine repeat (Nomura et al., 1993). The same group of investigators have mapped the inhibition of DNA binding to two nonoverlapping regions of the carboxyl terminus that flank but do not contain the heptad leucine repeat (Tanaka et al., 1997). On the other hand, other investigators have failed to observe inhibition of DNA binding by the carboxyl terminus (Krieg et al., 1995). Further experiments have suggested that there may be a direct regulation of the Myb DNA-binding domain by the carboxyl terminus. In particular, the amino and carboxyl termini of c-Myb were shown to score positively for protein-protein interaction in yeast two-hybrid and phage display assays (Dash et al., 1996; Kiewitz and Wolfes, 1997). Surprisingly, assays of transcriptional activation by various Myb proteins in budding yeast have demonstrated that the carboxyl terminus of c-Myb increases rather than inhibits transcriptional activation in this system (Chen and Lipsick, 1993; Seneca et al., 1993). These latter experiments have therefore led to a model in which negative regulation by the carboxyl terminus of c-Myb requires additional specific animal cell protein(s) not present in yeast. In support of such a model, other intestigators have reported that the carboxyl terminus of c-Myb can increase transcriptional activation in trans in some assays, presumably by titration of a limiting negative regulator (Vorbrueggenet al., 1994).

IV. REGULATION OF V-Myb AND c+Myb A. Sites of Phosphorylation The regulation of transcription factors by phosphorylation has been described for many different proteins (Karin, 1994). Both v- and c-Myb are phosphorylated at multiple sites in vivo, and at least some of these sites seem

42

Brigitte Ganter and Joseph S . Lipsick

\

I*

Fig. 4 Phosphorylation sites in c-Myb and v-Myb. The locations of potential phosphorylation sites in c-hilyb and v-Myb are indicated by asterisks. DBD, DNA-binding domain; R1, R2, and R3, Myb repeats; TA, transcriptional activation domain; NR, negative regulatory domain; HLR, heptad leiicine repeat; CK 11, casein kinase 11; PKA, protein kinase A; GSK 111, glycogen synthase kinase Ill; MAPK, mitogen-activated protein kinase. The dashed line indicates that a similar serine is present in v-Myb, but its phosphorylation by PKA has not yet been examined.

to be of functional importance (Fig. 4). At the N terminus of c-Myb, two ser-

ines (Ser-11 and Ser-12) have been mapped as in vivo phosphorylation sites and these two sites can be modified in vitro by casein kinase I1 (CKII) (Luscher eta/., 1990). Similar sequences are also found in Drosophila Myb and in vertebrate A-Myb, but not in B-Myb or urchin Myb (Fig. 2). Phosphorylation of Ser-1 1 and -12 was initially reported to reduce the DNA-binding activity of c-Myb, and substitution of these two serines by alanines resulted in a decreased cooperativity with NF-M (Luscher et al., 1990; Oelgeschlager et al., 1995). On the other hand, experiments with a shorter form of c-Myb that lacks the entire carboxyl terminus imply just the opposite, namely, that phosphorylation of Ser-11 and -12 by CKII increases DNA-binding activity (Ramsay et al., 1995).The same workers reported that in addition to the Nterminal CKII sites, Ser-116 within the second repeat of c-Myb can be phosphorylated by cyclic AMP-dependent protein kinase A (PKA) in vitro. Again it was suggested that phosphorylation of this site positively affects DNA binding by the c-Myb proteins. In general, it seems that CKII (Ser-11 and Ser12, absent in v-Myb) and PKA (Ser-116) potentially regulate c-Myb function through the N-terminal domain. However, no regulation of this phosphorylation has yet been reported in intact cells. Furthermore, mutation of the CKII sites does not cause oncogenic transformation in culture or in animals (Dini and Lipsick, 1993; Jiang et al., 1997). In addition to the amino-terminal phosphorylation sites (CKII and PKA sites), a number of other phosphorylation sites are present in c-Myb and in

Myb and Oncogenesis

43

v-Myb. Eight potential sites of phosphorylation by MAP kinases (proline-directed protein kinases) conserved between the avian, murine, and human Myb proteins are clustered in or near the carboxy-terminal negative regulatory domain of c-Myb (Aziz et al., 1993). Seven of these conserved sites are deleted in both the E26 and AMV viral oncoproteins. The p42""Pk kinase can phosphorylate avian c-Myb but not v-Myb in vitro on Ser-533, as analyzed by two-dimensional tryptic phosphopeptide mapping (Aziz et al., 1993).The same site is also phosphorylated in vivo and mutation of this serine causes increased transcriptional activation by c-Myb on some promoters, but not others (Aziz et al., 1995; Miglarese et al., 1996; Vorbrueggen et al., 1996). Therefore, it was suggested that modulation of c-Myb function might occur by phosphorylation of specific sites within the negative regulatory domain by MAP kinases. Because MAP kinases are localized both to the nucleus and to the cytoplasm, one might envision that MAP kinases regulate either the nuclear localization of c-Myb, alter the affinity of c-Myb for chromatin, or modulate the function of c-Myb by influencing inter- and intramolecular interactions of the carboxy-terminal negative regulatory domain. In vivo, other kinases, such as p44""pk and which show overlapping substrate specificity with p42""pk kinase (Hall and Vulliet, 1991),might also be involved in the phosphorylation and therefore regulation of c-Myb function. Consistent with this hypothesis, a mitosis-specific phosphorylation of c-Myb has also been reported (Luscher and Eisenman, 1992). For B-Myb the story is somewhat different. It was found that B-Myb is specifically phosphorylated during S phase (Robinson et al., 1996). Furthermore, the presence of ectopically expressed cyclin A stimulates transcriptional activation by B-Myb as well as its ability to promote the entry of cells into the S phase of the cycle (Ansieau et al., 1997; Lane et al., 1997; Sala et al., 1997; Ziebold et al., 1997). A similar increased transcriptional activation by A-Myb but not c-Myb in the presence of cyclin A has also been reported (Ziebold and Klempnauer, 1997).However, others have reported that exogenous cyclin A can stimulate both v-Myb and c-Myb transcriptional activation in a nonspecific fashion (Ganter et al., 1998). The mapping of cyclin A/cdk2 phosphorylation sites within these Myb proteins and an analysis of relevant mutations will therefore be of great interest. A cluster of serine and threonine phosphorylation sites was identified in AMV v-Myb (amino acids 267 to 303) by two-dimensional tryptic peptide mapping and antipeptide antibodies (Bading et al., 1989; Boyle et al., 1991). These sites can be phosphorylated efficiently in vitro by glycogen synthase kinase I11 (GSK-111)(Woodgett, 1991).However, although mutation of these phosphorylation sites greatly reduced the isoelectric heterogeneity of v-Myb, no alternations were observed in transcriptional activation or oncogenic transformation (Fu and Lipsick, 1996). The role of the homologous sites in c-Myb remains to be determined.

44

Brigitte Ganter and Joseph S. Lipsick

B. Interactions with Other Proteins A number of putative Myb-binding proteins (Fig. 5) have been identified. In this regard, several lines of evidence had suggested that specific proteinprotein interactions are important for the function of v-Myb and c-Myb. First, a series of deletions and linker insertions within v-Myb identified mutants that entered the nucleus, bound to DNA, but nevertheless failed to activate transcription or cause oncogenic transformation (Ibanez et a/., 1988; Ibanez and Lipsick, 1988, 1990; Lane et al., 1990). Second, mutations in both the DNA-binding and transcriptional activation domains of v-Myb are required to confer a monoblastic phenotype on transformed cells (Dini et a1.,1995; Introna et al., 1990). Three of the four amino acid substitutions within the v-Myb DNA-binding domain are predicted to lie on the surface of the protein that faces away from the DNA (Ogata et al., 1994). Third, although subsets of various transcriptional activation domains of v-Myb are sufficient for activating the expression of model reporter genes, the presence of all of these activation domains is required for oncogenic transformation (Chen et al., 1995; Fu and Lipsick, 1996). These results suggest that the regulation of multiple cellular target genes by distinct protein-protein interactions is likely to be required for transformation. Fourth, the observation that the carboxyl terminus of c-Myb functions as a negative regulator in animal cells but not in yeast suggests that specific animal cell proteins are required for this regulation (Chen and Lipsick, 1993; Seneca et al., 1993). Several proteins have been reported to interact directly with c-Myb. First, cyclin D was shown to inhibit transcriptional activation by v-Myb and oncogenic variants of c-Myb with similar amino-terminal truncations, but not by c-Myb (Ganter et a/., 1998). Truncation of the first Myb repeat ( R l ) was required for full inhibition by cyclin D. Surprisingly, this inhibition was CDK independent, a theme echoed by studies of a Myb-related cell cycle regulator DMP-1 that is also inhibited by cyclin D in a CDK-independent fashion (Hirai and Sherr, 1996; Inoue and Sherr, 1998). A second protein, Cyp40, was shown to inhibit DNA binding in vitro by c-Myb but not v-Myb (Leverson and Ness, 1998). This protein is a cyclophilin that contains a prolyl isomerase domain and a series of TPR repeats. Interestingly, the mutations present in the v-Myb DNA-binding domain specifically prevented inhibition of DNA binding by Cyp40. Because cyclosporin A inhibits the prolyl isomerase activity of Cyp40 and its ability to block DNA-binding by the c-Myb DNA-binding domain, it will be of interest to determine the effects of cyclosporin and other prolyl isomerase inhibitors on transcriptional activation by v-Myb and c-Myb. The central transcriptional activation domains of c-Myb and A-Myb have been reported to interact directly with the closely related CBP and p300 coactivators (Dai et al., 1996; Facchinetti et al., 1997; Kiewitz and Wolfes, 1997;

45

Myb and Oncogenesis

Chicken c-Myb

Domain of protein-protein interaction .Interaction with CBP -Interactionwith c y c h D

I

- Interaction with pl00

I

-Interaction with HF3 -Interaction with BS69

-

192

38

-Interaction with plM)

-Regionsof intramolecular interaction

317 I 342

192

38

192

-

192

426

-

-

561

375 I 405

513

563

Fig. 5 Sites of protein-protein interaction in c-Myb. Domains of c-Myb that have been reported to interact with other cellular proteins are indicated by black bars. The numbers indicate residues in chicken c-Myb without exon 9A.

Oelgeschlager et al., 1996).These proteins were initially discovered as PKAdependent coactivators of the CREB transcription factor and as cellular proteins that coprecipitated with the adenovirus E l A transforming protein (Goldman et al., 1997). Over the past few years a wide variety of sequencespecific transcription factors have been reported to bind directly to CBP and p300, including the retinoic acid receptor, the glucocorticoid receptor, Jun, Fos, STAT proteins, p53, Rb, and c-Myb. Interestingly, both CBP and p300 were reported to act as histone acetylases (Bannister and Kouzarides, 1996; Ogryzko et al., 1996). Various experiments have led to a model in which CBP/p300 are limiting coactivators in the cell and competition for them by different classes of activators (e.g., glucocorticoid receptor and Fos/Jun) results in inhibitory cross-talk between different signaling pathways (Kamei et al., 1996). Such a model is supported by the observation that CBP demonstrates haplo-insufficiency in humans-one mutant copy of the gene results in the multiple developmental defects that constitute the Rubinstein-Taybe syndrome (Petrij et al., 1995). In the case of c-Myb, it remains unknown whether CBP and p300 binding alone are necessary or sufficient for transcriptional activation or oncogenic transformation. In addition, it will be interesting to determine whether the previously reported inhibition of v-Myb transformation by liganded retinoic acid receptor is the result of such competition for CBP and/or p300 (Smarda et al., 1995). Several different proteins have been reported to bind directly to or mimic the carboxyl terminus of c-Myb. First, a protein that interacts with the heptad

46

Brigitte Canter and Ioseph S. Lipsick

leucine repeat of c-Myb was identified by pull-down assays and the relevant gene was later cloned (Favier and Gonda, 1994; Tavner et al., 1998). This p 160 protein appears to be present predominantly in the nucleolus and has some sequence similarity to known nucleolar proteins. However, Myb proteins appear to be specifically excluded from the nucleolus (Boyle et al., 1984; Klempnauer et al., 1984). In some hematopoietic cells, p160 is processed to a shorter cytoplasmic p67 form. In transient transfections, cDNAs encoding p67 but not p l 6 0 inhibited transcriptional activation by c-Myb. Second, a plOO protein that contains an EVES motif similar to that in the carboxyl terminus of c-Myb has also been reported to bind to the c-Myb DNA-binding domain and inhibit transcriptional activation by c-Myb (Dash et al., 1996).The EVES motif is a site for phosphorylation of c-Myb by MAP kinase, raising the possibility of a reversible and regulatable binding of plOO to c-Myb. The p100 protein was originally discovered in a yeast two-hybrid screen with the Epstein-Barr virus EBNAZ protein (Tong et al., 1995). In contrast to the results with c-Myb, p l 0 0 was reported to increase transcriptional activation by EBNA2. The EVES motif is not highly conserved among other Myb proteins, including A-Myb, B-Myb, and Drosophila Myb, implying that it may be a specific regulator for c-Myb alone. Third, the BS69 protein was recently identified in a yeast two-hybrid screen using the carboxyl terminus of c-Myb as bait (N. Collins and J. S. Lipsick, unpublished). This protein was initially identified as an E l A-binding protein than can inhibit transcriptional activation by E1A (Hateboer et al., 1995). We have found that BS69 can also inhibit transcriptional activation by fulllength c-Myb, but not by carboxy-terminal truncations of c-Myb. The vertebrate BS69 protein is present within the nucleus and contains several motifs common to other transcriptional regulators, including a PHD finger, a BKOMO-like domain, and a MYND domain that is also found in MTG8, Nervy, and DEAF-1. Furthermore, BS69 has been highly conserved during evolution, suggesting that perhaps it may regulate a variety of other Myb proteins via their highly conserved carboxyl termini, including A-Myb, BMyb, and Drosophila Myb (J. Manak and J. S. Lipsick, unpublished). The functional importance of all these putative Myb-binding proteins remains to be tested by appropriate genetic analyses.

V. TRANSCRIPTIONAL REGULATION BY V-Myb AND C-Myb A consensus DNA-binding sequence for the v-Myb protein was identified by analysis of a pool of random genomic DNA fragments that were selected with bacterially produced Ah4V v-Myb protein (Biedenkapp et al., 1988). Subse-

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quently several laboratories showed that concatemers of this consensus sequence PyAAC(T/G)Gcan confer v-Myb- and c-Myb-dependentinducibility to a variety of test promoters (Ibanez and Lipsick, 1990; Klempnauer et al., 1989; Nishina et al., 1989; Weston and Bishop, 1989). In addition, similar binding sites were found within the promoter of mim-I, a cellular gene that is directly regulated by the E26 and c-Myb proteins (Ness et al., 1989). However, v-Myb and c-Myb can also activate promoters that do not contain Myb-binding sites, presumably by more indirect mechanisms (Engelke et al., 1995b; Ibanez and Lipsick, 1990; Kanei-Ishii et al., 1994,1997; Klempnauer et al., 1989).

A. Genes Activated by vcMyb and e M y b Oncogenic transformation of myelomonocytic cells by v-Myb and truncated forms of c-Myb initially appeared to correlate well with their ability to activate transcription of model reporter genes (Hu et al., 1991; Lane et al., 1990), suggesting that cell transformation depends on the activation of crucial target genes. However, the v-Myb protein encoded by the naturally occurring isolate of AMV is an extremely weak transcriptional activator and stronger activation does not correlate well with transformation (Engelke et al., 1995b). On the contrary, mutants of AMV v-Myb have been identified that activate transcription better than the wild-type protein but fail to transform myelomonocytic cells oncogenically in culture (Chen et al., 1995). Transcriptional activation by mutants of c-Myb also does not strictly correlate with transformation (Dini and Lipsick, 1993).Furthermore, although the acidic region of the HSV VP16 protein can substitute for the acidic region of v-Myb in oncogenic transformation, a simple v-Myb-VP16 fusion protein that strongly activates transcription does not itself cause oncogenic transformation (Engelke et al., 1995b; Frampton et al., 1993). These results suggest a model in which v-Myb has multiple transcriptional regulatory domains that are required for the regulation of specific “target” genes, all of which are required for oncogenic transformation (Chen et al., 1995). Because the Myb proteins bind to a rather small recognition site (AACNG), such potential recognition sites’can be found in almost any random piece of DNA of 1 kb or longer. For example, the commonly used E. coli plasmid pUC contains several Myb-binding sites. Therefore, the presence of such sites alone within a promoter does not necessarily imply that the gene in question is really regulated by Myb even if transient transfection studies seem to support such a conclusion. Nevertheless, many publications have identified genes thought to be regulated by v-Myb and c-Myb solely on the basis of DNA-binding and transient transfection assays. A more complete listing and discussion of genes proposed to be regulated by Myb proteins is provided in a review by Ness (1996).We will limit our discussion here to those genes for

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which genetic evidence has been provided for direct regulation by Myb proteins. The first Myb-regulated gene to be identified was mim-1, a promyelocytespecific gene that is inducibly regulated by a temperature-sensitive mutant of the E26 Gag-Myb-Ets protein (Ness et al., 1989). This gene can also be induced by c-Myb but not by v-Myb of AMV, and it is not expressed in AMV- , transformed monoblasts, excepting for the AMV-transformed BM2 cell line (Dini et al., 1995; Queva et al., 1992). The promoter of the mim-1 gene contains three closely spaced Myb-binding sites and is strongly activated by vMyb and c-Myb in transient transfection assays. Only the strongest of these three Myb-binding sites is required for this activation in transient assays. Although c-Myb is expressed in many different types of hematopoietic and lymphoid cells, mim-1 gene expression is only detectable in granulocytic cells that constitute a small subset of those cell types that express c-My6 (Queva et al., 1992). Furthermore, some cells that express mim-1 in the developing embryo do not express c-My6. These observations were partially reconciled when it was shown that the myeloid-specific transcription factor NF-M, a B-ZIP protein that is the homolog of the mammalian C/EBP-P or NF-IL-6 protein (Sterneck et al., 1992), is required for the induction of mim-1 expression by Myb proteins (Ness et al., 1993). Remarkably, the introduction of c-Myb and NF-M into nonhematopoietic cells is sufficient to induce expression of the endogenous mim-1 gene (Burk et al., 1993; Ness et al., 1993). It has been reported that the Myb and C/EBP proteins interact directly via their DNA-binding domains even in the absence of DNA (Mink et al., 1996). Interestingly, proteins of the Myb and C/EBP families have also been reported to synergize in activating the promoters of other myeloid-specific genes (Burk et al., 1997). Another approach for identifying Myb-regulated genes has been to analyze a v-Myc-transformed chicken macrophage cell line that contains a hybrid E26/AMV v-Myb protein fused to the hormone-binding domain of the human estrogen receptor (Burke and Klempnauer, 1991). The molecular cloning of cDNAs that are differentially expressed in the presence or absence of estrogen has led to the identification of three additional Myb-regulated genes-chicken lysozyme, adenosine receptor 2B, and tom-1 (Burk et al., 1997; Worpenberg, et al., 1997). The latter gene is coregulated by Myb and C/EBP, as described above for mim-1. A similar approach has utilized a murine myeloid cell line transformed by a carboxy-terminal truncation of c-Myb fused to the hormone-binding domain of the estrogen receptor (Hogg et al., 1997). In these experiments the c-kit growth factor receptor gene was found to be a direct target of regulation by Myb. This is consistent with the presence of functional Myb-binding sites within the c-kit promoter region (Yamamoto et al., 1993). In contrast, the expression of c-Myc and Cdc2, which have also been proposed to be di-

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rectly regulated by c-Myb, was not altered by withdrawal of estrogen. Furthermore, Bcl2, an antiapoptosis gene also proposed to be directly regulated by c-Myb, was still expressed after withdrawal of estrogen and apoptosis did not occur. Myelomonocytic cells transformed by AMV v-Myb do not require exogenous chicken myeloid growth factor, whereas those transformed by v-Myb proteins lacking the amino acid substitutions in either the DNA-binding or transcriptional activation domains are growth factor dependent (Dini et al., 1995).Indeed, transformation by AMV v-Myb appears to drive an autocrine loop that results in the production of cMGF by the transformed cells themselves. These results suggested that AMV v-Myb might either directly or indirectly regulate the promoter of the cMGF gene. Recent studies have shown that the cMGF promoter is directly regulated by the homeobox-containing GBX2 protein, and that the G B X 2 gene is regulated by AMV v-Myb (Kowenz-Leutzet al., 1997). G B X 2 was directly regulated by an AMV-E26 Myb-estrogen receptor fusion protein that contains only a single amino acid substitution present in the DNA-binding domain of v-Myb, which by itself does not confer the full AMV v-Myb phenotype. In contrast, G B X 2 was not induced by c-Myb in the absence of additional signal transduction. The authors concluded that mutations in the DNA-binding domain of AMV v-Myb render it independent of signaling events that are normally required for cMyb to activate GBX2. The discovery of this Myb/GBX2/growth factor axis is the first example of gene regulation by v-Myb that clearly contributes to oncogenic transformation.

B. Transcriptional Repression by Myb Proteins The failure of transcriptional activation by v-Myb to correlate well with oncogenic transformation raises the possibility that repression as well as activation may be important in this process. An analysis of the effects of Dtype cyclins on transcriptional activation by v-Myb favors the importance of repression in oncogenic transformation. In particular, cyclins D1 and D2 specifically inhibit transcription when activated through the v-Myb DNAbinding domain, but not the c-Myb DNA-binding domain (Ganter et al., 1998). The D-type cyclins belong to a subfamily of cyclins, proteins that are thought to govern transitions through distinct phases of the cell cycle by regulating the activity of cyclin-dependent kinases (CDKs) (Sherr, 1993). The D-type cyclins are strongly implicated in controlling progression through the G,/G, phase of the cell cycle. However, the inhibition of v-Myb by D-type cyclins appears to be independent of CDKs. This was shown by utilizing a dominant negative CDK4 mutant and a cyclin D1 mutant that cannot bind to its cyclin kinase partners (Ganter et al., 1998).

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The biological relevance of these observations was supported by experiments showing that when a v-Myb-transformed monoblast cell line is induced to differentiate with phorbol esters, cyclin D levels go down and transcriptional activation by v-Myb goes up. These results suggested that repression rather than activation of some genes may correlate with v-Myb transformation. Consistent with this finding, a recent analysis of the Mybbinding sites within the N-Ras promoter revealed that both c-Myb and v-Myb can function as repressors by significantly reducing promoter activity in transient assays (Ganter and Lipsick, 1997). Furthermore, c-Myb has been shown to repress the c-ErbB2 promoter by direct competition with the TATA-binding protein in similar assays (Mizuguchi et al., 1995). The regulation of Myb-related proteins by D-type cyclins may be a more general phenomenon, because the distantly related Myb protein, DMPl, has recently been isolated in a yeast two-hybrid screen using cyclin D2 as the bait (Hirai and Sherr, 1996). Interestingly, cyclin D inhibits DNA binding by DMP1, also in a CDK-independent fashion (Inoue and Sherr, 1998). Furthermore, it has recently been shown that the estrogen receptor could be activated by cyclin D in a CDK- and ligand-independent fashion, arguing that cyclin D may have a wider role in transcriptional regulation than previously thought (Zwijsen et al., 1997).

VI. THE Myb-CHROMATIN CONNECTION Several additional transcriptional regulators have been identified that contain more distantly related Myb repeats (Table I). These Myb-repeat-containing proteins can be grouped into different families, including the telobox family (Bilaud et al., 1996), the SANT-domain family of transcriptional regulators (Aasland et al., 1996),and the transcription terminator family (Reeder and Lang, 1997). The telobox proteins include the recently identified human and mouse telomere-binding proteins TRFl and TRF2 (Bilaud et af., 1997; Broccoli et al., 1997; Chong et af., 1995), TBFl (Bilaud et al., 1996; Brigati et af., 1993), and the S. pombe Tazl protein (Cooper et al., 1997). Amino acid sequence comparison revealed that all of these proteins contain one or two distantly related Myb repeats. In addition, the three-dimensional X-ray structure of the major telomere-binding protein in budding yeast, RAPlp, unexpectedly revealed a Myb-related DNA-binding domain as a protein fold for telomeric DNA recognition (Konig et al., 1996; Konig and Rhodes, 1997). The SANT-domain-containing proteins include the SWISNF component SWI3 (Peterson and Herskowitz, 1992; Wang et al., 1996; Yoshinaga et al., 1992),the ISWI protein that participates in nucleosome remodelling and that also has significant similarity to SWI2 (Tsukiyama et al.,

Table I Myb Domain-Containing Proteins Involved in Transcription and Chromatin Remodeling" Protein family/name

Organism

Myb family A-Myb Mammals, birds, amphibians B-Myb Mammals, birds, amphibians c-Myb Mammals, birds, amphibians D-Myb Drosophila V-Myb AMV, E26 virus Telobox proteins Taz 1 Schizosaccharomyces pombe TBFl Saccharomyces cerevisiae TRFl Human, mouse TRF2 Human, mouse SANT-domain-containing proteins ADA2 Human S. cerevisiae B"

Saccharomyces cerevisiae

N-COR

Mouse

RSC-8 Saccharomyces Drosophila, human, S. cerevisiae I-SWI proteins SWI3 proteins Human, S. cerevisiae Transcription termination signals Rebl Saccharomyces cerevisiae, S. pombe TTFl Human, mouse Other proteins RAP1 SNAP190

Saccharomyces cerevisiae Human

Protein complex

Function

DNA binding

Transcriptional regulator, cell cycle control? Transcriptional regulator, cell cycle control? Transcriptional regulator, cell cycle control? Transcriptional regulator, cell cycle control? Leukemogenesis

Yes Yes Yes Yes Yes

Regulation of telomere length ? Regulation of telomere length Prevention of telomere fusion

Yes Yes Yes Yes

Transcriptional adapter, binds to histone acetyltransferase (GCNS) Subunit of TFIIIB

No No

Transcriptional corepressor

No

Chromatin remodeling Chromatin remodeling Chromatin remodeling

No No No

RNA pol I termination complex RNA pol I termination comp1ex

RNA pol I terminator

Yes

RNA pol I terminator

Yes

Telomere complex SNAPc complex

Regulation of telomere length, silencing snRNA transcriptional initiation by RNA pol I1 and I11

Yes Yes

Telomere protein Telomere protein Telomere protein Telomere protein

complex complex complex complex

ADA and SAGA complex RNA pol 111 initiation complex N-CoR/mSin3/mRPD3 corepressor complex RSC complex NURF complex SWI-SNF complex

"For a review of Myb-related proteins in plants, see Martin and Paz-Ares (1997).

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Brigitte Canter and loseph S . Lipsick

1995), the ADA component ADA2 that complexes with the GCN5 histone acetylase (Berger et al., 1992; Candau et al., 1996), the corepressor N-CoR (Horlein et al., 1995), and the B subunit of TFIIIB (Kassavetis et al., 1995), a component of the RNA polymerase I11 initiation complex. The transcription terminator signal proteins with Myb-related sequences include the yeast Rebl protein and the mouse mTTF1 (Evers etal., 1995;Ju etal., 1990; Lang and Reeder, 1993). Interestingly, although all of these proteins are either involved in transcriptional regulation or chromatin remodeling, some of them have no intrinsic DNA-binding activity. The conservation of the Myb domain during the evolution of different transcriptional regulators/chromatin effectors that do not bind to DNA suggests another function for the Myb domain such as in specific protein-protein interactions that alter DNA accessibility in chromatin. We believe that carefully designed experiments will reveal whether the Myb domain has such a general function in chromatin remodeling, telomere binding, and transcription. Such non-DNA-binding functions of the Myb domain are likely to be important in understanding oncogenic transformation by v-Myb and c-Myb, as well. We therefore look forward to learning what other secrets the Myb proteins still harbor.

ACKNOWLEDGMENTS We thank the members of our laboratory, past and present, and our colleagues in the world of My6 research for their scientific efforts and their helpful discussions. Research in our own laboratory was supported by the National Cancer Institute of the United States Public Health Service. BG was supported in part by the Swiss National Science Foundation.

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Torelli, G., Selleri, L., Donelli, A., Ferrari, S., Emilia, G., Venturelli, D., Moretti, L., and Torelli, U. (1985).Mol. Cell. Biol. 5,2874-2877. Torelli, G., \'enturelli, D., Colo, A., Zanni, C., Selleri, L., Moretti, L., Calabretta, B., and Torelli, U. (1987).Cancer Res. 47,5266-5269. Toscani, A., Mettus, R. V., Coupland, R., Simpkins, H., Litvin, J., Orth, J., Hatton, K. S., and Reddy, E. P. (1997).Nature (London) 386, 713-717. Toth, C. R., Hostutler, R. F., Baldwin, Jr., A. S., and Bender, T. P. (1995).J. Biol. Chem. 270, 7661-7671. Trauth, K., Mutschler, B., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and Klempnauer, K. H. (1994).EMBO]. 13,5994-6005. Travali, S., Reiss, K., Ferber, A., Petralia, S., Mercer, W. E., Calabretta, B., and Baserga, R. (1991).Mol. Cell. Biol. 11, 731-736. Tsukiyarna, T., Daniel, C., Tamkun, J., and Wu, C. (1995).Cell 83, 1021-1026. Urbanek, P., Dvorak, M., Bartunek, P., Pecenka, V., Paces, V., and Travnicek, M. (1988).Nucleic Acids Res. 16, 11521-11530. Vellard, M., Sureau, A., Soret, J., Martinerie, C., and Perbal, B. (1992).Proc. Natl. Acad. Sci. U.S.A. 89, 2511-2515. Vorbrueggen, G., Kalkbrenner, F., Guehrnann, S., and Moelling, K. (1994).Nucleic Acids Res. 22,2466-2475. Vorbrueggen, G., Lovric, J., and Moelling, K. (1996).Biol. Chem. 377, 721-730. Wallrapp, C . , Muller-Pillasch, F., Solinas-Toldo, S., Lichter, P., Friess, H., Buchler, M., Fink, T., Adler, G., and Gress, T. M. (1997).Cancer Res. 57, 313.5-3139. Wang, W., Xue, Y., Zhou, S., Kuo, A., Cairns, B. R., and Crabtree, G. R. (1996). Genes Dev. 10,2117-2130. Watson, R. J. (1988).Oncogene 2,267-272. Watson, R. J., Dyson, P. J., and McMahon, J. (1987).E M B O J . 6, 1643-1651. Watson, R. J., Robinson, C., and Lam, E. W. (1993).Ntrcleic Acids Res. 21,267-272. Westin, E. H., Gallo, R. C., Arya, S. K., Eva, A., Souza, L. M., Baluda, M. A,, Aaronson, S. A,, and Wong-Staal, F. (1982).Proc. Natl. Acad. Sci. U.S.A. 79,2194-2198. Weston, K. (1992).Nucleic Acids Res. 20, 3043-3049. Weston, K., and Bishop, J. M. (1989).Cell 58, 85-93. Wolff, L. (19961. Crit. Rev. Oncogen. 7,245-260. Wolff, L., Mushinski, J. E, Shen-Ong, G. I.., and Morse, H. C. D. (1988).J. Immunol. 141, 681-689. Woo, C. H., Sopchak, L., and Lipsick, J. S. (1998).]. Virol. 72,6813-6821. Woodgett, J. R. (1991).Trends Biochem. Sci. 16, 177-181. Worpenberg, S., Burk, O., and Klempnauer, K. H. (1997).Oncogene 15,213-221. Wu, H.-M., and Crothers, D. M. (1984). Nature (London) 308,509-513. Yamamoto, K., Tojo, X., Aoki, N., and Shibuya, M. (1993).Jpn.J.Cancer Res. 84,1136-1144. Yoshinaga, S . K., Peterson, C. I.., Herskowitz, I., and Yarnarnoto, K. R. (1992).Science 258, 1.598-1604. Ziebold, U., and Klempnauer, K. H. (1997).Oncogene 15,1011-1019. Ziebold, U., Bartsch, O., Marais, R., Ferrari, S., and Klempnauer, K. H. (1997).Curr. Biol. 7, 253-260. Zwicker, j., Liu, N., Engeland, K., Lucibello, F. C., and Muller, R. (1996). Science 271, 1595-1597. Zwijsen, R. M., Wientjens, E., Klompmaker, R., van der Sman, J., Bernards, R., and Michalides, R. J. (1997).Cell 88,405-415.

cmSrc, Receptor Tyrosine Kinases, and Human Cancer JacquelineS. BLcardi,*David A. Tice,* and Sarah J. Parsons? Department of Microbiology and Cancer Center University of Virginia Health Sciences Center Charlottesville, Virginia 22908

I. Introduction 11. Receptor Tyrosine Kinases and Human Cancers A. Hepatocyte Growth FactorlScatter Factor Receptor B. Colony-Stimulating Factor-1 Receptor C. Fibroblast Growth Factor Receptors D. Platelet-Derived Growth Factor Receptor E. Epidermal Growth Factor Receptor F. HER2lneu G. HER Family Members and Estrogen Receptor Interactions 111. c-Src and c-Src Family Members in Human Cancers A. c-Src Structure and Mechanisms of Regulation B. Evidence for the Involvement of c-Src in Human Cancers C. c-Src Family Members and Human Cancers D. Nonreceptor Tyrosine Kinases Related to c-Src Family Members and Human Cancers IV. Mechanisms of c-Src Action A. Evidence for Involvement of c-Src in Signaling through Receptor Tyrosine Kinases B. Targets of c-Src V. Potential Therapeutic Applications of c-SrclHER1 Interactions References

I. INTRODUCTION Since the discovery that tyrosine kinases are among the transforming proteins encoded by oncogenic animal retroviruses, it has been speculated that this family of enzymes may contribute to the development of human malignancies. However, evidence supporting that hypothesis has been slow to evolve, largely because early emphasis was placed on examining human tumors for genetic alterations in protooncogenes encoding these enzymes. Such alterations have proved rare or nonexistent. Instead, investigations have fo‘Equal contributions were made by these authors. tTo whom correspondence may be addressed. Advances in CANCER RESEARCH 0065-23OW99 $30.00

Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved.

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cused on determining levels of expression and posttranslational mechanisms of regulation of these proteins, particularly as they relate to signaling pathways that modulate growth, adhesion, invasion, and motility. Two classes of tyrosine kinases have emerged as potentially important players in promoting the evolution of human tumors: receptor kinases (RTKs) and nonreceptor tyrosine kinases of the c-Src family. Elevated levels of both these classes of tyrosine kinases can be found in a large number of tumors in a strikingly similar pattern of aberrant cooverexpression, suggesting that the two families may cooperate with one another during oncogenesis. Indeed, in model tissue culture systems, overexpression of receptor alone can result in malignant transformation when a continuous source of ligand is provided. However, overexpression of c-Src alone is non- or weakly oncogenic. These results indicate that c-Src, if it plays a role in tumorigenesis, most likely mediates its effects through RTKs. Demonstrations that c-Src physically associates with a number of RTKs in a ligand-dependent fashion provided some of the first evidence for functional cooperativity between these families of proteins. Subsequent studies showed that in complex, the two kinases reciprocally affect one another’s behavior, such that c-Src can be regarded both as a regulator of RTKs and as a cotransducer of signals emanating from them. c-Src is capable of physically associating with the receptors for platelet-derived growth factor (PDGF), prolactin, epidermal growth factor (EGF),colony-stimulating factor-1 (CSF-l), fibroblast growth factor (FGF), and hepatocyte growth factor/scatter factor (HGF/SF), as well as with the HER2/neu and Sky tyrosine kinases (this review and Toshima et al., 1995; Berlanga et al., 1995), all of which are postulated to play a role in the genesis and/or progression of various human cancers. Although c-Src and its family members are also known to participate in signaling events elicited by heterotrimeric G protein-coupled receptors (Malarkey et al., 1995) and neuronal ion channels (Ely et al., 1994; Holmes et al., 1996; Yu et al., 1997; van Hoek et al., 1997), this review focuses on the interactions of c-Src and Src family members with RTKs because of the growing documentation of the interactions between these proteins in human malignancies. First, a summary is presented, naming the RTKs that are most frequently implicated etiologically in human cancers and that have been shown to interact with c-Src. This summary includes a short review of the physical characteristics of the receptors, their molecular mechanisms of signaling, and their putative roles in specific cancers. Second, evidence is discussed for the involvement of c-Src and Src family members in human tumor development, and third, a synopsis is outlined showing the molecular mechanisms by which c-Src and its family members have been found to interact with receptors and other targets. Finally, we will speculate on the prospects for developing novel therapies based on these interactions.

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11. RECEPTOR TYROSINE

A N D HUMAN CANCERS

KINASES

Figure 1 depicts the structural features of several classes of RTKs that interact with c-Src. All consist of an extracellular ligand-binding domain that bears motifs characteristic of the type of receptor (e.g., repeated immunoglobulin-like motifs for the PDGF and FGF receptors or cysteine-rich motifs in the EGF family of receptors), a transmembrane segment, a tyrosine

Fig. 1 Structures of receptor tyrosine kinase families known to associate with c-Src. All receptors are transmembrane glycoproteins that function as receptors for polypeptide growth factors. Structurally, these molecules are composed of large, extracellular domains that exhibit characteristic ligand-binding motifs, as well as transmembrane, juxtamembrane, catalytic, and C-terminal domains. Ligand binding induces dimerization, enzymatic activation, and autophosphorylation on specific tyrosine residues in the C-terminal domains. These phosphorylated tyrosine residues serve as docking sites for signaling molecules that transmit biological signals from the extracellular milieu to the nucleus. In the platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) receptor families, the kinase domain is interrupted by an insert that contains additional docking sites. EGFR, Epidermal growth factor receptor; CSF-IR, colony-stimulating factor-1 receptor; HGF/SFR, hepatocyte growth factor/scatter factor receptor; HER, human epidermal growth factor receptor.

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kinase catalytic domain, and a carboxy-terminal region that contains sites of autophosphorylation. Binding of ligand causes dimerization of the receptor, activation of tyrosine kinase activity, and (trans) autophosphorylation of specific C-terminal tyrosine (Tyr) residues (reviewed in Heldin, 1996; Weiss et al., 1997), which in turn serve as docking sites for a variety of signaling molecules that contain SH2 domains (Pawson and Schlessinger, 1993), including phospholipase Cy (PLCy), phosphatidylinositol-3 kinase (PI-3) kinase), GTPase-activating protein of Ras (RasGAP), phosphotyrosine phosphatases (PTPases), Janus kinasedsignal transducers and activators of Transcription (JAK/STATS), adapter proteins (including Shc, Grb, Nck), and members of the c-Src family of tyrosine kinases (reviewed in Erpel and Courtneidge, 1995; Heldin, 1996). Signals are subsequently transmitted to the nucleus via several pathways, including the JAK/STAT and the Grb2/SOS/Ras/Raf/MEK/MAP kinase cascades (reviewed in Bonfini et al., 1996; Denhardt, 1996). Members of the STAT and MAP kinase families translocate from the cytoplasm to the nucleus and induce changes in gene expression, which bring about a variety of functional outcomes, such as mitogenesis, morphogenesis, and motility. The contribution of c-Src to downstream signaling from these RTKs has been the subject of growing interest, with emphasis on how c-Src may contribute to transformation and maintenance of the cancerous phenotype that is dependent on and induced by the receptors. In this treatise, a total of five RTK families and their putative roles in development of malignancy will be considered. The first four, receptors for HGFISF, CSF-1, FGF, and PDGF, are implicated as etiological agents in a wide variety of human cancers, and their ability to influence processes such as cytoskeletal changes, cell motility, and angiogenesis are thought to contribute to the metastatic potential of tumors. The fifth group, members of the EGF receptor family (HERl-4), will be discussed in the context of breast cancer, along with the estrogen receptor. This steroid hormone receptor plays a pivotal role in the etiology of breast cancer and growing evidence indicates its ability to reciprocally interact with c-Src and members of the HER family of RTKs.

A. Hepatocyte Growth FactodScatter Factor Receptor The Met tyrosine kinase is the receptor for hepatocyte growth factor/scatter factor (Bottaro etal., 1991; Naldini et al., 1991). This receptor was first identified as the product of the human oncogene, tpr-met, which was isolated from a chemically treated human cell line by the NIH3T3 gene transfer method (Cooper et al., 1984; Park et al., 1987). The normal cellular receptor is composed of two subunits, a 145-kDa p chain, which spans the cell

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membrane and possesses ligand-binding and tyrosine kinase activity, and a 50-kDa a chain, which resides extracellularly and is covalently bound to the p subunit through disulfide linkages (Gonzatti-Haces et al., 1988). Related family members include the Sea and Ron RTKs (Ronsin et al., 1993; Huff et al., 1993).Each member of the Met family possesses two tandemly arranged, degenerate YVH/NV motifs in the C-terminal tail of the receptor, which are capable of binding the SH2 domains of the signaling molecules PI-3 kinase, PTPase 2, PLCy, c-Src, and Grb2/Sos (Ponzetto et al., 1994). Mutations in these motifs (H1351N) result in increased transforming ability but decreased metastasis (Giordano etal., 1997), a phenomenon that is linked to the creation of an additional Grb2 binding site and hyperactivation of the Ras pathway. HGF/SF is produced by cells of mesodermal origin and acts on epithelial and endothelial cells, eliciting numerous biological responses, including cell motility, growth, morphogenesis, differentiation, and angiogenesis (Kan et al., 1991; Rubin et al., 1991; Halaban et al., 1992).Which response is elicited in part depends on the cell type, developmental stage, and tissue context (Weidner et al., 1993; Kanda et af., 1993; Zhu et al., 1994; Rosen and Goldberg, 1995; Grano et al., 1996). For example, Met signals through STAT3 to induce the formation of branched tubule structures in Madin-Darby canine kidney (MDCK) cells, a hallmark of angiogenesis (Boccaccio et ul., 1998). HGF binding to primary human osteoclasts and osteoblasts triggers receptor kinase activity and autophosphorylation in both cell types. However, in osteoclasts, HGF binding is accompanied by increased levels of intracellular calcium, activation of c-Src, changes in cell shape, stimulation of chemotaxis, and DNA replication, whereas osteoblasts respond simply by undergoing DNA synthesis (Grano et al., 1996). Furthermore, osteoclasts also express HGF, but osteoblasts do not. This finding suggests that an autocrine loop may be responsible for signaling in osteoclasts, whereas a paracrine mechanism is functional in osteoblasts. HGF/SF and the Met receptor have been implicated in several types of human cancer. Met is overexpressed in gastric, ileal, colorectal, and thyroid papillary carcinomas, as well as in osteogenic sarcoma (Di Renzo et al., 1991, 1992; Rosen et al., 1994; Grano et al., 1996). The level of Met expression, as measured by intensity of Met immunofluorescence, has also been shown to correlate with grade of malignancy in primary human brain tumors (Koochekpour et al., 1997). In the case of ovarian carcinoma, Met levels can be regulated by the cytokines interleukin la (IL-la),IL-6, and tumor necrosis factor a (TNFa),thereby providing a physiological mechanism by which overexpression of Met can be achieved (Moghul et al., 1994). Approximately 14% of patients with papillary renal carcinoma have germ-line alterations in the Met receptor (Schmidt et al., 1997). Receptors bearing these mutations have been shown in NIH3T3 cells to result in increased tyrosine kinase activity of the receptors and Met-mediated focus formation

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and tumors in nude mice, thus providing direct evidence for the ability of mutationally altered Met to function as an oncogene (Jeffers et al., 1997). The ability of HGF/SF to “scatter” cells and to increase their motility is strongly suggestive of a role for this ligand in tumor cell invasion. Indeed, several lines of evidence link HGF/SF to stimulation of the urokinase plasminogen activator (UPA) system, a cascade of proteases thought to promote release, extravasation, and migration of tumor cells. That the UPA cascade is critical for cell migration is supported by the findings that UPA -/- mice are unable to recruit migrating cells in response to inflammation (Gyetko et al., 1996), and do not support the growth and metastasis of experimental melanomas (Min et al., 1996). Shapiro et al. (1996) also showed that blocking interaction of UPA with its receptor results in decreased angiogenesis and tumor spread. The link between UPA and HGF was made when Jeffers et al. ( 1996b) reported that stimulation of the urokinase proteolytic system occurred concomitantly with HGF/SF-induced invasion and metastasis of human tumor cells. Rosen and Goldberg (1995) also demonstrated that HGF/SF is capable of stimulating angiogenesis in a rat cornea neovascularization assay. Together, these studies provide compelling evidence that HGF/SF are capable of promoting tumor progression by enhancing invasion and angiogenesis. Further evidence for a role for HGF/S/Met receptor in tumor invasiveness and angiogenesis comes from the findings that high titers of HGF/SF in invasive breast cancers are factors for relapse and death (Yamashita et al., 19941, that HGF/SF treatment of glioma cell lines stimulates proliferation and invasion (Koochekpour et al., 1997), and that invasive bladder carcinomas possess higher HGFISF titers than d o noninvasive cancers (Joseph et al., 199s). In addition, the Met receptor is overexpressed in several types of tumor stroma, including bladder wall, vascular smooth muscle, and vascular endothelial cells (Rosen and Goldberg, 1995),suggesting a paracrine signaling mechanism between tumor cells and the underlying stroma. Thus, HGF/SF and Met interactions may promote metastasis by enhancing proliferation via autocrine or paracrine routes, stimulating the expression of plasminogen activators, and triggering angiogenesis. (Rong et al., 1992; Kanda et al., 1993; Bellusci et a/., 1994; Jeffers et al., 1996a,b).

B. Colony-Stimulating Factor- I Receptor c-Fms, the cellular homolog of the viral oncogene v-Fms (Sherr et al., 198S), is the receptor for colony-stimulating factor-1, which stimulates the proliferation and differentiation of macrophages, osteoclasts, and placental trophoblasts (Sherr, 1990; Roth and Stanley, 1992; Insogna et al., 1997). That CSF-1 is critical for the development of mononuclear phagocytes was

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shown by studies in mi,ce that fail to express functional CSF-1: these mice exhibit an osteopetrotic phenotype and lack osteoclasts and macrophages (Wiktor-Jedrzejczaket al., 1990, 1991). The CSF-1 receptor is expressed in placenta (Pollard et al., 1987; Regensstreif and Rossant, 1989; Hume et al., 1997),osteolasts (Insogna etal., 1997), and cells of monocyte lineage (Woolford et al., 1985),whereas the ligand, CSF-1, is produced by fibroblasts, myoblasts, osteoblasts, bone marrow stromal cells, and endothelial cells (Sherr, 1990; Roth and Stanley, 1992). Such independent distribution of ligand and receptor underlies the importance of cell-cell interactions in regulating receptor function. c-Fms bears sequence and structural similarity to the steel receptor, c-Kit, and to the receptors for FGF, PDGF, and Flt3/FLK2 (Hanks etal., 1988; Rosnet and Birnbaum, 1993).The unique feature of this group is that each member possesses an “insert” region within its kinase domain. The downstream targets of c-Fms include PI-3 kinase, STAT 1, and PLCy, all of which bind to phosphorylated tyrosine residues within the kinase insert portion of the molecule (Varticovskiet al., 1989; Shurtleff et al., 1990; Reedijk et al., 1990; Novak et al., 1996; Bourette et al., 1997). Bourette et al. (1997)have shown that sequential activation of the PI-3-kinase-dependent and PLCy-dependent signaling pathways is required to initiate the differentiation process of myeloid cells. c-Src has also been shown to associate with c-Fms and to be activated on binding of CSF-1 to the receptor. Complex formation between c-Src and c-Fms is thought to occur via the SH2 domain of c-Src and a juxtamembrane phosphotyrosyl residue on the receptor (Courtneidge et al., 1993; Alonso et al., 1995). In osteoclasts, phosphorylation of c-Src in response to CSF-1 stimulation occurs concomitantly with rearrangements of the actin cytoskeleton and spreading of the cells, suggesting that c-Src may be involved in regulating these processes (Insogna et al., 1997). Overexpression of c-Fms in NIH3T3 or Rat2 fibroblasts or in various tumor cells results in transformation, growth in soft agar, and tumor formation in nude mice (Rettenmeier et al., 1987; Taylor et al., 1989; van der Geer and Hunter, 1989; Favot et al., 1995). These findings demonstrate the oncogenic potential of overexpressed c-Fms. As described above, c-Fms and CSF1 are normally not expressed in the same cell type. However, coexpression is seen in tumors of the pancreas, endometrium, stomach, lung, and breast, and in acute myeloid leukemia, hairy cell leukemia, and Hodgkin’s lymphoma (Rambaldi et al., 1988; Kacinski et al., 1990; Paietta et al., 1990; Baiocchi et al., 1991; Kauma et al., 1991; Bruckner et al., 1992; Filderman et al., 1992; Storga et al., 1992; Tang et al., 1992; Leiserowitz et al., 1993; Till et al., 1993; Burthem et al., 1994; Berchuck and Boyd, 1995). Coexpression correlates with poor patient prognosis, most likely due to the establishment of an autocrine loop (Kacinski et al., 1990; Tang et al., 1992). Evidence suggests that such an autocrine loop contributes not only to tumor

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cell proliferation but also to invasiveness (Bruckner et al., 1992; Filderman et al., 1992; Burthem et al., 1994). In this regard, coexpression of c-Fms and its ligand in endometrial cancers correlates with a more advanced stage and with increased myometrial invasion (Leiserowitz et al., 1993). Moreover, CSF-1 stimulation results in the expression of UPA in lung tumors, Lewis lung carcinoma cells, and in NIH3T3 cells transfected with c-Fms (Filderman et al., 1992; Favot et al., 1995; Stacey etal., 1995). Together, these findings suggest that, like HGF/SF/MetR, deregulation of c-FmsKSF-1 interactions has the potential of contributing to the metastatic process in a variety of human cancers.

C. Fibroblast Growth Factor Receptors The FGF receptors comprise a large family that is encoded by four separate genes, each oi which can be alternatively spliced. Each receptor is also capable of binding several different ligands, resulting in a complex array of possible receptor/ligand pairs (Johnson and Williams, 1993). All receptors for FGF possess extracellular ligand-binding domains, which contain immunoglobulin-like repeats, and bipartite, intracellular tyrosine kinase domains (Lappi, 1995).Which signaling molecules are recruited varies with cell type and receptor/ligand pair. For example, in NIH3T3 cells (Zhan et al., 1994) FGFR 1 and c-Src physically associate following ligand binding, and activation of the receptor triggers the c-Src-dependent phosphorylation of the actin-binding protein, cortactin. Because cortactin is localized to cortical actin, particularly at the leading edge of a migrating cell (Wu et al., 1991; Maa et al., 1992; Wu and Parsons, 1993), its phosphorylation is speculated to influence cell motility and invasiveness. In other studies, ligand stimulation of FGFR 1 and FGFR 3 on C6 rat myoblasts results in activation of the p21Ras and MAPK pathway (Klint et al., 1995; Kanai et al., 1997). In these same cells, activation of the FGFR 3 receptor alone causes an increase in phosphorylation of PLCy but a decrease in c-Src phosphorylation (Kanai et al., 1997). FGF receptors are ubiquitously expressed during embryogenesis, but their presence is restricted after birth (Wanaka et al., 1991; Peters et al., 1992, 1993; Pastone et al., 1993). As a family, FGFs have mitogenic, nonproliferative, and antiproliferative effects. Which response is elicited is determined by the ligand, the type of cell exposed to the ligand, and the particular isoform of the receptor expressed on that cell (Schweigerer et al., 1987; Sporn and Roberts, 1988). For example, FGF 2 promotes survival of cultured neurons (Walicke, 1988), whereas FGF 1 and FGF 2 stimulate growth of fibroblasts, oligodendrocytes, astrocytes, smooth muscle cells, endothelial

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cells, and retinal epithelial cells (Burgess and Maciag, 1989). FGFs can also act as chemotactic factors for fibroblasts and glial cells (Senior et af., 1986). Basic FGF (bFGF, or FGF 2) induces neurite outgrowth in embryonic chick ciliary ganglion cells (Schubert et al., 1987) and can mediate cellular migration in experimental systems (Sato and Rifkin, 1988).Treatment of cultured vascular endothelial cells with FGF 2 induces the formation of blood capillary-like tubules, a finding that suggests FGFs may play a role in angiogenesis (Montesano et al., 1986; Slavin, 1995). In this regard, a large literature is beginning to accumulate in support of a role for FGFs in angiogenesis, because they have been demonstrated to stimulate endothelial cell division, migration, release of proteolytic enzymes, and capillary formation (Slavin, 1995). In addition to these functions in normal cells, FGFR family members are implicated in the progression of a variety of human cancers. FGFs are thought to act as autocrine growth factors for melanomas, gliomas, and meningiomas (Lappi, 1995), and their levels are elevated in many different tumor types (Nguyen et al., 1994).FGF receptors are also overexpressed in human tumors. For example, 10% of human breast tumors exhibit amplifications of chromosomal regions encoding FGF receptors (Adnane et af., 1991), and FGFR 4 mRNA levels are frequently elevated in breast cancer cells as compared to normal tissue (Lehtola et af., 1993; Ron et al., 1993; Penault-Llorca et al., 1995). Some evidence also suggests that differential expression of FGFR isoforms can influence the propensity of a cell to undergo malignant transformation. In normal fetal and mature brain, FGFR 1, which possesses three immunoglobulin-like extracellular repeats, is expressed. However, in astrocytic tumors, an increase in the expression of an FGFR with two immunoglobulin-like domains is observed. This form has increased affinity for acidic and basic FGF (Shing et al., 1993). Changes in FGFR expression also occur during the conversion of normal or hyperplastic prostatic epithelium to malignant tumor tissue, where the increased expression of an alternatively spliced form of FGFR 2, which has a higher affinity for bFGF, appears to create an autocrine stimulatory loop (Wang et af., 1995). FGFs, along with other factors, are often secreted by tumors, and their increased extracellular abundance is linked to enhanced invasiveness (Klagsbrun et af., 1976; Libermann et af., 1987; Wadzinski et af., 1987; Folkman et al., 1988). In breast tumor cells, FGFR 4 activation results in membrane ruffling, a morphological change that is associated with metastasis (Johnston et al., 1995). In in vitro invasion assays, FGF 2 induces the migration of bovine capillary endothelial cells through placental tissue in a dose-dependent manner (Mignatti et al., 1989), and bFGF stimulates production of metalloproteinases in human bladder cancer cell lines, an event associated with increased invasiveness of the cells (Miyake et al., 1997). Moreover,

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bFGF-dependent, sustained activation of MAPK correlates with the scattering of neuroepithelioma cells (van Puijenbroek et al., 1997).Together, these studies suggest that FGFs and FGFRs play important roles in human cancer progression by promoting the metastatic process.

D. Platelet-Derived Growth Factor Receptor The PDGFR has two isoforms, a and p, which differ in their preferences for binding homo- or heterodimers of the A and B forms of the PDGF ligand (Yarden et al., 1986; Claesson-Welsh et al., 1989; Claesson-Welsh and Heldin, 1989; Heldin and Westermark, 1990; Ross et al., 1990; Matsui et al., 1993). Both receptor isoforms consist of an extracellular domain that contains immunoglobulin-like motifs, transmembrane and juxtamembrane regions, a catalytic domain with an insert, and a C-terminal tail (Heldin and Westermark, 1990; Ross et al., 1990). Signaling molecules, which include PI-3 kinase (Kazlauskas and Cooper, 1989; Auger et al., 1989; Coughlin et al., 1989), PLCy (Kumjian et al., 1989; Meisenhelder et al., 1989; Wahl et al., 1989; Morrison et al., 1990), RasGAP (Molloy et al., 1989; Kaplan et al., 1990; Kazlauskas et al., 1990), and the Src family members c-Src, Fyn, and c-Yes (Kypta et al., 1990) (Twamley et al., 1992), bind phosphorylated tyrosine residues in the C-terminal tail, the kinase insert, and the juxtamembrane region via their SH2 domains. Interestingly, the same downstream effectors in different cell types can elicit different cellular responses. For example, in human hepatoma cell lines, PLCy and PI-3 kinase can independently transmit mitogenic signals (Valius and Kazlauskas, 1993), whereas in C H O cells a precise balance exists between migration-promoting signaling via PLCy and PI-3 kinase and migration-inhibitory signaling via RasGAP (Kundra et al., 1994). Phosphorylation of Tyr-988 in the carboxy terminus of the 01 receptor is associated with induction of chemotaxis, whereas phosphorylation of Tyr-768 and Tyr-1018 negatively regulates this process (Yokote et al., 1996). These results suggest that the different phosphorylation sites serve as binding sites for unique signaling molecules that influence cellular behavior in different ways. This hypothesis is further supported by studies in smooth muscle cells showing that PDCF-induced activation of PLCy is associated with actin disassembly and chemotaxis, whereas an independent signaling pathway, probably involving small GTPases such as Rho, appears to mediate the proliferative effect of PDGF in this system (Bornfeldt et al., 1995). PDGF receptors and their ligands regulate a wide spectrum of normal cellular processes in cells of mesenchymal and endothelial origin. These processes include differentiation, proliferation, survival, and migration. For example, the receptor for PDGF 01 is necessary for the development of neur-

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a1 crest cells (Soriano, 1997) and alveolar branching in the lung (Souza et al., 1995);the PDGF p receptor is required for proper development of the cardiovascular and renal systems (reviewed in Betsholtz, 1995). The PDGF p receptor is also found in mesenchymal tissue of the developing trachea and intestine and in the endothelium of blood vessels, where it is thought to play a role in regulating mesenchymal-epithelial interactions (Shinbrot et al., 1994).In addition, the PDGF 01 receptor is required for the maximal chemotactic effect of PDGF on lung fibroblasts (Osornio-Vargas et al., 1996). Numerous studies suggest that various PDGF and PDGFR isoforms are also involved in the genesis or maintenance of human cancers. The PDGFR is overexpressed in human pancreatic cancer (Ebert et al., 1995), primary and metastatic melanomas (Barnhill et al., 1996), and in mesothelioma cell lines (Versnel et al., 1994; Langerak et al., 1996).PDGFR expression is also seen in many neural crest-derived human tumors, including neuroblastoma and Ewing’s sarcoma (Matsui et al., 1993), in basal cell carcinoma (Ponten et al., 1994), and in tumors of the lung and pituitary (Leon et al., 1994; Vignaud et al., 1994). PDGF and its receptors are not normally expressed in epithelial cells, but their aberrant expression in tumors of this origin suggest that they could be involved in the oncogenic process. The situation is made more complex by the fact that some tumors express one or both forms of the ligand and no receptor(s) or vice versa, suggesting that both autocrine and paracrine signaling loops are involved in PDGF-mediated growth of tumors. For example, autocrine signaling loops have been shown to contribute to the growth of human esophageal carcinomas (Juang et al., 1996), mesotheliomas (Langerak et at., 1996), malignant melanomas (Barnhill et ul., 1996), gliomas, and glioblastomas (Potapova et al., 1996). However, results from Coltrera et al. (1995) show that PDGF may also function in a paracrine fashion in some human breast tumors. Their studies revealed that PDGF p is expressed in breast epithelium and tumor tissues, and the receptor is present in stromal fibroblasts. A similar situation appears to exist in ovarian cancer (Versnel et al., 1994), in lung tumors (Vignaud et al., 1994), and in basal cell carcinomas (Ponten et al., 1994). The ability of PDGF to induce chemotaxis may also play a role in tumor cell metastasis. For example, expression of the receptor for PDGF 01 in Lewis lung carcinoma cells increases their metastatic potential, whereas expression of the receptor truncated at the kinase domain reverses this effect (Fitzer-Attas et al., 1997). Potapova et al. (1996) demonstrated that in human glioblastoma cells, which express both the PDGF p receptor and its ligand, further expression of PDGF p results in tumor formation in nude mice and increased metastasis. These examples support the idea that in addition to mediating normal cell migration, aberrant expression or activation of PDGF receptors in tumor cells can contribute to their proliferative and invasive properties.

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E. Epidermal Growth Factor R e c e p t o r The human epidermal growth factor receptor, HER1, belongs to a family of human RTKs that includes HER2/neu, HER3, and HER4 (Ullrich and Schlessinger, 1990). All members of this family are transmembrane tyrosine kinases that possess an extracellular domain with two cysteine-rich repeats, an intact catalytic domain, and a C-terminal tail that binds SH2-containing signaling effectors on activation of the receptor. Ligands for HERl include epidermal growth factor (EGF), transforming growth factor-a (TGF-a), betacellulin (Riese et al., 1996), and epiregulin (Komurasaki et al., 1997). No specific ligand for HER2 has yet been defined, but HER3 and HER4 can be activated by a family of alternatively spliced ligands, called heregulins (HRG) (reviewed in Hynes and Stern, 1994). Each member of the HER family is capable of heterodimerizing with other members of the family, thereby providing a means by which HER2, although it lacks a ligand, can signal. Such dimerization appears to occur in a hierarchical order, wherein the HER2/3 interaction is the most preferred and the HER1/4 interaction the least preferred (Pinkas-Kramarski et al., 1996). Studies in 32D hematopoetic cells, which do not express any HER family members, show that heterodimers have more potent mitogenic activity than do homodimers and that HER3 heterodimers are the most transforming. However, when HERl is present, signaling through this receptor dominates over other members of the family (Pinkas-Kramarski et al., 1996). The focus of our discussion will be on the roles of HERl and HER2 in human cancer, because large bodies of literature exist for each. HER3 and HER4 are more recent additions to the family, and characterization of them with respect to their possible involvement in human cancers is just beginning. However, it is important to note that overexpression of HER3 has been detected in some breast cancers (Lemoine et al., 1992) and in papillary thyroid carcinomas (Faksvag et al., 1996). Thus, the possibility exists that homo- or heterodimerization of HER3 or HER4 with HERl or HER2 mediates tumorigenic signaling in a manner similar to that of HERl and HER2. A major role for HERl is its involvement in normal human development. It affects many stages, from postfertilization to sexual maturation. For example, HERl and its ligand, TGF-a, control proliferation of blastocoel cells as well as embryoluterine signaling and implantation (Rappoll et al., 1988; Dardik and Schultz, 1991; Arnholdt et al., 1991; Zhang et al., 1992).HERl is also necessary for development of embryonic lung, skin, palate (Lee and Han, 1990), and hair follicles (Hansen et al., 1997). During puberty, HERl and the estrogen receptor together regulate the differentiation of normal breast epithelium and uterine and vaginal growth (Nelson et al., 1991; Ignar-Trowbridge et al., 1992). Loss of control of these interactions is thought to play a role in the genesis of human tumors, and the diversity of tissues

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that are regulated developmentally by HERl is reflected in the spectrum of tissues and cell types in which HERl is thought to play an oncogenic role. That HERl can function as an oncoprotein was demonstrated by the ability of NIH3T3 cells, engineered to overexpress HERl and held in the continual presence of EGF, to become transformed and develop tumors in nude mice (Velu et al., 1987; DiFiore et al., 1987a; Di Marco et al., 1989). HERl effector substrates include the adaptor proteins Shc (Pelicciet al., 1992; RuffJamison et al., 1993)and Grb2, which feed into the well-defined Ras/MAPK signaling pathway (Li et al., 1993; Egan et al., 1993; Rozakis-Adcock et al., 1993), as well as PLCy (Rhee, 1991), c-Cbl (Levkowitz et al., 1996), eps 8, and eps 15 (Fazioli et al., 1992,1993aYb). Reports from several laboratories show that on activation, HERl physically associates with the c-Src nonreceptor tyrosine kinase in both normal fibroblasts and in a variety of tumor cell lines (Luttrell et al., 1994; Maa et al., 1995; Sat0 et al., 1995; Stover et al., 1995; Biscardi et al., 1998a). Complex formation with c-Src occurs concomitantly with enhanced phosphorylation of receptor substrates, suggesting that c-Src may act to increase the receptor’s tyrosine kinase activity, thus enhancing the potential for cellular transformation and tumorigenesis (Maa et al., 1995; Tice et al., 1998; Biscardi et al., 1998a,b). This hypothesis was tested directly using a panel of C3HlOT1/2 murine fibroblasts that were engineered to overexpress HERl and c-Src,-either alone or in combination. Cells overexpressing both HERl and c-Src were found to produce synergistically larger and more numerous tumors in nude mice and colonies in soft agar than those produced by cells overexpressing either HERl or c-Src alone (Maa et al., 1995). These findings represent the first causal evidence for cooperativity between c-Src and HERl in tumorigenesis. What is the evidence for involvement of HERl in the genesis of human tumors? Aberrant expression, overexpression, or truncation of HERl has been demonstrated to occur in a variety of human cancers, including benign skin hyperplasia, glioblastoma, and cancers of the breast, prostate, ovary, liver, bladder, esophagus, larynx, stomach, colon, and lung (Harris et al., 1992; Khazaie et al., 1993; Scambia et al., 1995). In patients with ovarian cancer, overexpression of HERl correlates with a decreased response to chemotherapy and decreased survival (Scambia et al., 1995; Fischer-Colbrie et al., 1997), suggesting that HERl plays a proactive role in ovarian tumor progression. HERl overexpression also appears to play a role in the etiology of glioblastomas. Forty percent of glioblastomas exhibit amplification of the HERl gene (Khazaie et al., 1993), but in these tumors, overexpression is not the only abnormality regarding HER1. An alternatively spliced form of the receptor, termed EGFRvIII, is also frequently observed (Libermann et al., 1985; Yamazaki et al., 1988; Tuzi et al., 1991; Chaffanet et al., 1992). This form of the receptor lacks nucleotides 275-1075, which encode a large

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portion of the extracellular domain (Humphrey et al., 1990; Ekstrand et al., 1992; Wong et al., 1992, and displays constitutive activity, perhaps due to its inability to be controlled by ligand (Ekstrand et al., 1994). Existing evidence suggests that EGFRvIII signals differently than wild-type receptor, prefering the PI-3 kinase pathway (Moscatello et al., 1998) to the Ras/MAPK pathway (Montgomery et al., 1995; Moscatello et al., 1998).In addition to glial tumors, one study showed that EGFRvIII is present in 16% of non-small-cell lung carcinomas (Garcia et al., 1993) as well as in 86% of medulloblastomas, 78% of breast cancers, and 73% of the ovarian cancers examined (Moscatello et al., 1995). In contrast, EGFRvIII has not yet been detected in normal tissue, a finding that provides compelling evidence for an oncogenic role for this form of the receptor. A link between HERl and breast cancer has also emerged in recent years. Amplification or overexpression of the genes encoding one or more of the HER family members is estimated to occur in approximately 67% of human breast cancers (Harris et al., 1992), with overexpression of HERl detected in approximately 30% of patients (Battaglia et al., 1988; Delarue et al., 1988; Bolla et al., 1990; Koenders et al., 1991; Toi et al., 1991; Harris et al., 1992). Elevated levels of HERl are also associated with loss of estrogen-dependent growth (Klijn et al., 1993), suggesting a role for HERl in the later stages of tumor progression. In addition to transformation and proliferation, studies from several laboratories suggest that HERl also enhances the invasive potential of tumor cells. Overexpression of HERl has been shown to result in an increased ability of rat mammary carcinoma cells to migrate through matrigel (Lichtner et al., 1995; Kaufmann et al., 1996), and higher levels of HERl are found in tumor tissue at metastatic sites as compared to primary sites (Sainsbury et al., 1987; Toi et al., 1991). Both these findings are supportive of a role for HERl in metastasis.

E HER2lneu Like HERl, activated HER2 possesses an intracellular tyrosine kinase domain as well as C-terminal phosphotyrosines that are capable of binding downstream substrates, such as PLCy, PI-3 kinase, Grb7, p120 RasGAP, p190, RhoGAP, c-Src, Shc, PTPlD, PTPlB, eps-8, and Tob, an antiproliferative protein (Hynes and Stern, 1994; Matsuda et al., 1996; Liu and Chernoff, 1997).Because both HERl and HER2 appear to activate similar downstream signaling pathways in experimental cell systems, it is unclear how specificity of signaling is achieved. The most likely explanation is that activation of a particular signaling pathway is dependent on cell type and on the

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subset of HER family members and effector molecules available at any given time. However, a few examples of specific substrates have been reported, such as the c-Cbl adaptor protein for HERl (Levkowitz et al., 1996) and paxillin and a protein of 23 kDa (p23) for HER2 (Romano et al., 1994). HER2 is expressed in all tissues except the hematopoietic system (De Potter et al., 1990; Press et al., 1990). Studies using mice that are deficient in HER2, HER4, or the HER3/4 ligand, HRG, demonstrate that signaling through HER2 heterodimers is necessary for proper cardiac and neural development (Meyer and Birchmeier, 1995; Gassmann et al., 1995; Lee et al., 1995). A great deal of evidence from both experimental systems and human patients also points to the involvement of HER2 in malignant transformation. In certain tumors, it has been found that HER2 can be overexpressed up to 100 fold, due to gene amplification (Hynes and Stern, 1994).This finding, coupled with the fact that overexpression of HER2 alone, without the addition of agonist for HER family members, can induce focus formation in cultured fibroblasts (Hudziak et al., 1987; DiFiore et al., 1987b) suggests that overexpression of HER2 is capable of inducing oncogenic activity in the human. In addition, overexpression of HER2/neu in PC-3 prostate cancer cells has been shown to result in an increased incidence of metastasis after orthotopic introduction (Zhau et al., 1996). Whereas amplification of the gene encoding HER2 is found in 10-30% of breast, ovarian, and gastric tumors (Hynes and Stern, 1994), tumors of the lung, mesenchyme, bladder, and esophagus contain high levels of HER2 protein but no gene amplification, suggesting that both transcriptional and posttranscriptional mechanisms are responsible for increased HER2 levels (Kraus et al., 1987; Hynes et al., 1989; King et al., 1989; Kameda et al., 1990). HER2 is apparently involved in the genesis of many types of human tumors, but its role has been most well-characterized in breast cancer. Increased levels of HER2 protein appear to correlate with poor patient prognosis (Slamon et al., 1987, 1989; Paik et al., 1990; Gusterson et al., 1992) and a loss of responsiveness to the,antiestrogen, tamoxifen (Nicholson et al., 1990; Wright et al., 1992; Klijn et al., 1993). In transgenic mouse models, HER2/neu was demonstrated to induce mammary tumors when expression was targeted to the mammary gland by the use of the murine mammary tumor virus promotor (Muthuswamy et al., 1994). These HER2/neu tumors contain increased levels of c-Src and c-Yes kinase activity as compared to normal, surrounding tissue (Muthuswamy et al., 1994; Muthuswamy and Muller, 1995). Furthermore, c-Src was found to coimmunoprecipitate with HER2/neu (Muthuswamy et al., 1994), suggesting that c-Src cooperates with HER2 as well as with HERl in regulating malignant progression. Because HER2/neu is most frequently localized to the primary tumor mass in the murine model and is found in earlier stage in situ carcinomas in humans

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(van de Vijver et al., 1988; Paik et al., 1990; Lin et al., 1992; Barnes et al., 1992), it is speculated that this molecule is involved in earlier stages of breast cancer than is HER1.

G. HER Family Members a n d Estrogen Receptor Interactions Increasingly compelling data are accumulating that point to interactions among the estrogen receptor (ER), HER1, HERZ, and c-Src as being major factors in the development of human breast cancer. The ER is a steroid hormone receptor of 6 7 kDa that dimerizes and becomes activated as a transcription factor on binding of estrogen (Mangelsdorf et al., 1995). Functional domains of the ER include an amino-terminal A/B region, which is responsible for ligand-independent transcriptional activation; a central DNA-binding domain; and a carboxy-terminal E/F hormone-binding domain, which is responsible for estradiol-induced transcription (Tsai and O’Malley, 1994; Beato et al., 1995). In addition to the well-characterized ER 01 isoform, a p isoform, which has differing transcriptional properties and expression patterns, has been discovered (Paech et al., 1997). Loss of ER responsiveness in human breast tumors correlates with overexpression of HERl and with a poorer patient prognosis (Fitzpatrick et al., 1984; Sainsbury et al., 1985; Dauidson et al., 1987; Nicholson et al., 1988). The mechanism by which a breast tumor cell loses responsiveness to estrogen is unclear, but this event may be regulated in part by interactions with HER family members and/or c-Src. Cross-talk between growth factor receptor tyrosine kinases and the ER was first demonstrated by IgnarTrowbridge and co-workers (1992, 1993), who showed that treatment of cells with EGF activates the transcriptional activity of the ER and that this effect is dependent on the amino-terminal A/B domain of the ER. The ER also appears to have the ability to affect expression of the EGF receptor. In ER-positive breast cancer cells, estradiol treatment increases HERl mRNA levels (Yarden et al., 1996). This effect may be directly mediated by the ER, because the HERl promoter has sequences that share loose homology with the estrogen response element (ERE)and can bind human ER (Yarden et al., 1996). Conversely, HERl can affect ER expression. Overexpression of TGF-cx in the ER-positive ZR75-1 breast cancer cell line, along with prolonged treatment of these cells with antiestrogens, results in loss of the ER, whereas treatment of parental ZR75-1 cells with antiestrogens alone has little effect (Clarke et al., 1989; Agthoven et al., 1992). These results are interpreted to mean that continual and concomitant stimulation of HERl and ER can cause a reduction in ER expression. In this regard, it is through that expres-

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sion of HERl and ER and mutually exclusive, because most breast tumors that overexpress HERl lack functional ER (Fitzpatrick et al., 1984; Sainsbury et al., 1985; Davidson et al., 1987; Nicholson et al., 1988). Because breast tumors that do not show this inverse expression tend to be HERl/ER positive rather than HERUER negative, it has been suggested that overexpression of HERl precedes loss of the ER (Koenders et al., 1991; Dittadi et al., 1993; Chrysogelos and Dickson, 1994). It is unclear how overexpression or activation of HERl leads to loss of ER expression. One possible mechanism may involve signaling to MAP kinase. HERl activation results in the phosphorylation of the ER on Ser-118, a phosphorylation that is required for hormone-independent transcriptional activity of the ER (Kato et al., 1995). Ser-118 is also thought to be a target for MAP kinase, because studies using dominant negative Ras and MEK demonstrated a loss of this phosphorylation concomitantly with a loss of EGF-dependent transcriptional activation (Bunone et al., 1996). Autocrine stimulatory loops involving TGF-a and HERl are known to exist in breast cancer, thus it is speculated that the continued stimulation of the ER via the HERl/MAP kinase pathway leads to its down-regulation and eventual loss. Estradiol is also known to induce phosphorylation of the ER (Auricchio et al., 1987).In addition, Arnold et al. (1995a)reported that the ER is basally phosphorylated on Y537 in vivo. The role of the Y537 phosphorylation is controversial. Early studies showed that tyrosine phosphorylation of the ER activates its hormone-binding activity (Migliaccio et al., 1989) and that phosphorylation of Y537 is required for binding of the ER to the ERE (Arnold and Notides, 1995; Arnold et al., 1995b). Further in vitro studies demonstrated that c-Src is able to phosphorylate Y537 and that this phosphorylation is necessary for homodimerization of the ER and for binding of estradiol (Arnold et al., 1995a, 1997).In agreement with these findings, Castoria et al. (1996) reported that a non-hormone-binding form of the ER found in mammary tumors can be converted to a hormone-binding form by irt vitro phosphorylation with a calcium/calmodulin-regulated kinase, which is thought to be a c-Src family member. However, additional studies in which Y537 was mutated to various amino acids suggest that phosphorylation of Y537 per se is unnecessary for estradiol-mediated activation of the ER but may be important in ligand-independent (i.e., growth factor-mediated) activation (Weis et al., 1996; Lazennec et al., 1997). Although c-Src is capable of phosphorylating the ER, the ER may also influence c-Src activity. Estradiol treatment has been shown to increase c-Src tyrosine phosphorylation and kinase activity in MCF7 breast cancer cells (Migliaccio et al., 1993, 1996) and to stimulate kinase activity of c-Src and its related family member, c-Yes, in colon carcinoma cells (Di Domenico et al., 1996). Ligand-independent down-regulation of the ER may also be mediated by

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HER2 signaling pathways. Pietras et al. (1995) showed that overexpression of HER2 in MCF7 cells leads to estrogen-independent growth and ERE transcriptional activation. Furthermore, treatment of these cells with HRG, which stimulates HER2-dependent signaling via HER2/3 heterodimers, induces tyrosine phosphorylation and down-regulation of the ER. Other investigators have shown that HRG treatment inhibits the expression of ER in ER-positive breast cancer cells and can revert the estradiol-mediated decrease in HER2 expression (Grunt et al., 1995).Taken together, these results suggest that in experimental cell systems, HER2 and the ER are expressed in a mutually exclusive manner. However, in human breast tumors, the situation is less clear, with some reports indicating an inverse correlation between HER2 and ER expression (Adnane et al., 1989; Borg et al., 3 990) and others indicting no such correlation (Slamon et al., 1989; Bacus et al., 1996).

111. c4rc AND c-Src FAMILY MEMBERS IN HUMAN CANCERS

A. c-Src Structure and Mechanisms of Regulation c-Src is the cellular, nontransforming homolog of v-Src, the oncoprotein encoded by the chicken retrovirus, Rous sarcoma virus. c-Src is a 60-kDa tyrosine kinase that is composed of six domains: an N-terminal membraneassociation domain, a “Unique” domain, SH3 and SH2 domains, a catalytic domain, and a negative regulatory domain (Fig. 2, see color plate). Although c-Src is cytosolic, it localizes to intracellular membranes, including the plasma membrane and membranes of endosomes and secretory vesicles within the cytosol (Parsons and Creutz, 1986; Kaplan et al., 1992; Resh, 1994). It is tethered to these membranes by the combined action of an N-terminal, covalently linked myristate moiety, salt bridges between basic amino acids in the N terminus and phosphates of the lipid backbone, and noncovalent interactions with integral or associated membrane proteins (Resh, 1994).Membrane localization of c-Src is required for its ability to participate in growth factor receptor-mediated signaling in normal cells (Wilson et al., 1989). The function of the Unique domain is not well-defined. However, based on the fact that it exhibits the greatest sequence divergence among family members of all the domains (Brown and Cooper, 1996), it is speculated to specify protein-protein interactions that are unique to individual Src family members. The SH3 and SH2 domains mediate the binding of c-Src with other signaling proteins through proline-rich or phosphotyrosine-containing regions on target proteins, respectively (Pawson and Schlessinger, 1993). The major regulatory region of the enzyme is a short domain at the extreme C terminus of

Fig. 2 Structure of c-Src. c-Src is the prototype of a large family of cytoplasmic tyrosine kinases that associate with cellular membranes through lipid modifications at their N termini. As a linear molecule, the relationship between the various domains can be seen: an N-terminal membrane association domain that contains the site of myristylation, a Unique domain that exhibits the widest sequence divergence among family members of any of the domains, an Srchomology-3 (SH3) domain that binds poly(pro1ine) motifs on target molecules, an Src-homology-2 (SH2) domain that binds phosphotyrosine residues on target molecules, an SH2/kinase linker, the catalytic domain, and the negative regulatory domain that contains the predominant site of tyrosine phosphorylation on the inactive molecule (Y527 in chicken, Y531 in human). The three-dimensional orientation of the molecule, lacking the membrane-association and Unique domains, is depicted as a ribbon diagram. Reprinted with permission from Nature, Xu et al. (1997), and from Michael Eck (configured from the atomic coordinates provided on the Web). Copyright 1997 Macmillan Magazines Limited. The enzymatic activity of c-Src is regulated by the coordinated effects of target proteins binding to or covalent, posttranslational modifications of the SH3, SH2, and negative regulatory domains on the catalytic domain, as described in the text.

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the molecule, which harbors a Tyr residue that becomes phosphorylated (Y530 in human c-Src; Y527 in chicken c-Src) by a C-terminal Src kinase, CSK (Okada et al., 1991). Phosphorylated Y527/530 (pY527) is capable of binding its own SH2 domain in a manner that inhibits kinase activity without physically blocking the catalytic site, as shown in Fig. 2 (Yamaguchi and Hendrickson, 1996; Sicheri et al., 1997; Xu et al., 1997). Binding of tyrosine-phosphorylated cellular proteins to the SH2 domain is thought to destabilize the intramolecular pY527/SH2 domain interaction and induce a conformational change that results in enzymatic activation. Structural studies have revealed that the SH2 and SH3 domains collaborate in their binding of respective protein partners, thereby cooperatively influencing the activity of the enzyme (Eck et al., 1994). Furthermore, crystallographic analysis has shown that sequences just N terminal to the catalytic domain (termed the SH2-kinase linker) comprises a loop structure that functions as a “pseudo” SH3 binding site (Yamaguchi and Hendrickson, 1996; Sicheri et al., 1997; Xu et al., 1997). Together, the intramolecular phosphotyrosine/SH2 and linkerlSH3 interactions direct a conformation that presses the linker against the backbone of the catalytic domain and renders the protein inactive. As with the SH2 domain, binding of signaling proteins to the SH3 domain is thought to release the constraints of the linker/SH3 interaction on the kinase domain, resulting in activation of catalytic activity. Mutation of Y527 to F or deletion of the C-terminal regulatory domain (as in v-Src) results in a constitutively active protein that phosphorylates target proteins in an unregulated fashion and induces cellular transformation and oncogenesis (Cartwright et al., 1987; Kmiecik and Shalloway, 1987; Piwnica-Worms et al., 1987; Reynolds et al., 1987). In normal cells, c-Src is nononcogenic or only weakly so, even when it is overexpressed (Shalloway et al., 1984; Luttrell et al., 1988). However, under certain conditions (growth factor stimulation or translocation; outlined below), the enzyme can become activated, either via dephosphorylation of pY527 or by binding of signaling proteins to the N-terminal half of the protein. Activation is most frequently a transient event, and c-Src, in contrast to v-Src, is thought to respond to negative control by rephosphorylation of Y527 or by the release of binding proteins and the resumption of intramolecular interactions. It has been the conjecture of many investigators that the transient nature of c-Src activation often prevents its detection. In fact, the possibility exists that little or no activation above basal levels is necessary for catalysis, if the substrate is properly positioned near the catalytic cleft. Thus, another “regulator” of c-Src activity may well be its intracellular localization and, at a finer level, its appropriate juxtaposition to substrate within a signaling complex. Identification of c-Src substrates and proteins that bind its SH2 and SH3 domains is now critical for further understanding of the role c-Src and its family members play in biological processes.

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The majority of the studies leading to the above model have been conducted in animal tissue culture systems and are just now being applied to the study of c-Src in human tumors. In the following section evidence is presented for the involvement of c-Src in the genesis of human tumors, with particular emphasis on its putative role in colon, breast, lung, and myeloid tumors.

B. Evidence for the Involvement of c-Src in Human Cancers Like the RTKs, many lines of evidence are suggestive of a role for c-Src in the genesis and progression of multiple types of human cancer. This evidence is both genetic and biochemical in nature and has been generated by studies of cultured tumor cell lines and surgically generated tumor tissue. Together these studies have implicated c-Src as an etiological agent for the development of neuroblastomas, myeloproliferative disorders (including myeloid leukemia), and carcinomas of the colon, breast, lung, esophagus, skin, parotid, cervix, and gastric tissues. Interestingly, although alterations of cSrc have been described at both the gene and protein levels in various cancer tissues, the changes are quite variable and include both increases and decreases in gene copy number and in protein levels and specific enzyme activities. Taken at face value, these findings suggest multiple ways in which c-Src can contribute to the oncogenic process, both as a dominantly acting oncogenic protein and as a negatively acting tumor suppressor. However, the multitude of changes could also reflect fortuitous alterations that do not contribute to the ultimate malignant phenotype. There may also be technical reasons for the variability in the findings, such as the different probes used for genetic analysis and the different antibodies and cell extraction conditions used for biochemical analysis. It is clear that further work needs to be done to clarify these issues and attempts made to minimize technical problems. Of particular importance to future studies will be the development and characterization of good animal and tissue culture models to test the hypotheses derived from analyses of human tumor tissues, whereby the contribution of individual genes or proteins can be evaluated for their oncogenic potential against a normal cell background rather than against a heterogeneous background of unknown numbers and types of genetic alterations that occur in every human tumor. 1 . GENETIC EVIDENCE

With the identification of the first protooncogenes came a plethora of studies examining the genomic content of multiple human tumors for deletions, amplifications, and diverse rearrangements in chromosomes containing pro-

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tooncogenes. For the most part, these studies identified few if any gross changes in the c-Src gene, which maps to the q arm of chromosome 20. Furthermore, gene expression studies, employing a variety of techniques to measure steady-state levels and newly synthesized mRNA have also revealed few changes in c-Src-specific mRNA (Bishop, 1983; Slamon et al., 1984). These findings led many investigators to conclude that c-Src played a minor (if any) role in the genesis of human tumors. Not until researchers began examining protein levels and specific enzyme activities did evidence for the involvement of c-Src begin to emerge. However, there were a few exceptions to the general rule described above, and one in particular is noteworthy. Four groups have identified a deletion of 16-21 cM in the long arm of chromosome 20 [del(20q)] as a recurring, nonrandom abnormality in malignant myeloid disorders, including nonlymphocytic leukemia and polycythemia (Simpson, 1988; Roulston et al., 1993; Hollings, 1994; Asimakopolous et al., 1994). This deletion maps between 20q11.2 and 20q13.3, a region that encodes the c-Src protooncogene (Hollings, 1994). The notion that deletion of a chromosomal region is signatory for a tumor suppressor gene suggests that, if c-Src is a critical gene in this deletion, it behaves as a negative regulator of cell growth, not as a dominant oncogene, as is commonly believed. That c-Src may have some tumor suppressor-like characteristics in myeloid cells is supported by the finding of several groups (Barnekow and Gessler, 1986; Gee et al., 1986) that c-Src expression levels increase during myeloid differentiation. If c-Src plays a critical role in promoting differentiation and maintaining the postmitotic state, then loss of such an activity might permit cells to once again acquire proliferative activity-the hallmark of a tumor suppressor.

2. BIOCHEMICAL EVIDENCE By far the bulk of evidence supporting a role for c-Src in the development of human tumors comes from biochemical studies, wherein the levels of cSrc protein and tyrosine kinase activity have been examined in hundreds of human tumors and compared to normal tissue controls. As will be discussed in more detail below, in some tumor specimens, high enzymatic activity is accompanied by high protein level, yielding little or no change in specific activities, whereas in others, protein levels are only slightly or modestly elevated, and the specific activity of the enzyme is increased. In yet other examples, high protein levels are accompanied by low enzymatic activity. However, the overall conclusion is that in a very high percentage (>50% and approaching 100% in some studies) of human tumors of many different tissue types, c-Src activity is altered (usually elevated) and that this alteration occurs in early to middle stages of tumor progression and is maintained or increased throughout progression to metastasis.

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These findings raise questions as to the mechanism of c-Src activation and the mechanisms by which protein levels are elevated (especially in light of the few instances of increases in c-Src-specific mRNA production). The consensus at the present time is that changes in c-Src specific activity in human tumors are due to posttranslational events and not to mutations of the gene. Using RNase protection and restriction fragment-length polymorphism assays to detect activating mutations of c-Src in a spectrum of human tumors, Wang et al. (1991)were unable to detect mutations at codons known to contribute to the oncogenicity of v-Src and c-Src (namely, codons 98, 381,444, and 530 in the human c-Src sequence). These findings led the investigators to conclude that mutational activation is not the mechanism of enhancement of c-Src-specific kinase activity. On the other hand, DeSeau et al. (1987) described differential activation of c-Src in normal colon cells versus colonic tumor cells depending on the conditions of extract preparation, i.e., whether the lysis buffers contained the proteins tyrosine phosphatase inhibitor, vanadate, and/or high concentrations of ionic and nonionic detergents. From these results, one could deduce that tyrosine phosphorylation of c-Src or other cellular proteins and proteidprotein interactions play a role in regulating not only c-Src activity but also its stability and abundance. Indeed, structural studies on the c-Src molecule described above would support this notion. However, so as not to think that the issue is resolved, studies by Watanabe et af. (1995) indicate that in 18 cancer cell lines, elevated activities of c-Src and c-Yes (a Src-related family member) are accompanied by correspondingly elevated levels of C-terminal Src kinase, the protein that phosphorylates Y530 in human c-Src and negatively regulates c-Src kinase activity. These findings suggest that CSK may not have an antioncogenic role to play in tumor progression or that dephosphorylation of Y530 is not required for activation of c-Src. Here the focus is on three different carcinomas-colon, breast, and lungfor which substantial amounts of data are accumulating to indicate a role for c-Src in their development. That these represent three of the four most common forms of cancer in adults (prostate cancer being the fourth) suggests that c-Src may be a more formidable player in tumorigenesis than had previously been appreciated.

3 . COLON CANCER Utilizing c-Src-specific antibodies and an immune complex-based tyrosine kinase assay, a number of investigators have reported that c-Src-specific tyrosine kinase activity (total activity relative to total c-Src protein in an immune complex) is elevated in colon cancer. In panels of colon cancers examined by Rosen et al. (1986), Bolen et al. (1987a,b), and Cartwright et al.

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(1989),c-Src was found to exhibit elevated kinase activity, ranging from -2to 40-fold above that found in normal colon tissues or cultures of normal colon mucosal cells. In some cases this increase could be accounted for by increases in protein levels, but in other instances it could not, indicating an increase in specific kinase activity. These results suggest that either elevation in c-Src protein and/or activation of c-Src may contribute to the genesis of human colon tumors. Indeed, additional studies by Lundy et al. (1988) and Cartwright et al. (1990,1994) demonstrated increased kinase activity in premalignant epithelia of ulcerative colitis and in early-stage colonic polyps as compared to adjacent normal mucosa. In the latter study, activity was highest in malignant polyps and in >2-cm benign polyps that contained villous structure and severe dysplasia. Thus, c-Src activity is found to be elevated in early stages of colon cancer and this elevation is associated with those polyps that are at greatest risk for developing cancer. Talamonti et al. (1993) also demonstrated incremental increases in c-Src activity and protein level as the tumors progressed, with the greatest increases seen in metastatic lesions. Increases in specific kinase activity were also observed, with liver metastases exhibiting an average increase of 2.2-fold over normal mucosa, whereas extrahepatic metastases demonstrated an average 12.7-fold increase. These results support the idea that c-Src may play multiple roles in tumor progression. A number of studies have been done to determine if c-Src indeed plays a causal role in tumor development. Herbimycin A, an inhibitor of Src family kinases, was shown to inhibit the growth in monolayer of seven colon tumor cell lines as compared to one cell line from normal colonic mucosa, CCL239 (Garcia et al., 1991). In another study, blockage of the myristylation modification of Src family members in a panel of human colon adenocarcinoma tumor cell lines by N-fatty acyl glycinal compounds was shown to prevent localization of c-Src to the plasma membrane and to depress colony formation of these cell lines in soft agar and cell proliferation assays (Shoji et al., 1990). Tumor necrosis factor (TNF-a-mediated growth inhibition of human colorectal carcinoma cell lines was accompanied by a reduction in the activity of s-Src (Novotny-Smith and Gallick, 1992).And last, using an antisense expression vector specific for c-Src, Staley et al. (1997) demonstrated that expression of c-Src antisense in HT29 human colon adenocarcinoma cells resulted in slower proliferation and slower growing tumors in nude mice as compared to the parental control. Together, these studies are consistent with a causative role for c-Src in colon cancer progression. How could c-Src be functioning to promote progression of colonic tumors? Using an in vitro progression model based on the PC/AA premalignant colonic adenoma cell line, Brunton et al. (1997) demonstrated that in the conversion from adenoma to carcinoma, levels of both the EGF receptor and FAK protein increased, while the expression and activity of c-Src were

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unaltered. However, EGF induced motility in the carcinoma cells, but not in the adenoma cells, and this increase was accompanied by an EGF-induced increase in c-Src kinase activity, relocalization of c-Src to the cell periphery, and phosphorylation of FAK. The authors interpret these findings to indicate that c-Src is not the driving force for tumor progression, but cooperates with other molecules (such as EGFR and FAK) in the process. Other investigators have observed that adhesion of HT29 human colon carcinoma cells to E-selectin results in a decrease in c-Src activity (Soltesz et al., 1997), suggesting that, on release from substratum restrictions, c-Src activity is restored or elevated. In a related study, Empereur et al. (1997)generated evidence for cooperativity between c-Src and HGF/SF in developing invasive properties of the PC/AA cell line. Specifically, introduction of activated c-Src or polyoma middle-T antigen (which requires c-Src for oncogenic activity) into the adenoma PC/AA cell line induced conversion of the adenoma to carcinoma, overexpression of the HGF receptor, and an invasive capacity in the presence of HGF. Thus, current evidence suggests that one mechanism by which c-Src promotes colonic tumor progression is by cooperating with components of the cell adhesion/motility machinery. Similar conclusions were reached by Mao et al. (1997), who demonstrated activation of c-Src in response to EGF or HGF treatment of human colon cancer cells with high metastatic potential. 4. BREAST CANCER As with colon cancer, a number of early investigations reported elevated c-Src activity in human breast cancers (Jacobs and Rubsamen, 1983; Rosen et al., 1986; Lehrer et al., 1989). In several reports the elevation in activity was not accompanied by elevated levels of c-Src protein, suggesting an activation of the protein. However, Koster et al. (1991), using a screening method based on in vitro synthesis of cDNA copied from total cellular RNA of tumor tissue, found that 25-30% of the analyzed tumors showed significant elevations in expression of several protooncogenes, including c-Src. Using immune complex kinase assays, immunoblotting, and immunohistochemical approaches, Verbeek et al. (1996) and Biscardi et al. (1998a) demonstrated that increases in c-Src kinase activity are almost invariably accompanied by increases in c-Src protein levels and little if any change in specific kinase activity. Interestingly, the immunohistochemical studies of Verbeek et al. (1996) showed that in malignant cells, the majority of c-Src appeared to be concentrated around the nucleus, whereas in normal cells, it is distributed more evenly in the cytoplasm. The discrepancies between the more recent data and the earlier data may reflect changes in the quality of the antibodies and the more quantitative analyses performed in the recent studies. In total, the current evidence indicates that few “activations” of c-

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Src occur in breast tumor cells; rather, elevations in protein levels appear to be the major cause of the increases in c-Src kinase activity. In a recent study involving 72 breast cell lines and tumor biopsies, tyrosine kinase activity was found to be elevated in 100% of the samples, as compared to normal tissue controls, and c-Src tyrosine kinase accounted for 70% of the total cytosolic activity (Ottenhoff-Kalff et al., 1992). The same group performing that study had previously found that the level of cytosolic protein tyrosine kinase activity parallels the malignancy in breast tumors (Hennipman et al., 1989) and that the majority of this activity is precipitated by anti-c-Src antibodies. These results provide compelling correlative evidence that c-Src plays a key role in the development of breast cancer. In agreement with this conclusion, Lehrer et al. (1989) and Koster et al. (1991) also note that elevated c-Src kinase activity is most frequently found in tumors that are progesterone receptor negative. Because loss of progesterone receptor is a histochemical marker for later stage tumors, c-Src activity appears to increase as the tumor progresses in severity. To directly assess the effect of mammary gland-specific expression of c-Src, Webster et al. (1995) established transgenic mice that carried a constitutively activated form of c-Src under the transcriptional control of the murine mammary tumor virus long terminal repeat. Female transgenic mice exhibited a lactation defect and frequently developed mammary epithelial hyperplasias, which occasionally progressed to frank neoplasias. The authors interpret these results to mean that expression of activated c-Src in the mammary gland is not sufficient for induction of mammary tumors-that some other event must take place for frank neoplasias to occur. That c-Src can play more than a bystander role in tumor development, however, was demonstrated by the experiments of Guy et al., (1994),wherein mice transgenic for the polyoma virus middle-T antigen under the control of the murine mammary tumor virus long terminal repeat developed tumors when in a genetic background positive for c-Src, but not when in a background null for c-Src. Similar results were obtained by Amini et al. (1986a), who used c-Src antisense expression vectors to demonstrate that c-Src is required for transformation of rat FR3T3 cells by polyoma middle-T antigen in tissue culture. Together, these studies indicate that c-Src is necessary but not sufficient for tumor development in the mammary gland. 5. LUNG CANCER

Lung cancer is the leading cause of cancer death in the United States. Small cell lung cancer (SCLC) accounts for 20-25% of all bronchogenic carcinoma and is associated with the poorest 5-year survival of all histologic types. c-Src expression was found to be elevated in 60% of all lung cancers (Mazurenko et al., 1991b), when biopsy material of tumors, metastases, and

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“normal” surrounding tissues from patients with different histological types of stomach and lung cancer, melanoma, and other malignancies were analyzed by immunoblotting and immunohistochemistry. A breakdown of the lung histologic types exhibiting increased c-Src expression revealed that cSrc protein was elevated in SCLC and atypical carcinoid tumors, as well as in non-small-cell tumors, such as adenocarcinoma, bronchoalveolar, and squamous cell lung cancer (Mazurenko et al., 1991a). In these studies no analysis of c-Src kinase activity was reported. Somewhat contrasting results were reported by authors of a study in which 60 human cell lines used by the National Cancer Institute for the random screening of potential anticancer drugs were analyzed for c-Src kinase activity. In this study SCLC-derived cell lines had a low activity, whereas non-small-cell lung tumors exhibited activity that was greater than that observed in colon cancer cells, which are considered to have high c-Src activity (Budde et al., 1994). The findings from these studies are strongly supportive of other investigations, concluding that c-Src is frequently overexpressed in SCLC and other types of lung cancer (Cook et al., 1993).

6 . OTHER CANCERS Many other tumor types exhibit elevations in c-Src kinase activity or proteinlmRNA levels, including neuroblastomas (Bjelfman et al., 1990)and carcinomas of the esophagus (Jankowski et d.,1992; Kumble et d.,1997), gastric tract (Takekura et al., 1990), parotid gland (Bu et al., 1996), ovary (Budde et al., 1994), and skin (Kim et al., 1991). With regard to skin cancers, a study carried out in a mouse model of epidermal tumor promotion described activation of erbB2 and c-Src in phorbol ester-treated mouse skin as a possible mechanism by which phorboi esters promote skin tumors in mice. Activation of erbB2 and c-Src kinase is also observed in the epidermis of TGFa transgenic mice, where expression of human TGFa was targeted to basal keratinocytes (Xian et al., 1997). In cervical cancer, evidence is beginning to emerge for cis activation of cellular protooncogenes (including c-Src) by integration of human papillomavirus DNA into the genome of cervical epidermal cells (Durst et al., 1987). In tissue culture studies using primary hamster embryo cells, infection with other DNA tumor viruses, such as SV40, adenovirus, or bovine papillomavirus, also results in increases in the specific activity of c-Src (Amini et al., 198613).

C. c-Src Family Members and Human Cancers c-Src is the prototype for a family of nonreceptor protein tyrosine kinases, for which novel members are regularly being identified. Current members

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include c-Src, Fyn, c-Yes, Lck, Hck, Lyn, c-Fgr, Blk, and Yrk (Brickell, 1992; Sudol et al., 1993; Brown and Cooper, 1996). All members have the same overall structure and minimally contain Unique, SH3, SH2, and kinase domains. The greatest sequence divergence occurs in the Unique domain, thus its name. Not all members are linked to lipids at the N terminus, nor are all negatively regulated by a C-terminal domain that includes the Tyr-530 homolog of human c-Src. c-Fgr, Lck, Hck, and Blk are expressed predominantly in cells of hematopoietic lineage, whereas c-Src, c-Yes, Fyn, Lyn, and Yrk are more ubiquitous. All members have been implicated in various signal transduction pathways, and with the mounting evidence for involvement of c-Src in the genesis of multiple human cancers, the question arises as to whether close relatives of c-Src may also be implicated in these diseases. If so, there are other questions that warrant investigation: Is more than one family member involved in the genesis of the same tumor type? Do c-Src family members fulfill overlapping or unique functions in promoting tumor formation and progression? Are there members of this family that are expressed exclusively in tumors as compared to normal tissue? A review of the literature reveals a paucity of information with regard to any of these issues. It is not clear whether this paucity reflects the unavailability of useful and appropriate reagents to investigate the questions, or whether studies have been conducted and few have uncovered evidence for c-Src family member involvement. Although the following description is not meant to be comprehensive, it does suggest that family members in addition to c-Src may be involved in the genesis of human and certain animal tumors. 1. GENETIC EVIDENCE

In humans, sequences related to the human c-Yes gene were found to be amplified in a single primary gastric cancer out of 22 cases that were examined (Seki et al., 1985). The sequences were amplified four- to fivefold, but normal stomach tissue adjacent to the tumor tissue in the same patient showed no amplification. In the dog, a protooncogene related to the human c-Yes gene was detected as restriction fragment-length polymorphisms (RFLPs) in a Southern blot analysis of genomic DNA from six canine primary mammary tumors in a screen employing seven protooncogene probes. These RFLPs were 0.1 to 1.0 kb shorter than the normal gene, suggesting the occurrence of chromosomal rearrangements and possible deregulation of gene expression, leading to tumorigenesis (Miyoshi et al., 1991). Melanoma formation in the fish Xiphophorus is a genetic model for the function of tyrosine kinases in tumor development. In malignant melanomas from these fish, elevated levels of c-Yes and Fyn activity have been detected as compared to normal tissue (Hannig et al., 1991). Fyn has also been found to coprecipitate with the Xiphophorus melanoma receptor kinase (Xmrk),the molecule

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that is responsible for the formation of hereditary malignant melanoma in this lower vertebrate (Wellbrook et al., 1995). These results suggest that Xmrk may function at least in part through Fyn in melanoma formation.

2. BIOCHEMICAL EVIDENCE In studies similar to those conducted for c-Src, evidence for the involvement of other c-Src family members in the etiology of human cancers is emerging, but at a much slower pace than that for c-Src. Elevated c-Yes tyrosine kinase activity has been detected in premalignant lesions of the colon that are at greatest risk for developing cancer (Pena et al., 1995). In this study, the activity of c-Yes in such adenomas was 12- to 14-fold greater than activity in adjacent normal mucosa. Similar results were obtained when mRNA levels of nine protooncogenes in colonic tissue from patients with inflammatory bowel disease (IBD) were measured. The steady-state level of cYes-encoded mRNA was considerably higher in IBD patients resected for colon cancer than in patients resected for active chronic IBD or in controls (Alexander et al., 1996). These results suggest that expression of this gene may be a marker for development of colon cancer in IBD. Finally, in rodents, the action of the transforming proteins of mouse and hamster polyomaviruses (middle-T antigens) is mediated in part through c-Src family kinases, with preferential action of hamster T antigen for Fyn (Brizuela et al., 1995). c-Src family members have also been implicated in the genesis of diseases involving Epstein-Barr Virus (EBV), such as Burkitt’s lymphoma, Hodgkin’s disease, and nasopharyngeal cancer. All of these diseases involve abnormal proliferation of B cells. EBV encodes two transformation-associated proteins, LMPl and LMP2, that are integral membrane proteins. LMP2 mRNA is the only EBV-specific message detected in B lymphocytes from individuals harboring EBV latent infections. LMP2 protein also associates with c-Src family tyrosine kinases, LMPl, and other unidentified proteins, suggesting that the association of these two EBV-encoded membrane proteins could create a macromolecular complex mediating constitutive B lymphocyte activation through normal cell signal transduction pathways (Longnecker, 1994). In human malignant melanoma and other cancers, aberrant expression of basic fibroblast growth factor (bFGF) causes constitutive autocrine activation of its cognate receptor and autonomous growth of tumor cells in culture (see above). Expression of a dominant-negative mutant of the FGF receptor (lacking the kinase domain) was found to suppress tumor formation in nude mice and markedly reduce c-Src family kinase activity in melanoma cells (Yayon et af., 1997). Together these studies suggest that c-Src family kinases play an important role in maintenance and/or progression of malignant melanoma.

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D. Nonreceptor Tyrosine Kinases Related to c-Src Family Members and Human Cancers Several new nonreceptor tyrosine kinases have been isolated from human breast cancer cells. A cDNA encoding a 54-kDa hhosphoprotein, called Rak, was cloned from human breast cancer cells (Cance et al., 1994). This protein shares 51% identify with c-Src and contains SH3,SH2,kinase, and negative regulatory domains. However, it has some properties that are distinct from c-Src, such as its predominant expression in epithelial cells, its lack of a myristylation site, and its almost exclusive localization to the nucleus. However, like c-Src, Rak is overexpressed in subsets of primary human epithelial tumors, suggesting that it may play a role in development of human cancer. Another protein, named Brk (breast tumor kinase), appears to be expressed exclusively in breast tumor tissue as opposed to normal mammary epithelium (Barker et al., 1997). Approximately two-thirds of breast tumors express appreciable levels, and 27% of these overexpress Brk 5- to 40-fold or more. When overexpressed in fibroblasts or mammary epithelial cells, Brk sensitizes cells to the action of EGF and also induces a partial transformed phenotype (Kamalati et af., 1996). These findings suggest that Brk is a functionally important factor in the evolution of breast cancer.

IV. MECHANISMS OF c-Src ACTION A. Evidence for Involvement of c-Src in Signaling

through Receptor Tyrosine Kinases The elevated levels of c-Src expression and/or activation in a wide spectrum of human tumors suggest that c-Src is contributing in some way to the neoplastic phenotype. That c-Src is overexpressed in many of the same tumors in which specific RTKs are also often overexpressed suggests that the two classes of tyrosine kinases may functionally interact to promote tumorigenesis. Many of the RTKs are oncogenic when overexpressed or inappropriately expressed, as described above. The question that follows is whether c-Src is required for the oncogenic capabilities of overexpressed RTKs, or whether c-Src enhances or contributes to RTK-mediated oncogenesis by any means. This latter question was in part addressed when it was shown that cooverexpression of c-Src with HER1 in a mouse fibroblast model resulted in synergistic increases in tumor volume, as compared to tumors developed by cells overexpressing only one of the pair of kinases (Maa et al., 1995). These results provided direct evidence for the enhancing effect of cSrc on receptor-transforming ability, and suggested that a similar synergism

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may be occurring in human tumors that cooverexpress c-Src and the EGFR or other RTKs. Targets of c-Src action can be inferred from an analysis of its known intracellular substrates, which, besides the cell surface receptors, are almost exclusively proteins that regulate actin cytoskeleton dynamics. Thus, c-Src appears to have the capability of affecting both mitogenic growth pathways and morphogenic pathways that influence cell/matrix and cell/cell interactions, motility, invasiveness, and metastasis. Here the focus is on studies that are beginning to reveal the molecular interactions between c-Src and its substrates (as they relate to malignancy) and the effects phosphorylation by c-Src have on their functions. It is becoming clear that c-Src is an obligate partner in mediating mitogenic signaling of at least two RTKs, specifically the PDGF and EGF receptors, and that in the case of EGFR, c-Src mediates tumorigenic signaling as well. This new information, in turn, can be used to design novel diagnostics and therapeutics to interdict the symbiotic relationship between c-Src and the RTKs. A number of different growth factor receptors that have been shown to associate with or activate c-Src or Src family members were enumerated in Section I. These included receptors for PDGF, CSF-1, HGF/SF, and EGF, as well as HER2. With the exception of the PDGFR and EGFR, little is known about the role of c-Src in signaling through these receptors, other than the fact that c-Src either associates with the receptor or is activated following specific ligand stimulation. Therefore, we focus our discussion on c-Src interactions with the PDGF and EGF receptors. The data implicating c-Src in PDGF-dependent signaling will be briefly summarized, this being the subject of several other reviews. The bulk of our attention will then be focused on the mechanism of interaction between c-Src and EGFR family members. 1 . ROLE FOR

c-Src IN SIGNALING FROM THE PDGFR

The first evidence that c-Src participates in PDGFR signaling came from the work of Ralston and Bishop (1985), who first observed that c-Src becomes activated on PDGF stimulation. Kypta et al. (1990) later demonstrated that c-Fyn and c-Yes are also activated in a PDGF-dependent manner. Activation of c-Src was shown to be accompanied by a translocation of c-Src from the plasma membrane to the cytosol (Walker et al., 1993), a process that may be linked to internalization of the receptor. PDGF stimulation was also shown to stimulate transient association of Src family members with the PDGFR (Kypta et af., 1990). Association between Src family members and the receptor is believed to involve phosphotyrosine-SH2 interactions, because the SH2 domain of c-Fyn is required for binding to the receptor in vitro (Twamley et al., 1992) and mutation to phenylalanine of Y579 and Y581 in the juxtamembrane region of the receptor results in a decrease in both PDGF-induced c-Src activation and binding to the receptor in

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vivo (Mori et al., 1993). These data and results from in vitro peptide binding studies (Alonso et al., 1995) suggest that Y579 and Y581 directly mediate binding of Src family members to the PDGFR. Interaction of c-Src with the PDGFR appears to have consequences for both c-Src and the PDGFR. Hansen et al. (1996) have shown that Y934 in the kinase domain of the PDGFR is phosphorylated by c-Src both in vitro and in vivo. Expression of a receptor harboring a phenylalanine substitution at residue 934 in intact cells results in a decreased mitogenic signal and an increase in chemotaxis and motility, along with enhanced PLCy tyrosine phosphorylation. These data suggest that phosphorylation of Y934 by c-Src positively regulates mitogenesis, while negatively regulating cell motility, possibly via a PLCy-mediated pathway. Activation of c-Src by PDGF is also accompanied by the appearance of novel phosphorylations on c-Src, including two serine phosphorylations, S12 and an unidentified S residue (Gould and Hunter, 1988)), and one tyrosine phosphorylation, Y138 (Broome and Hunter, 1997). Y138 is located in the SH3 domain of c-Src, and phosphorylation of this residue diminishes the ability of peptide ligands to bind the SH3 domain in vitro. Mutation of Y138 or Y133 to phenylalanine or complete deletion of the SH3 domain reduces the mitogenic effect of PDGF (Erpel et al., 1996; Broome and Hunter, 1996). The hypothesis that Src family members are required for PDGF-dependent signaling is supported by the inhibitory effects of kinase-inactive c-Src or an antibody specific for the C-terminal domain of Src family members on PDGF-induced BrdU incorporation into newly synthesized DNA (Twamley-Steinet al., 1993).

2. ROLE OF c-Src IN SIGNALING FROM EGFR In our laboratory, initial attempts to detect EGF-induced alterations in cSrc kinase activity or physical association between c-Src and the EGFR in a panel of nontransformed avian and rodent cell lines were negative, or yielded inconsistent results (Luttrell et al., 1988). Therefore, a direct test of the involvement of c-Src was undertaken, in which wild-type (wt) and mutational variants of c-Src were overexpressed in C3HlOT1/2 mouse fibroblasts, and the effect of overexpression of these variants on EGF-induced [3H]thymidine incorporation was examined. Overexpression of wt c-Src resulted in a two- to fivefold increase in [3H]thymidine incorporation above Neo-only controls (Luttrell et al., 1988), whereas overexpression of c-Src harboring inactivating mutations in the kinase, SH2, or rnyristylation domains resulted not only in a reduction in the enhanced effect of overexpressed wt c-Src but also in a dominant negative effect on endogenous, EGFinduced DNA synthesis (Wilson et al., 1989). These results indicated not only that c-Src is required for mitogenesis stimulated by EGF, but also that c-Src kinase activity, an intact SH2 domain, and membrane association are

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necessary to fulfill the role of c-Src in the process. These findings were corroborated by studies in NIH3T3 cells, in which a decrease in EGF-induced BrdU incorporation was observed on microinjection of antibodies to c-Src family members or introduction of a kinase-inactive c-Src cDNA into cells (Roche et al., 1995). c-Src was also shown to affect EGF-induced tumorigenesis (Maa et al., 1995). In C3H10T1/2 cells, coexpression of c-Src and the HERl results in synergistic increases in proliferation, colony formation in soft agar, and tumorigenicity in nude mice, as compared to cells overexpressing c-Src or HERl alone. Furthermore, under conditions of receptor and c-Src overexpression, an EGF-inducible complex between the proteins can be detected. Enhanced tumor growth correlates with the ability of c-Src to associate stably with the receptor, the appearance of two novel tyrosine phosphorylation sites on the receptor, and enhanced phosphorylation of the receptor substrates, Shc and PLCy. These findings suggest that c-Src association with and phosphorylation of the receptor results in hyperactivation of the receptor and enhanced mitogenic signaling to downstream effectors. Subsequent investigations have revealed that the kinase activity of c-Src is required for the biological synergy between c-Src and overexpressed HERl (Tice et al., 1998). Kinase-defective c-Src, when expressed in a cell line overexpressing HER1, acts in a dominant negative fashion to inhibit EGF-dependent colony formation in soft agar and tumorigenicity in nude mice. The effects of both wt and kinase-defective c-Src are very striking, with the single wt c-Src or HERl overexpressors forming barely detectable tumors in nude mice (<<300 mm3) and the c-Src/HERl double overexpressor forming large tumors (- 1600 mm3). In contrast, the HERl/kinase-defective c-Src overexpressors form no palpable tumors. Thus, the extent of tumor inhibition by kinase-defective c-Src is complete in this model system, and the results suggest that the catalytic activity of c-Src may be a fruitful target for human tumor therapy. Interestingly, expression of c-Src variants that bear mutations in either the SH2 or myristylation domains augments, rather than inhibits, tumor formation of cells overexpressing the receptor (D. A. Tice, unpublished). These results are in surprising contrast to those observed when the same c-Src variants are expressed in cells containing normal levels of EGFR (see above and Wilson et al., 1989). The mechanism by which tumor growth is enhanced by these variants is not known. The mechanism of synergy between wt c-Src and HERl is beginning to be elucidated. Several groups have now demonstrated an EGF-dependent complex formation between c-Src and the HERl (Luttrell et al., 1994; Maa et al., 1995; Stover et al., 1995) and an EGF-induced activation of c-Srcspecific kinase activity (Osherov and Levitzki, 1994; Oude Weernink et al., 1994). In all instances, these events are seen in cells overexpressing one or both partners, suggesting that the interaction is either transient or low affin-

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ity. In addition, there is evidence for phosphorylation of HERl by c-Src on EGF stimulation. In the C3HlOT1/2 murine fibroblasts, two sites of tyrosine phosphorylation on c-Src-associated HERl have been identified both in vitro and in vivo as Y845 and YllOl (Biscardiet al., 1998b). Y845 has also been identified as a c-Src-specific phosphorylation site in A431 cells (Sato et al., 1995) and in MDA-MB-468 breast cancer cells (Biscardiet al., 1998b), whereas two other nonautophosphorylation sites, Y891 and Y920, were identified on the receptor from MCF7 cells (Stover et al., 1995). Tice et al. (1998) have shown in 10T1/2 cells that Y845 is the only phosphorylation that is completely dependent on c-Src kinase activity, implicating a direct phosphorylation of the receptor by c-Src at this site, and suggesting that phosphorylation of other c-Src-dependent sites may involve a third component. Y845 is located in the activation loop of the kinase domain and is highly conserved among all tyrosine kinases, receptor and nonreceptor alike. Its homolog in c-Src is Y416. Phosphorylation at the homologous site in other kinases is required for full enzymatic activation, through ATP and substrate accessibility (Ellis et al., 1986; Fantl et al., 1989; Knighton et al., 1991; van der Geer and Hunter, 1991; Longati et al., 1994; Kato et al., 1994; Russo et al., 1996; Yamaguchi and Hendrickson, 1996; Mohammadi et al., 1996; Hubbard, 1997).In 10T1/2 cells the presence of this phosphorylation on the HERl correlates with an increase in tyrosine phosphorylation of receptor substrates Shc and PLCy, and enhanced growth and tumor formation (Maa et al., 1995),consistent with hyperactivation of the receptor. Conversely, the absence of this phosphorylation (in 10T1/2 cells overexpressingreceptor and kinase-defectivec-Src) correlates with reduced growth and tumor formation. Thus, phosphorylation of Y845 appears to be required for the oncogenic capabilities of the receptor. Interestingly, in all other kinases but HER1, the Y845 homolog is an autophosphorylation site. That Y845 has not been identified as such for the HERl may be due to the high lability of the phosphorylation (Biscardi et al., 1998b), or to the fact that c-Src appears to be the kinase that phosphorylates it (Tice et al., 1998). Phosphorylation of Y845 also appears to be critical for normal signaling through the receptor. This is supported by recent findings that a Y 845F mutation completely ablates EGF or serum-induced DNA synthesis, either in the presence or absence of overexpressed c-Src (Tice et al., 1998). Thus, the ability of c-Src to phosphorylate Y845 is critical for manifestation of both the mitogenic and tumorigenic properties of the receptor.

a. EGF Receptor Internalization Based on evidence that implicates the actin cytoskeleton as critical for EGFR internalization (Lamaze et al., 1997), and the involvement of c-Src and c-Src substrates in actin dynamics, as well as the localization of c-Src to

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membranes of intracellular vesicles (Parsons and Creutz, 1986; Kaplan et al., 1992), it is reasonable to speculate that c-Src enhances EGF-dependent signaling by influencing receptor internalization. One hypothesis is that c-Src enhances mitogenesis and tumorigenesis by inhibiting internalization and prolonging receptor signaling at the plasma membrane. In surprising contrast to this hypothesis, however, it was found in studies of 10T1/2 cells that c-Src overexpression enhances rather than inhibits receptor internalization by increasing steady-state pools of internalized, activated receptors (Ware et al., 1997). Receptor recycling rates are not altered. The kinase activity of cSrc is required for the increase, because overexpression of kinase-deficient cSrc exhibits basal or slightly reduced internalization rates. How might the increased internalization contribute to the enhanced cell proliferation and tumorigenic potential seen in cells overexpressing wt c-Src? Recent evidence indicates that receptor/SHC/GRB2/SOS complexes are present in endosomes (DiGuglelmoet a/., 1994), suggesting that EGFlEGFR complexes continue to signal in the endosomal compartment (Baass et al., 1995; Bevan et al., 1996). Because c-Src overexpression increases the steadystate pool of internalized, activated receptors, c-Src may enhance mitogenic and tumorigenic signaling by promoting the frequency of interactions between receptor complexes in the endosomes and Ras at the plasma membrane. Although the mechanism by which c-Src affects EGFR internalization is unknown, several possibilities are plausible. First, c-Src may increase the rate of association of the EGFR with components of the endocytic pathway, such as the adaptins (Sorkin and Carpenter, 1993), or Grb2 (Wang and Moran, 19961, which are thought to recruit activated receptors into clathrin-coated pits. Interestingly, Grb2 associates with dynamin (Gout etal., 1993),a GTPase that is critical for the formation and release of the endosome from the plasma membrane. The c-Src SH3 domain is also reported to activate the GTPase activity of dynamin rn vitro (Herskovits et af., 1993). These considerations suggest that as a second mechanism, overexpression of c-Src could result in the activation or recruitment of a pool of dynamin larger than that in cells expressing normal levels of c-Src. A third mechanism by which c-Src may affect EGFR internalization is through processes that do not involve clathrin-coated pits, such as through caveolae. Caveolae are small invaginations of the plasma membrane that have been implicated in the transcytosis of macromolecules across capillary endothelial cells, the uptake of small molecules, interactions with actin-based cytoskeleton, and the compartmentalization of certain signaling molecules, including G-protein-coupled receptors, H-Ras and Ras-related GTPases, and members of the Src family of tyrosine kinases (Li et al., 1996a,b). Caveolae are enriched for a specific protein, caveolin, which is a substrate for v-Src (Li et al., 1996a,b), and has also been shown to copurify with c-Src in normal

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cells (Lisanti et al., 1994; Henke et al., 1996; Li et al., 1996a,b). Caveolin normally acts as a scaffolding protein to bind inactive signaling molecules, such as G a subunits, Ras, EGFR, and c-Src (Sargiacomo et al., 1993; Lisanti et al., 1994; Chang et al., 1994; Li et al., 1995; Couet et al., 1997). It has also been shown that caveolin expression is down-modulated in cells transformed by various oncogenes (Koleske et al., 1995), and that reexpression of caveolin in v-abl- and H-ras-transformed cells will abrogate anchorageindependent growth in these cell lines (Engelman et al., 1997). Caveolin expression has also been shown by differential display and subtractive hybridization techniques to be down-regulated in human mammary carcinomas and several breast tumor cell lines compared with normal breast epithelium (Sager et al., 1994). This evidence suggests that caveolin is inhibitory for transformation and that overexpression of c-Src may be deactivating caveolin through phosphorylation, leading to increased transformation.

b. Evidence for the HERl/c-Src Synergy Model in Human Breast Cancer Simultaneous overexpression or activation of HERl and c-Src in a significant portion of human breast tumors suggests that the two molecules might functionally interact in human tumors as they do in the 10T1/2 murine fibroblast model. This question was examined by Biscardi et al. (1998a,b), who analyzed a panel of 14 breast tumor cell lines and over 20 tissue samples for levels of HERl and c-Src overexpression, association between c-Src and HER1, phosphorylation of Y845 and YllOl on the receptor in complex with c-Src, increases in Shc phosphorylation, MAP kinase activation, and increases in tumor formation in nude mice. A direct correlation was found between the expression levels of c-Src and HERl and the ability to detect stable interactions between the two kinases, the presence of the novel phosphorylations on the receptor, enhanced phosphorylation of downstream substrates, and tumor formation. Although not direct proof, results from these studies are consistent with those in the 10T112 model and suggest that c-Src and HERl can functionally synergize to promote tumor progression when cooverexpressed in human tumors.

3. c-SrclHERZlneu INTERACTIONS Because HER2/neu is so abundantly and frequently overexpressed in human tumors (particularly in breast cancers) and is oncogenic when overexpressed in cultured fibroblasts (DiFiore et al., 1987b),an important question arises as to whether c-Src acts as a cotransducer to tumorigenic signals through HER2 as it does through HER1. Luttrell et al. (1994) showed that HER2 can be precipitated by the GSTc-SrcSH2 fusion protein from a hu-

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man breast cancer cell extract, suggesting that stable complexes may also form between c-Src and HER2 in vivo. c-Src association with and activation by HER2 was also shown in mammary tumors from HER2 transgenic mice (Muthuswamy et al., 1994; Muthuswamy and Muller, 1995). Moreover, in coimmunoprecipitation studies our laboratory has detected c-Src in association with HER2 in 3 of 14 human breast tumor cell lines and in 3 of 13 tumor tissues (Belsches and Parsons, 1998). Cell lines exhibiting this complex respond to HRG mitogenically and tumorigenically, as measured by cell growth assays and colony formation in soft agar, in contrast to those cell lines that express HER2 but form no complex with c-Src. Interestingly, in contrast to the HER1, overexpression of neither HER2 nor c-Src is a prerequisite for detecting association between the two proteins. These data suggest that c-Src may potentiate HER2-dependent tumorigenicity through mechanisms similar as well as dissimilar to those described for HER1.

B. Targets of c-Src 1. TARGETS WHOSE EXPRESSION LEVELS ARE AFFECTED BY c-Src

The preceding discussion provides compelling evidence that RTKs can be direct targets of c-Src. Phosphorylation of specific sites by c-Src appears to regulate the shift from motility to mitogenesis in the case of the PDGFR (Hansen et al., 1996) and the entrance into S phase of the cell cycle in the case of the EGFR (Fig. 3 ) (Tice et al., 1998). Is c-Src capable of contributing to the malignant phenotype in ways other than through direct regulation of growth factor receptors? One alternative is the ability of c-Src to regulate gene transcription. Barone and Courtneidge (1995) showed that Myc was required to overcome a block of PDGF-induced DNA synthesis by kinasedeficient c-Src, suggesting that Src kinases control the transcriptional activation of Myc, which in turn can induce a program of gene transcription that c

Fig. 3 Targets of c-Src and their potential roles in transformation. c-Src associates with and phosphorylates the ligand-activated EGF receptor, thereby potentiating downstream signaling from the receptor. This is manifested by increased levels of phosphorylated receptor substrates and augmented steady-state pools of internalized, activated receptors. In a reciprocal fashion, activated receptors can mediate activation and translocation of c-Src to the cytoskeleton, where it phosphorylates several substrates, including cortactin, pl30CAS, and pl9ORhoCAP. These substrates are central to regulation of actin cytoskeleton rearrangements and thus signals that control morphological transformation and migration. c-Src also contributes to neoplastic development through cell-cell adhesion signaling and up-regulation of gene transcription. Similar types of interactions are thought to occur with other receptor tyrosine kinases known to associate with c-Src.

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is required for growth. c-Src and v-Src have also been shown to up-regulate transcription of vascular endothelial growth factor (VEGF) (Rak et al., 1995; Mukhopadhyay et al., 1995a,b; Weissenberger et al., 1997). VEGF is a multifunctional cytokine that alters the pattern of gene expression and stimulates the proliferation and migration of endothelial cells that line the walls of microcapillaries. VEGF treatment also renders these same cells hyperpermeable, thereby allowing plasma proteins access to the extracapillary space. This process, in turn, leads to profound alterations in the extracellular matrix that favor angiogenesis (reviewed in Klagsbrun and D’Amore, 1996). Another potent modulator of angiogenesis (reviewed in Tkachuk et al., 1996) and metastasis (reviewed in Andreason et al., 1997) is urokinase-type plasminogen activator (UPA),whose expression is up-regulated by v-Src (Bell et al., 1990, 1993) and whose receptor is found in complex with c-Src family members (Bohuslav et al., 1995). The ability of c-Src to influence gene transcription is a new and emerging question that is receiving considerable attention. However, most of the investigations that focus on the role of c-Src in neoplastic transformation have focused on substrates of c-Src and their contributions to development of the malignant phenotype. c-Src has a number of characterized substrates, most of which have functional connections to the actin cytoskeleton. These different substrates and their potential roles in transformation are discussed below. 2. TARGETS THAT SERVE AS SUBSTRATES OF c-Src

a. Focal Adhesion Kinase Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that localizes to focal adhesions and contributes to the processes of integrin-mediated cell spreading and migration through regulation of actin cytoskeleton remodeling (reviewed in Parsons and Parsons, 1997). FAK becomes tyrosine phosphorylated in response to various environmental stimuli, such as extracellular matrices and polypeptide and neuropeptide growth factors (reviewed in Schaller and Parsons, 1994). c-Src is intimately involved in FAK-mediated signaling. Activated c-Src (Y527F) is complexed with FAK through binding of the c-Src SH2 domain to Y397, the FAK autophosphorylation site (Schaller et al., 1994; Cobb et al., 1994). In addition, wt c-Src appears to be required for the FAK-mediated, integrin stimulation of mitogen-activated protein kinase (MAPK) (Schlaepfer and Hunter, 1997). Introduction of the amino-terminal half of c-Src, which lacks the kinase domain, reconstitutes integrin-induced MAPK activation in c-Src -/- fibroblasts, thus the process appears to be independent of c-Src kinase activity (Schlaepfer et al., 1997). This finding suggests a potential role for c-Src as a docking protein. The role of c-Src in cell spreading also appears to be independent of the kinase domain, because defects in spreading of fibroblasts derived from c-Src null mice can be restored by the SH2 and SH3 domains but not by the catalytic do-

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main of c-Src (Kaplan et al., 1994, 1995). These findings suggest that even though c-Src is activated in response to motogenic factors such as EGF and HGF, it may require only the redistribution of c-Src to focal adhesions to stimulate motility and focal adhesion turnover. Given that FAK is known to transduce signals involved in the regulation of cell adhesion and motility as well as the anchorage-independent growth of transformed cells, it would not be unexpected to find aberrant expression of FAK in human tumors. Indeed, increased expression or activation of FAK is observed in a number of human tumors, including sarcomas and carcinomas of the breast, prostate, and colon (Weiner et al., 1994; Owens et al., 1995; Withers et al., 1996; Tremblay et al., 1996). As might be expected with involvement of FAK in motility, the highest levels of FAK are seen in metastatic or invasive lesions (Weiner et al., 1994; Owens et al., 1995; Tremblay et al., 1996). An increase in FAK phosphorylation also correlates with increased migration and invasiveness of squamous cell carcinoma cells treated with HGF (Matsumoto et al., 1994).Together, the evidence suggests that signaling through FAK/c-Src complexes in normal and malignant cells is bidirectional. Integrins activate FAK, which can signal through c-Src to activate ERK2, or conversely, engaged growth factor receptors activate c-Src, which can then signal through FAK to mediate motogenic or cytoskeletal responses.

b. pl30CAS pl30Cas (CAS) was first identified as a highly tyrosine-phosphorylated protein in cells transformed by a variety of oncogenes (Mayer and Hanafusa, 1990; Auvinen et al., 1995; Salgia et al., 1996) and in normal cells following activation of integrins (Nojima et al., 1995; Petch et al., 1995; Vuori et al., 1996) and stimulation with mitogenic neuropeptides, such as bombesin, vasopressin, and endothelin (Zachary et al., 1992; Seufferlein and Rozengurt, 1994). The role of CAS in integrin or growth factor-mediated signaling is not understood. However, recent evidence indicates that CAS functions like an adapter molecule, binding a number of signaling molecules that participate in cell adhesion, such as FAK (Polte and Hanks, 1995; Harte et d., 1996), PTP-PEST (Garton et al., 1997), and Src family kinases (Polte and Hanks, 1995; Nakamoto et al., 1996).Tyrosine phosphorylation of CAS is increased on adhesion and is largely dependent on c-Src (Vuori et al., 1996; Hamasaki et al., 1996). Thus, the link between cell adhesion, the actin cytoskeleton, and tumorigenesis is repeated, and the common involvement of c-Src in both processes suggests that c-Src may be a critical factor that links them together.

c. Cortactin The v-Src and c-Src substrate, p75lp80lp85 cortactin, is an actin-binding protein that contains five tandem repeats in the N terminus and an SH3 domain in the extreme C terminus (Wu et al., 1991; Maa et al., 1992; Wu and

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Parsons, 1993). The N terminus is responsible for the in vitro binding to actin. In vivo phosphoamino acid analysis of cortactin from primary chick embryo cells reveals that it contains only serine and threonine phosphorylations, whereas it exhibits constitutive phosphorylation on tyrosine in addition to serine and threonine in cells transformed by activated c-Src (Y527F) (Wu et al., 1991). In immortalized murine 10T1/2 fibroblasts, cortactin has a low basal level of tyrosine phosphorylation that is increased on both EGF stimulation and c-Src overexpression (Wilson and Parsons, 1990; Maa et al., 1992). Cortactin has also been shown to be tyrosine phosphorylated in response to fibroblast growth factors (Zhan et al., 1993, 1994).These observations suggest that cortactin may be a substrate of growth factor receptors as well as of c-Src or that c-Src mediates growth factor-induced tyrosine phosphorylation of cortactin. The findings that cortactin associates with the Src SH2 domain and colocalizes with v-Src in transformed cells (Okamura and Resh, 1995), and that increased tyrosine phosphorylation of cortactin is seen in CSK-deficient cells, favor the notion that c-Src and/or its family members are responsible for phosphorylating cortactin (Nada et al., 1994; Thomas et al., 1995).Interestingly, two phases of EGF-induced cortactin tyrosine phosphorylation can be observed in lOTl/2 cells, one occurring within 2-10 min following stimulation and another occurring later in GI, with the maximum level seen approximately 9 hr posttreatment. In both cases, the level of phosphorylation is increased by overexpression of c-Src (Maa et al., 1992). These observations raise the question of whether cortactin may function in mid-late G, as well as in immediate-early GI. Indirect immunofluorescence microscopy of lOTl/2 cells reveals that cortactin is localized within the cytoplasm to punctate sites that are concentrated around the nucleus and colocalized with actin at the plasma membrane and peripheral adhesion site (Maa et al., 1992). This pattern is not altered on EGF treatment or c-Src overexpression in 10T1/2 cells. However, in v-Src- or Y527F-c-Src-transformed cells, cortactin is localized to modified focal adhesions, termed podosomes. The appearance of podosomes is associated with loss of adhesive properties (Marchisio et al., 1987; Wu et al., 1991). Cortactin is also localized to podsome-like cell matrix sites in human tumors that overexpress the protein (including carcinomas of the breast and head and neck) (Schuuring et al., 1992; Brookes et al., 1993; Williams et al., 1993; Schuuring et al., 1993; Meredith et al., 1995; Campbell et al., 1996; van Damme et al., 1997).From these observations, it follows that abnormal subcellular distribution of cortactin in human carcinomas may play a role in deregulating important protein-protein interactions that may be required for the proper formation of cell matrix contact sites. In support of this hypothesis is the correlation of cortactin overexpression with increased invasiveness, metastasis, and a poorer patient prognosis (Williams et al., 1993; Meredith et al., 1995; Takes et al., 1997).

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d. p 190RhoGAP pl9ORhoGAP was first identified as a tyrosine-phosphorylated protein that coprecipitates with pl20RasGAP from v-Src-transformed Rat-2 cells (Ellis et al., 1990). p190 has an N-terminal domain that binds GTP (Settleman et al., 1992b; Foster et d., 1994) and a C-terminal Gll’ase-activating domain (GAP) that is specific for small GTP-binding proteins of the Rho family (Settleman et al., 1992a). p190 is functionally linked to the actin cytoskeleton through its ability to stimulate the conversion of Rho-GTP (which stimulates stress fiber formation) to Rho-GDP (which permits actin disassembly) (Ridley and Hall, 1992). Two phosphorylation sites, Y1087 and Y1105, in the middle portion of the molecule are postulated to mediate binding to the two SH2 domains of pl20RasGAP (Bryant et al., 1995; Hu and Settleman, 1997), although of the two sites, only Y1105 phosphorylation can be detected in vivo. Overexpression of c-Src in 10T1/2 cells results in an increase in the basal tyrosine phosphorylation of p190, specifically at Y1105 (Roof et al., 1998). This evidence, along with the findings that overexpression of kinase-deficient c-Src decreases the phosphorylation at Y1105 and that c-Src phosphorylates Y1105 in vitro, suggests that c-Src is directly responsible for phosphorylation of this residue. Levels of p190 tyrosine phosphorylation are generally correlated with levels of the p190/p120RasGAP complex that can be detected in vivo, suggesting that high levels of p190 tyrosine phosphorylation could bind more RasGAP, thereby sequestering RasGAP away from Ras and permitting Ras to remain in the active, GTP-bound state longer. This scenario is consistent with the role of c-Src as a comitogenic signaling partner of growth factor receptors. Although EGF treatment of 10T1/2 cells does not cause a further increase in tyrosine phosphorylation of p190, it does cause a rapid (seconds to minutes) and transient redistribution of p190 from a diffuse cytoplasmic localization into concentric arcs that radiate away from the nucleus with a time course that mimics EGF-stimulated actin dissolution (Chang et al., 1995). Overexpression of wt c-Src expands the window of time in which EGF-induced actin dissolution and p190 arc formation occur, whereas overexpression of kinase-deficient c-Src contract the window. These results correlate with the level of p190 tyrosine phosphorylation and implicate another role for c-Src in regulating cytoskeletal reorganization, possibly by inactivating Rho through activation and redistribution of pl9ORhoGAP.

e. Platelet Endothelial Cell Adhesion Molecule Platelet endothelial cell adhesion molecule-1 (PECAM-1)is a 130-kDa glycoprotein of the immunoglobulin gene superfamily that localizes to points of contact between confluent endothelial cells (Newman et al., 1990; Tanaka et al., 1992). On induction of endothelial sheet migration, PECAM-1 becomes diffusely organized within the cytoplasm, and ectopic expression of

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the gene inhibits cell migration, suggesting that translocation from the periphery to the cytoplasm is a mechanism by which the inhibitory action of PECAM-1 is relieved (Schimmenti et al., 1992). The cytoplasmic tail of PECAM-1 is critical for cell surface activity (DeLisser et al., 1994; Yan et a!., 1995). It contains immunoreceptor tyrosine-based activation motifs (ITAMS) that are phosphorylated by c-Src in vitro and in vivo and bind cSrc SH2 domains in vitro (Lu et al., 1997). Several lines of evidence suggest that tyrosine phosphorylation is involved in transducing cell migration signals through this molecule (Lu et al., 1996; Pinter et al., 1997). Tyrosines 663 and 686 appear to be the major sites of tyrosine phosphorylation, because mutation of either residue results in a drastic reduction in tyrosine phosphorylation, and mutation of Y686 is associated with a reversal of the PECAM-1-mediated inhibition of cell migration (Lu et al., 1996). Again, phosphorylation by c-Src is a potential mechanism of regulation of a molecule involved in cell-cell contacts and migration, pointing to a role for both PECAM-1 and c-Src in angiogenesis and metastasis.

f. Other Substrates Additional reports implicate still other c-Src substrates in cell-cell adhesion. Syndecan-1 is a cell surface proteoglycan that interacts with extracellular matrix molecules and growth factors to maintain epithelial cell morphology, anchorage-dependent growth, and inhibition of invasiveness in cell culture assays. The absence of this molecule correlates with a higher grade of transformation and poorer patient prognosis (Inki and Jalkanen, 1996). Its expression is negatively regulated at the level of translation on transformation by polyoma virus middle-T antigen. The effects of middle-T antigen are dependent on association with and activation of c-Src (Levyet al., 1996). c-Src may also function in processes other than those related to cytoskeletal or adhesion dynamics but that still lead or contribute to a transformed phenotype. For example, a role for c-Src in mitosis has been implicated through the identification of a 68-kDa RNA-binding protein, called Sam68 (Srcassociated in mitosis), that binds the SH3/2 domains of c-Src (reviewed in Courtneidge and Fumagali, 1994) and is postulated to act through c-Src to regulate microtubule dynamics via association with (Abu-Amer et al., 1997) and phosphorylation of (Matten et al., 1990) tubulin.

V. POTENTIAL THERAPEUTIC APPLICATIONS OF c-Src/HERl INTERACTIONS Along with the first evidence for possible roles for receptor and nonreceptor tyrosine kinases in human tumorigenesis has come the development of strategies to inhibit the functions of these classes of enzymes. A plethora of

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inhibitors based on various structural and functional characteristics of the enzymes have been developed. First and foremost among these are inhibitors of catalytic activity. Among these inhibitors are the tyrphostins (Burke, 1992; Levitzki and Gazit, 1995), which compete with the protein substrate for access to the catalytic site; genistein (Akiyama et al., 1987), a competitive inhibitor of ATP; lavendustins A and B (Onoda et al., 1989); erbstatin (Imoto et al., 1987); and herbimycin A (Uehara et al., 1986, 1989a,b), which has been shown to promote the ubiquitin-based degradation of the TKs (SeppLorenzino et al., 1995).Other inhibitors are designed to prevent interactions mediated through SH2 and SH3 domains (peptidomimetics) (Smithgall, 1995; Plummer et al., 1996,1997) or to prevent myristylation (N-fatty acyl glycinal compounds) (Shoji et al., 1990). In numerous cases these reagents have been demonstrated to be antiproliferative (Clark et al., 1996; Traxler et al., 1997; Hartmann et al., 1997).In other instances membrane penetrance of the drug has been a problem in testing their efficacies in tissue culture and animal models (Gilmer et al., 1994). Studies characterizing the molecular interactions between c-Src and HERl have revealed an additional target for drug design, specifically the sequences surrounding Y845 of HER1. Phosphorylation of this site by c-Src appears to be required for the mitogenic and tumorigenic aspect of receptor function, as shown by the inability of kinase-defective c-Src to phosphorylate Y845 and the nonfunctionality of the mutant Y845F receptor. In human tumors that overexpress c-Src and HERl, inhibiting the ability of c-Src to phosphorylate Y845 might reduce the tumorigenic potential of the overexpressed receptor as well as the ability of c-Src to synergize with the receptor. Such inhibition might be accomplished by a Y 845 peptidomimetic. The advantages appear to be that this inhibition targets an enzymehubstrate interaction that occurs to the greatest degree in those cells that overexpress both players (c-Src and HER1, respectively), namely, cancer cells. In no normal cells are these two molecules known to be simultaneously overexpressed. For example, in the adult, the highest levels of c-Src are found in platelets (Golden et al., 1986) and in cells of the nervous system, whereas high levels of HERl are found in the liver and kidney (Nexo and Kryger-Baggesen, 1989).In theory, therefore, the Y845 peptidomimetic might be more likely to target the tumor cells than the normal cells, thus providing a potential “tumor-specific” drug for cancers such as carcinomas of the colon, breast, and lung.

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Epidemiology of Kaposi’s Sarcomawhsociated Herpesvirus/Human Herpesvirus 8 Thomas F. Schulz Molecular Virology Group Department of Medical Microbiology and Genitourinary Medicine The University of Liverpool Liverpool L69 3GA, United Kingdom

I. Introduction 11. KSHV Phylogeny and Molecular Epidemiology 111. Geographic Distribution A. PCR-Based Studies B. Serological Studies C. Prevalence of KSHV and Incidence of Classic or Endemic KS in Different Geographic Regions IV. KSHV Prevalence in Risk Groups for HIV-1 Transmission A. PCR Studies B. Serological Studies C. KSHV Prevalence and Risk for KS in Different Risk Groups for HIV Transmission D. KSHV Prevalence in Transplant Recipients V. Transmission of KSHV A. Sexual Transmission B. Transmission in Childhood C. Parenteral Transmission VI. Association of KSHV with Disease A. Kaposi’s Sarcoma B. Body Cavity-Based LymphomalPrimary Effusion Lymphoma C . Castleman’s Disease and Related Lymphoproliferative Disorders D. Unconfirmed Links between KSHV and Other Diseases VII. Conclusion References

I. INTRODUCTION Very quickly after its discovery by Chang and co-workers in 1994, the strong association of human herpesvirus 8 (HHV 8) with Kaposi’s sarcoma was confirmed in several studies, and the original designation as Kaposi’s sarcoma-associated herpesvirus (KSHV)appeared justified (Chang et al., 1994). Yet, skeptical views as to its role in the pathogenesis of Kaposi’s sarcoma perAdvances in CANCER RESEARCH 0065-23OW99 $30.00

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sisted (Levy, 1995; Cohen, 1995); these related to epidemiological considerations, such as the typical rapid spread and wide distribution of other herpesviruses in infected populations, which appeared incompatible with the postulated sexual transmission of the “KS agent.” The skeptical views were fueled by some reports claiming a wide distribution of KSHV and association with other diseases in developed countries. Although some controversial issues remain, many others have been, or are close to being, resolved. What is known about the epidemiology of KSHV supports its causative role in the pathogenesis of Kaposi’s sarcoma. At the same time, the present state of knowledge points to an involvement of other cofactors in the pathogenesis of this unusual tumor.

KSHV PHYLOGENY AND MOLECULAR EPIDEMlOLOGY

11.

Phylogenetic analysis of the KSHV viral genome reveals regions that appear to be conserved in other herpesviruses (Moore et al., 1996a), placing KSHV in the yz subgroup of herpesviruses (rhadinoviruses). This is exemplified in Fig. 1 by a comparison of glycoprotein B genes of several herpesviruses. Rhadinoviruses include several representatives capable of causing lymphoproliferative disease (Herpesvirus saimirz, alcephaline herpesvirus, and murine herpesvirus 68) and others that have not yet been clearly associated with any disease (equine herpesvirus 2). Three rhadinoviruses found in different macaque species presently appear to be the closest “relatives” of KSHV, and may in fact define a new subgroup of rhadinoviruses (Rose et ai., 1997; Desrosiers et al., 1997). Of these, rhesus rhadinovirus (RRV) has not so far been linked to any disease (Desrosiers et al., 1997), whereas two others, retroperitoneal fibromatosis virus of Macaca mulatta (RFmm) and retroperitoneal fibromatosis virus of Macacu nemestrina (RFmn) were first identified in animals coinfected with simian retrovirus 2 (SRV-2) and suffering from retroperitoneal fibromatosis (Rose et al., 1997). Additional evidence suggests that another closely related rhadinovirus is present in African Green monkeys (Greensill et al., 1998). KSHV occurs in several variants, defined on the basis of sequence variability at the left and right end of the viral genome (all other parts of the viral genome are highly conserved) (Russo et al., 1996; Lagunoff and Ganem, 1997; Neipel et al., 1997a,b; Nicholas et al., 1998). At the left genomic end, the first viral gene, open reading frame (orf) K 1 is highly variable (14-35% protein sequence difference) and phylogenetic analysis based on this region reliably distinguishes four major KSHV “subtypes” with high bootstrap values (>85%) (Nicholas et a/., 1998; Cook et al.,1998; Russo et al., 1996;

Gamma 2 herpesviruses (‘Rhadinoviruses’) KSHV/HHV-S

Gamma 1 heruesviruses EBV

H I

HHV-6

-

I

CMV

0.1

Beta herpesviruses

Fig. 1 Phylogenetic relationship of KSHVIHHV-8 to other herpesviruses. The glycoprotein B sequence of different herpesviruses was aligned by Clustal, and the alignment analyzed by the Neighbor Joining method. For a and p herpesviruses, only human viruses are shown. HSV, Herpes simplex virus; VZV, varicella zoster virus; CMV, cytomegalovirus; HHV-6, HHV-7, human herpesviruses 6 and 7; EBV, Epstein-Barr virus; EHV-2, equine herpesvirus 2; HVS, Herpesvirus saimiri; RRV, Rhesus rhadinovirus; MHV-68, murine herpesvirus 68.

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Thomas F. Schulz

Lagunoff and Ganem, 1997; Neipel et al., 1997a,b; Nicholas et al., 1998; Lee et al., 1998; Cook et al., 1998). Variability in orf K1 appears to be driven by selective pressure in that there is a consistently high ratio of nonsynonymous to synonymous nucleotide changes throughout the entire K 1 region (Nicholas et al., 1998; Cook et al., 1998). Three of these four subtypes have also been defined on the basis of the minimal sequence variation in orfs 26 and 75 (Boshoff et al., 1995a; Huang et al., 1995; Marchioli et al., 1996; Luppi et al., 1997; Zong et al., 1997). However, because of the high degree of sequence conservation in these regions, phylogenetic analysis is much less robust that if orf K1 is used. Within each of the four major subtypes, further lineages can be distinguished that do not however separate as reliably in phylogenetic analysis as the major subtypes, and some of these may also be associated with certain geographic regions or populations (Nicholas et al., 1998; Cook et al., 1998). At the right genomic end, two highly divergent virus variants have been recognized (Nicholas et al., 1998) that, unlike orf K1, do not appear to be under any selective pressure (Nicholas et al., 1998). Recombination between the left and the right end of the viral genome appears to occur occasionally (Nicholas et al., 1998). An initial comparison of sequence variants in the orf 26/75 region had suggested that variant “A” may predominate in Mediterranean “classic” KS, with variants “B” and “C” possibly more common in Africa (Zong et al., 1997), or among patients with lymphoproliferative disease (Luppi et al., 1997), and all three had been found among AIDS KS patients in the United States and the United Kingdom (Zong et al., 1997; Boshoff et al., 1995a; Marchioli et al., 1996). More recent studies on the variability of orf K1 indicate that subtype B is mainly found in Africa, in AIDS KS patients as well as in HIV-infected asymptomatic of HIV-uninfected individuals (Nicholas et al., 1998; Zong et al., 1998; Cook et al., 1998). Both A and C strains also occur in Africa (Cook et al., 1998). In Europe, subgroups A and C are found most frequently in AIDS KS, HIV-negative “classic” KS and HIV-uninfected individuals (Cook et al., 1998).

Ill. GEOGRAPHIC DISTRIBUTION A. PCR-Based Studies To study its distribution in asymptomatic individuals from different geographic regions, several groups have used nested polymerase chain reaction (PCR) to detect KSHV DNA in peripheral blood mononuclear cells (PBMCs) and semen samples (Collandre et al., 1995; Whitby et al., 1995;

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125

Ambroziak et al., 1995; Howard et al., 1997; Monini et al., 1996a,b; Moore et al., 1996b; Marchioli et al., 1996; Humphrey et al., 1996; Lefrkre et al., 1996; Heredia et al., 1996; Harrington et al., 1996; Brambilla et al., 1996; Viviano et al., 1997; Blum et al., 1997). As discussed in more detail below, this technique can detect KSHV in PBMCs and semen samples of KSHVinfected patients. Most larger studies reported KSHV detection rates of 45-67% in PBMCs from AIDS KS and classic KS patients (Whitby et al., 1995; Moore et al., 1996b; Lefrkre et al., 1996; Humphrey et al., 1996; Bigoni et al., 1996; Brambilla et al., 1996; Marchioli et al., 1996; Ariyoshi et al., 1998). The rate of detection may depend on the extent of clinical disease (Noel et al., 1996; Aluigi et al., 1996; Blum et al., 1997). Because all KS patients can be assumed to be infected with KSHV (see below), these results indicate that nested PCR on PBMC samples is not sufficiently sensitive to derive exact prevalence rates of KSHV infection in HN-uninfected healthy individuals. However, studies of this kind have pointed to geographic regions where KSHV may be more prevalent. Thus, with one exception (Decker et al., 1996), KSHV DNA was not found in PBMCs from donor blood in the United Kingdom, United States, Spain, or France (Whitby et al., 1995; Humphrey et al., 1996; Marchioli et al., 1996; Collandre et al., 1995; Heredia et al., 1996),whereas it was found infrequently (8-11%) in some (Bigoni et al., 1996; Viviano et al., 1997) but not other (Luppi et al.,1996a) studies on Italian control subjects. Likewise, KSHV was detected in 10% of HIV-negative antenatal mothers from The Gambia (Ariyoshi et al., 1998), and in 8% of febrile Zambian children (Kasolo et al., 1997). Similarly,KSHV has been found in semen and prostate of some KS patients (Corbellino et al., 1996a,b; Howard et al., 1997; Gupta et al., 1996; LebbC et al., 1997b; reviewed in Blackbourn and Levy, 1997). Attempts have been made to estimate the rate of KSHV infection in the general population by analyzing semen samples from healthy donors. KSHV DNA was not detected in semen or prostate samples from healthy semen donors from the United Kingdom (Howard et al., 1997), the United States (Gupta et al., 1996), or France (LebbC et al., 1997b). One United States study that initially reported the detection of KSHV DNA by nested PCR in a significant proportion of healthy donors (Lin et al., 1995) has since been withdrawn. By in situ hybridization KSHV DNA was found in a significant proportion of prostate biopsies, collected in the United States (Staskus et al., 1997). However, in view of the undefined provenance of these samples, these authors refrained from drawing conclusions as to the epidemiological significance of these findings (Staskus et al., 1997). In Italy, one study from Sicily (Viviano et al., 1997) found 3/25 (13%) semen samples from healthy donors to contain KSHV DNA, whereas in Ferrara, Northeastern Italy, Monini et al. (1996a,b) reported initially a very high positivity rate (900/,)that decreased to 23% in

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Thomas E Schulz

a second report (Monini et al., 1996b). In contrast, no KSHV DNA was found in semen or prostate of HN-uninfected patients from Milan, in Northwestern Italy (Corbellino et al., 1996a,b). While partially confusing, and at times very controversial, most of these results fit with more recent seroepidemiological findings (see below). KSHV appears to be more prevalent in Southern Italy and at least some regions of Northeastern Italy than in Milan or Northern Europe and the United States. However, given the low detection rate of KSHV in semen samples from KS patients reported in most larger studies (Howard et al., 1997; Gupta et al., 1996),this approach would not appear to be suited to determine exact prevalence rates for KSHV in the general population.

B. Serological Studies 1 . SEROLOGICAL METHODS

Several serological assays, detecting antibodies to both latent and lytic (i.e., structural proteins expressed during lytic viral replication) KSHV antigens, have been developed. As shown in Table I, some of these assays give very different seroprevalence rates in blood donors from different geographic regions. A consensus has not yet been reached on which assay, or combination of assay and antigens, produces the best specificity and/or sensitivity. However, some of the antigens discussed below are likely to be part of the next generation of assays. The most prominent latent serological antigen appears to be the highmolecular-weight (224-236 kDa) nuclear protein, LNA (Gao et al., 1996a), encoded by open reading frame (orf) 73 of KSHV (Rainbow et al., 1997; Kellam et al., 1997). Antibodies to this latent nuclear antigen can be detected by Western blot (Gao et al., 1996a) or immunofluorescence on latently (persistently) KSHV-infected B cell lines established from body cavity-based lymphomas/primary effusion lymphomas (Kedes et al., 1996; Gao et al., 1996b; Simpson et al., 1996). LNAlorf73 appears to be the main component of the speckled nuclear immunofluorescence pattern, termed “latencyassociated nuclear antigen” (LANA), seen using serum from KSHV-infected individuals (Kedes et al., 1996; Gao et al., 1996b; Simpson et a/., 1996; Rainbow et al., 1997; Kellam et al., 1997). As in the case of the EBV-encoded EBNAs, other nuclear KSHV proteins could conceivably contribute to LANA (Rainbow et al., 1997; Kellam et al., 1997), but none has so far been identified. The sensitivity and specificity of LANA immunofluorescence assays and LNA Western blot are high: antibodies to LANA are detected in 80-85% of patients with AIDS-associated KS, >90% of those with classic (HIV-negative) KS, but only in 0-3% of blood donors from the United States

Table 1 Serological Assays for KSHV Antibody detection rate (%) Blood donors Antigen

Assay

Latent antigen LANA IFA (latency-associated nuclear antigen)

LNA WB (2261234-kda nuclear protein) Structural (lytic) antigens vp19 (orf65) ELISANW

vp23 (orf26)

Classic KS

AIDS KS

UK

USA

85-94

71-88

3

0-9

100

80

-

0

91

75-84

1.7

5

40-60 -60

ELISA

-

90-97

Lytically infected cells

IFA

90-100

20-29

IFA IFA

100 100

11 0-8

92

11

93

Ref.

7-25

Gao et al. (1996a,b); Kedes et al. (1996), Simpson et al. (1996), Whitby et al. (1998), Lennette et al. (1996), Rabkin et al. (1998), Calabrb et al. (1998) Gao et al. (1996a)

9-23

Simpson et al. (1996), Lin et al. (1997), Calabri, et al. (1998) AndrO et al. (1997), Davis et al. (1997), Rabkin et al. (1998) Raab et al. (1998), Chandran et al. (1998b) Lennette et al. (1996) Rabkin et al. (1998) Ablashi et al. (1997) Smith et al. (1997), Chandran et al. (1998b) Ablashi et al. (1997), Chatlynne et al. (1998)

-11 0

ELISAJWB

ELISA

Germany

20-54

or& 8.1

Purified virus

Italy

-

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Thomas E Schulz

or United Kingdom (Kedes et al., 1996; Gao et al., 1996a,b; Simpson et al., 1996; Rabkin et al., 1998). There is no homolog of orf73/LNA in EBV, eliminating the possibility of antigenic cross-reactivity with this very prevalent y, herpesvirus. To address the possibility that the distribution of antibodies to this latent antigen may underestimate the true prevalence of KSHV infection, intensive efforts have been made to devise assays based on lytic (structural) viral proteins. Because most structural KSHV proteins have more or less homologous counterparts in other y herpesviruses (Russo et al., 1996; Moore et al., 1996a), the challenge is to avoid any cross-reactivity with homologous proteins in other herpesviruses, in particular Epstein-Barr virus (EBV). Treatment of primary effusion lymphoma (PEL)-derived cell lines with phorbol esters or butyrate induces the expression of late and structural (lytic) viral genes (see above) and allows the detection of antibodies to the respective proteins by immunofluorescence (Miller et al., 1996,1997; Lennette et al., 1996; Smith et al., 1997; Ablashi et al., 1997). Depending on the experimental conditions used (serum dilution, counterstain with Evans blue to reduce background staining, signal enhancement by including a monoclonal antibody to human immunoglobulin), these immunofluorescence assays (IFAs) have been reported to detect antibodies in between 0% (Smith et al., 1997)and 25% (Lennette et al., 1996)of United States blood donors. In view of the high homology of some KSHV structural proteins to their counterparts in EBV [e.g., the major capsid protein orf25, which is about 50% identical to EBV BcLFl (Moore et al., 1996a)], and the documented example of serological cross-reactivity between KSHV major capsid protein and EBV (AndrC et al., 1997),the specificity of these assays is still under investigation. Using low serum dilutions (<1:40) may increase the potential for cross-reactivity (Smith et al., 1997). As far as this has been reported, sera with hightiter antibodies to EBV viral capsid antigen (VCA) d o not appear to react more frequently in these lytic IFAs than do other sera (Lennette et al., 1996; Smith et al., 1997), and sera from New World monkeys infected with Herpesvirus saimiri ( H V S ) , a closely related rhadinovirus, do not appear to react in these assays (Ablashi et al., 1997). However, serological cross-reactivity between H V S and a KSHV-related macaque rhadinovirus (RRV) has been described (Desrosiers et al., 1997).Also, a few sera from African Green monkeys reacted in a lytic KSHV IFA in a typical pattern, suggesting cross-reactivity with yet another KSHV-related virus, which has recently been found in one of these animals (Greensill et al., 1998). N o such cross-reactivity was observed with a LANA IFA. Unlike the LANA IFA, lytic IFAs may therefore detect cross-reacting antibodies against closely related rhadinoviruses, but whether this is a concern for their diagnostic or seroepidemiological use in humans is still unclear. After chemical induction with 12-0-tetradecanoylphorbol-13-acetatesome body cavity-based lymphoma (BCBL) cell

Kaposi’s Sarcoma-AssociatedHerpesvirus

129

lines produce enough virions to allow their purification and use in wholevirus enzyme-linked immunoassays (ELISAs) (Whitman et al., 1997; Chatlynne et al., 1998). Antibodies to several lytic antigens have also been detected by Western blot or radioimmunoprecipitation of chemically induced PEL cell lines (Miller et al., 1996, 1997; Smith et al., 1997; Chandran et al., 1998a). Prominent structural proteins, which are reactive with patient sera, include glycoproteins of 116, 110, 90, 70, 55, and 50 kDa, as well as other bands of 155, 105, 38, 40, 29, and 23 kDa (Miller et al., 1996, 1997; Smith et al., 1977; Chandran et al., 1998a). Several groups have generated defined recombinant structural antigens. A capsid-related protein, vp19, encoded by orf65, detects antibodies in 75-80% of AIDS KS and 85-90% of classic KS sera, in -1-3% of United Kingdom blood donors, but in a slightly higher percentage of (5-10%) United States donors, compared to LANA immunofluorescence (Simpson et al., 1996; Lin et al., 1997; Rabkin et al., 1998). Assays based on a minor capsid protein, encoded by orf26 (a recombinant protein or peptides), have been reported to detect antibodies in -40-60% of KS sera and 20% of blood donor sera (Andre et al., 1997; Davis et al., 1997).However, in a blind comparison of different serological assays, a recombinant orf26 protein did not discriminate between KS cases and blood donors (Rabkin et al., 1998). The orf25encoded major capsid protein is also immunogenic (AndrCet al., 1997; Chandran et al., 1998a), but a recombinant orf25-derived protein did not appear to be specific for KSHV (AndrC et al., 1997). More recently, a recombinant protein based on orf K 8.1 has been reported to be highly reactive with patient sera but not with blood donor sera and provides another recombinant antigen with high sensitivity and specificity (Raab et al., 1998; Chandran et al., 1998a,b). In addition to these viral proteins, Chandran et al. (1998a) identified cDNAs derived from orf6 (ssDNA binding protein), orf8 (glycoprotein B), orf9 (DNA polymerase), orf39, orf59 (DNA replication protein), and orf68 as encoding immunoreactive KSHV structural proteins. Several studies (Lennette et al., 1996; Simpson et al., 1996; Rabkin et al., 1998) have noted that sera reactive in one of these assays do not always react in another assay. Rabkin et al. (1998)found a fair agreement among different assays [latent IFA, lytic IFA, orf65lvpl9 enzyme immunoassay/Western blot (ELISA/WB),whole virus ELISA] and laboratories for sera from KS patients, but generally only poor agreement for blood donor sera. The pattern of disagreement did not suggest that any one assay only detected a subset of sera that were reactive in another assay. This suggests that not every KSHV-infected individual, including patients with KS, mounts an antibody response to all immunogenic KSHV proteins, suggesting that a combination of antigens is likely to be required. In addition, there may be still significant disagreement between different laboratories using the same assay (LANA

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IFA) (Rabkin et al., 1998).Thus details of technical protocol and, for the immunofluorescence assays, interobserver variability may affect the results reported with these assays. In spite of these limitations, some of these assays have been used successfully to investigate the epidemiology of KSHV.

2. SEROPREVALENCE OF KSHV IN DIFFERENT GEOGRAPHIC REGIONS As a result of the different assays used (see above), and because only a relatively limited number of studies have been carried out so far, the exact prevalence rates in different countries are still uncertain. However, most groups agree that the prevalence of KSHV varies widely in different countries and geographic regions, being lower in Northern Europe and the United States than in Southern Europe, and (so far) highest in several parts of Africa. As summarized in Tables I and 11, prevalence rates among United Kingdom blood donors are below 3 % for antibodies to both LANA And orf65 (Simpson et al., 1996). A similarly low rate was found among a group of 50 Danish railway workers who served as a control for a study of KSHV transmission among Danish homosexual and bisexual men (Melbye et al., 1998). A study of 162 Swedish (Stockholm) blood donors found seroprevalence rates of 3% for LANA, and 7% for orf65, but 19% for lytic IFA antigens (Enbom et al., 1998).Among United States blood donors prevalence rates for antibodies to LANA range from 0 to 3%; for antibodies to orf65lvpl9, from 5 to 10%; for antibodies to gp35/37 (orfK 8.1) around 90-97%; and for antibodies to undefined structural antigens (IFA),from 0 to 25% (Kedes et al., 1996; Gao et al., 1996a,b; Simpson et al., 1996; Lennette et al., 1996; Smith et al., 1997; Ablashi et al., 1997; Whitman et al., 1997; Rabkin et al., 1998; Chandran et al., 1998b; Raab et al., 1998; Chatlynne et al., 1998). In Greece, -10% of control subjects in a case-control study for classic KS had antibodies to LANA and/or orf65/vp19 (Simpson et al., 1996). In Italy, there appears to be extensive regional variation in KSHV antibody prevalence, with the lowest rates seen for northwestern Italy (Milan) and two prealpine/alpine regions of the north, higher at the lower end of the Po Valley than further up the river valley, intermediate rates in central Italy (Tuscany, Umbria), and the highest rates for the south of mainland Italy, Sicily, and Sardinia (Gao et al., 1996a,b; Whitby et al., 1998; Calabro et al., 1998; Mitterer et al., 1998).These findings are summarized in Table I1 and Fig. 2, and further discussed in the next section. In Italy, Calabro et al. (1998) also found that KSHV prevalence, as measured by two KSHV antigens, was significantly lower in a transfusion center located close to, and recruiting its donors from, a prealpine region, compared to another transfusion center (Padova) in the Po Valley. This suggests that KSHV may be more prevalent in low-lying parts of the country. In keeping with this observation, Mitterer found a seroprevalence of only 5%, compa-

131

Kaposi’s Sarcoma-Associated Herpesvirus

Prevalence of antibodies to KSHV LANA and 0 6 6 protein

Incidence of classic KS

England

England

Denmark

Denmark

Sweden

Sweden

USA

USA

Greece

Greece

Sardinia

Sardinia

Central Italy

Central Italy

Northern Italy

Northern Italy

Italy

Italy 0

5

10

15

20

25

X seroposltlve blood donors

0

0.5 I incldence/100,000

1.5

Fig. 2 KSHV seroprevalence and incidence of classic KS in different countries. Because of the different seroprevalence rates obtained with different assays, and by different groups, only results from one group (Simpson et al., 1996; Calabrb et al., 1998; Melbye et al., 1998; Rabkin et al., 1998; Enbom et al., 1998) were used to compile this comparison. Incidence rates for classic KS before 1980 are from Geddes et al. (1994, 1995), Franceschi and Geddes (1995), and Hjalgrim et al. (1998). A similar relationship between KSHV seroprevalence (antibodies to LANA) and incidence of classic KS within Italy has been reported by Whitby et al. (1998).

rable to that in northern Europe, in the alpine region of South Tyrol (Mitterer et al., 1998). Why KSHV should be less widespread in the prealpine and alpine regions of Italy is presently unclear, but this observation fits with the previously reported (Geddes et al., 1994, 1995; Franceschi et al., 1995) higher risk for classic KS associated with birth in low-lying parts of the country (see below). Calabro et al. (1998) also noted that the seroprevalence of KSHV is significantly higher in elderly individuals in all the regions examined (Fig. 3). Because the incidence of classic KS in Italy may have been declining in the last two decades (Geddes et al., 1994) it is conceivable that this represents a cohort effect, i.e., that infection with KSHV was more common among those born before the second World War. However, this age distribution in Italian blood donors could also be explained by a preferential reactivation of KSHV in elderly individuals. Whitby et al. (1998) noted that, in addition to a higher KSHV seroprevalence in southern Italy, antibody titers to LANA were also higher in this part of the country. In contrast, the distribution of absorbance values obtained in the orf65lvpl9 ELISA by Calabro et al. (1998) did not differ between regions with a high or low KSHV prevalence.

I

W N

Table II KSHV Seroprevalence (?A) in Different Geographic Regions Seroprevalence by assay (%)

Region

Country

Americas

USA

USA USA USA USA USA

USA

Europe

USA USA Haiti Haiti Dominican Republic Guatemala UK UK Italy Italy Italy France France Sweden

SampIes Blood donors Blood donors Blood donors Blood donors Blood donors Blood donors HIV-negative men Blood donors HIV-negative women HIV-negative women Unknown Unknown Unknown Blood donors M yelomakontrol patients Blood donors Blood donors NHL patients Transplant recipients Myeloma/ lymphoma patients Blood donors

LANA IFA 0

1 0 0 1-9" -

1

13 0

0 0 0 3

Orf65 EIA

K8.1

Whole-virus Ref. Gao et al. (1996a,b) Kedes et al. (1996) Simpson et al. (1996) Lennene et al. ( 1 996) Smith et al. (1997) Rabkin etal. (1998) Chandran et al. (1998b) Chatlynne et al. (1998) Kedes et al. (1997) Goedert et al. (1997) Lennette et al. ( 1996) Lennette et al. ( 1 996) Lennette et a/. (1996) Simpson et al. ( 1996) MacKenzie et al. (1997)

14b 13 5

Calabri, et al. (1998) Whitby et al. (1998) Whitby et al. (1998) Farge et a1. (1998)

3

Marcellin et al. (1997)

0.5-3

Enbom et al. (1998)

18b

Asia

Middle East Africa

Denmark Greece

Railway workers Control patients

Thai1and

Blood donors

Philippines India Malaysia Israel Saudi Arabia Uganda

Blood donors Blood donors Blood donors Blood donors Hospital controls HIV-negative patients

-

Uganda Gambia Gambia Cameroon Zaire Nigeria Zimbabwe Ivory Coast South Africa

Unknown Antenatal mothers Unknown HIV-negative women Unknown Unknown Unknown Unknown Hospitalized adults

11 47 29 43 25 6 11 43 14

0 20

1

53

~~

=Different results obtained on the same sera by different laboratories. bAverage values across different Italian regions. Both studies noted a marked regional variation in KSHV seroprevalence.

L

W W

Melbye et al. (1998), Simpson et al. (1996) Hatzakis et al. (1998) Jaromanokul et al. (1998) Ablashi et al. (1997) Constantine et al. (1998) Ablashi et al. (1997) Ablashi et al. (1997) Constantine et al. (1998) Qunibi et al. (1998) Simpson et al. (1996), Gao et al. (1996a,b) Lennette et al. (1996) Ariyoshi et al. (1998) Lennette et al. (1996) Bestetti et al. (1998) Lennette et al. (1996) Lennette et al. (1996) Lennette et al. (1996) Lennette et al. (1996) Wilkinson et al. (1998)

Thomas F. Schulz

134

age (yean)

Fig. 3 Age distribution of antibodies to KSHV among Italian blood donors. Data shown here are those reported by Calabro et al. (1998)for five different blood banks in different regions of Italy. Reproduced with permission of the publisher, Lippincott Williams & Wilkins.

In Africa antibodies to LANA have been reported in 6 4 3 % of tested samples (Lennette et al., 1996; Gao et al., 1996a,b; Simpson et al., 1996; Ariyoshi et al., 1998; Mayama et al., 1998; Bestetti et al., 1998), also with considerable regional variation. Antibodies to LANA are very prevalent in East, West, and South Africa, with prevalence rates ranging from 14 to approximately 50% (Table 11).Similarly, antibody prevalence rates for orf65lvpl9 ranged from 35 to approximately 50%, and for lytic IFA antigens from 32 to higher than 80% (Lennette et al., 1996; Gao et al., 1996b; Simpson et al., 1996; Ariyoshi et al., 1997; Mayama et al., 1998; Wilkinson et al., 1998). In one study (Lennette et al., 3 996) no antibodies to LANA were found in a small number of sera from a few Central American and Caribbean countries, but 10-29% of these sera contained antibodies to lytic IFA antigens (Lennette et al., 1996). In contrast, 13% of HIV-negative women in Haiti had antibodies to LANA in another study (Goedert et al., 1997), i.e., a significantly higher proportion than found by the same investigators among women in the United States (Kedes et al., 1997), and comparable to some Italian regions (see above and Table I1 and Fig. 2), but not as high as in many parts of Africa (Simpson et al., 1996; Gao et al., 1996b; Ariyoshi et al., 1998; Mayama et al., 1998; see Table 11).

Kaposi’s Sarcoma-Associated Herpesvirus

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KSHV infection appears to be uncommon in one region of Thailand with a 0.5% antibody prevalence rate for LANA and 2% for orf65hpl9 (Jaromanokul et al., 1998). Preliminary reports, based on a relatively small number of sera from Indian, the Phillipines, and the Middle East (Ablashi et al., 1998; Constantine et al., 1998; Qunibi et al., 1998), would suggest that KSHV may be uncommon in Asia and of intermediate prevalence in Israel and Saudi Arabia (Table 2), but more systematic studies are clearly needed before firm conclusions can be reached.

C. Prevalence of KSHV and Incidence of Classic or Endemic KS in Different Geographic Regions In spite of the remaining uncertainty regarding the “true” prevalence rates for KSHV in different geographic regions, there appears to be a strong correlation between KSHV seroprevalence rates in different countries, if compared with the same assays and by the same groups, and incidence rates for classic KS. This correlation is, however, not without interesting exceptions. The incidence of classic KS in Italy had long been known to be higher in the south of mainland Italy, and on the islands of Sicily and Sardinia, than in central Italy or the north (Geddes et al., 1994, 1995; Francheschi et al., 1995).The pattern of regional KSHV prevalence in Italy therefore fits at least partially that for the incidence of classic KS (Fig. 2) (Calabrb et al., 1998; Whitby et al., 1998). The previously noted (Geddes et al., 1994, 1995; Francheschi et al., 1995) association between classic KS and birth in low-lying parts of the country is also mirrored by the marked differences in KSHV prevalence in blood donor populations from the lower end of the Po Valley and nearby prealpine or alpine regions (Calabrb et al., 1998). A possible exception to this is the previously reported relatively high incidence of classic KS in the area around Trieste (Geddes et al., 1994),where KSHV prevalence appears to be low (Whitby et al., 1998). However, the location and catchment area of the different blood transfusion centers from where the blood donor sera were collected to obtain KSHV seroprevalence data (Calabrb et al., 1998; Whitby et al., 1998) were not matched exactly to the different tumor registries in Italy, and this could easily explain such discrepancies. KSHV seroprevalence rates for Italy as a whole, and for the United Kingdom, United States, and Sweden, obtained with the same assays, have been compared to population-based incidence rates for classic KS in these countries (Gao et al., 1996a,b; Kedes et al., 1996; Simpson et al., 1996; Calabrb et al., 1998; Whitby et al., 1998; Enbom et al., 1998; Rabkin et al., 1998) (see Fig. 2). Such a comparison also suggests an association between KSHV prevalence and KS incidence: in Italy as a whole, Calabrb et al. found an approximately 7- to 10-fold higher KSHV prevalence (average -24%) than

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previously reported by the same group (Simpson et al., 1996) for the United Kingdom, which is in keeping with the 8-fold higher incidence for classic KS in Italy compared to the United Kingdom (see Fig. 2) (Calabrb et al., 1998; Whitby et al., 1998; Grulich et al., 1992; Geddes et al., 1994). Comparisons between Italy and the United States, or Italy and northern European countries for which suitable data are available (Fig. 2), suggest a similar correlation. However, only in Italy have systematic surveys of KSHV prevalence in blood donor populations from different regions been carried out (Whitby et al., 1998; Calabro et al., 1998), whereas the available prevalence rates for most other countries (see Table 11) are based only on relatively few studies. Further detailed surveys are therefore required to strengthen this association and to investigate regional variations in KSHV seroprevalence in countries such as Sweden where, similar to Italy, there is strong regional variation in the incidence of classic KS (Hjalgrim et al., 1996). Seroprevalence studies in Israel (7% of blood donors with antibodies to orfbS), in comparison to results obtained by the same group (Constantine et al., 1998) for United States blood donors (0% with antibodies to orf65), are also in line with the slightly higher rates for classic KS in Israel (1.5/ 100,000; data for 1970-1979) (Landmann et al., 1984) compared to rates in the United States (0.3/100,000 for men; 0.07/100,000 for women; data 1973-1979) (Biggar et al., 1994). In spite of KSHV seroprevalence rates of 20-35% in Italy, classic KS is a relatively rare tumor, with population-based incidence rates of 0.7-3/100,000, even in Sicily and Sardinia, where KSHV appears to be most common. Classic KS would therefore seem to be a relatively infrequent outcome of infection with KSHV in an otherwise healthy individual. Whether the higher rate of classic KS in men than in women (Geddes et al., 1994,1995; Franceschi et al., 1995) is explained by a higher prevalence of KSHV infection in men is still unclear; although Calabro et al. (1998) found no difference between men and women for antibodies to LANA and orf65/vp19 protein, Whitby et al. (1998) reported a 1.7-fold higher prevalence rate for antibodies to LANA in men than in women, and Angeloni et al. also reported a two- to threefold higher prevalence of antibodies to orf65 protein in men than in women (Angeloni et al., 1998). Although endemic KS has long been known to be more common in East and Central Africa than in the rest of the continent, KSHV appears to be as common in West Africa (Ariyoshi et al., 1998) and South Africa (Wilkinson et al., 1998) as it is in East Africa (Simpson et al., 1996; Lennette et al., 1996; Mayama et al., 1998). This could suggest the involvement of additional cofactors, and/or the existence of KSHV variants with different pathogenic potential. Because classic KS is a relatively rare tumor even in the regions of Italy where KSHV is endemic, it is conceivable that rare cases of classic, indolent KS could have been missed in

Kaposi’s Sarcoma-Associated Herpesvirus

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some parts of Africa. If so, this would point to the involvement of a cofactor in the pathogenesis of endemic KS in East Africa, which could be responsible for its more aggressive clinical features. A precedent for such a scenario has recently emerged from a study in West Africa. In a retrospective analysis of KS patients attending the hospital at the Medical Research Council (MRC) laboratories in Fajara, The Gambia, Ariyoshi e t al. (1998) found that all KS cases occurred in AIDS patients, 14/16 AIDS KS cases in HIV-1-infected individuals, 1/16 in an HIV-2 infected patient, and 1/16 in a dually infected patient, in spite of comparable rates of KSHV infection in HIV-1 and HIV-2-infected patients in The Gambia. This would suggest that HIV-1 infection, rather than, or more effectively than, HIV-2 infection, represents a cofactor for the development of KS. If HIV-2- and HIV-1-infected individuals in this study were adjusted for CD4 counts, AIDS KS was still much more common in the HIV-1-infected group (odds ratio, 12.4; 95% CI, 1.5-101), suggesting that the cofactor role of HIV-1 infection is not limited to immunosuppression. How HIV-1 infection might act as a cofactor in the pathogenesis of KS is discussed below. Another still unexplained aspect of KSHV epidemiology relates to the observation that, as an AIDS-definingillness, KS is as common among HIV-infected patients with hemophilia, intravenous drug users, and heterosexually infected women of KSHV endemic countries as it is in countries where KSHV infection is rare (Casabona et al., 1991). It is conceivable that sexual transmission of KSHV (this appears to be common among homosexual men) may be associated with a higher risk for the development of KS than infection in childhood (this may be the predominant route of infection in endemic countries) or that the timing of KSHV infection relative to HIV-1 infection may play a critical role (see below).

IV. KSHV PREVALENCE IN RISK GROUPS FOR HIV-1 TRANSMISSION That AIDS KS is much more common among homosexual and bisexual men than among those who contracted HIV through contaminated clotting factors, blood transfusion, or intravenous drug use, or among heterosexually infected women, had long been one of the key arguments in favor of the involvement of a sexually transmissible agent in the pathogenesis of KS (Beral et d., 1990, 1992; Schechter e t al., 1991; Archibald e t d., 1992). If the distribution of KSHV among risk groups for HIV transmission were to follow this predicted pattern this would strengthen the case for it being the “KS agent.”

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A. PCR Studies Comparing the detection of KSHV DNA in PBMCs of homosexual men and blood donors, in the United Kingdom Whitby et al. (1995)found KSHV in 11/143 HIV-infected homosexual men without KS compared to 0/134 blood donors and 0/26 oncology patients ( p < 0.05), and LebbC et al. (1997a)reported KSHV in 114 French HIV-uninfected homosexual men versus 0/20 blood donors. In a similar comparison in the United States, Moore et al. (1996b) detected KSHV DNA in 3/23 homosexual/bisexual AIDS patients without KS and 0/19 AIDS patients with hemophilia. In a large study investigating the presence of KSHV in semen samples, Howard et al. (1997) detected KSHV in 3/9 HIV-infected homosexual men without KS, compared to 0/115 HIV-uninfected semen donors ( p < 0.0005). Gupta et al. (1996) found KSHV in 2/24 HIV-infected men with KS, compared to 0/10 HIV-infected men without KS. In view of the much larger differences in KSHV prevalence between these groups reported in serological studies (see Section IV,B), these small differences highlight the lack of sensitivity of PCR on PBMCs or semen samples of KSHV-infected asymptomatic persons, as discussed above. In addition, because KSHV viral load in peripheral blood is related to tumor burden (Brambilla et al., 1996; Blum et al., 1997; Farge et al., 1998), it is possible that homosexual men with KSHV DNA in their peripheral blood already have subclinical disease, and, on their own, these results cannot be taken to infer a higher prevalence of KSHV infection among homosexual men. This caveat may, however, not apply to the detection of KSHV in semen, because Howard et al. (1997) noted an association between the presence of KS and the rate of KSHV detection in peripheral blood, but not in semen.

B. Serological Studies Several studies, using different KSHV antigens and serological assays, agree that KSHV antibodies are significantly more common among HIV-infected homosexual/bisexual men than among the other HIV transmission groups (Fig. 4). In the United States Kedes et al. (1996)found antibodies to LANA in 35% of HIV-infected homosexual men compared to 5% of HIV-infected transfusion recipients and 3 % of HIV-infected patients with hemophilia. Gao et al. (1996a,b) found antibodies to LANA by immunofluorescence, and the predominant 220- to 230 kDa LNA protein, the main component of LANA (Rainbow et al., 1997), in 30% of HIV-infected homosexual men compared to 0% of HIV-infected patients with hemophilia. Lennette et al. (1996) detected antibodies to LANA in 22% of HIV-infected homosexuaUbisexua1

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Rate of KS in UK AIDS patients

Hemophilia patients intravenous drug users 0

5

10

15

20

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35

X with KS

Prevalence of KSHV infection in HIV risk groups from the UK T

Hemophilia patient. intravenous drug usera

I 0

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X with KSHV antibodies

Fig. 4 Comparison of KSHV prevalence and proportion of AIDS cases with KS as a defining diagnosis in the United Kingdom risk groups for HIV transmission [data from Simpson et al. (1996) and IARC (1996)l.

men, compared to 0% of HIV-positive intravenous drug users. In an immunofluorescence assay for lytic (structural) antigens, Lennette et al. (1996) found KSHV antibodies in 100% of HIV-infected homosexual men, compared to 23% of HIV-infected drug users and 21% of HIV-infected women. In the United Kingdom, Simpson et al. (1996) found 30% of HIV-infected homosexual men with antibodies to both LANA and the orf65 structural protein, compared to 0% of HIV-infected patients with hemophilia, 2% of HIV-uninfected patients with hemophilia, and 0-5% of HIV-infected or uninfected intravenous drug users. In Denmark, 21% of a cohort of Danish gay men had antibodies to either LANA or orf65 in 1981, with the prevalence rising to 39% in 1996 (Melbye et d., 1998), compared to 2.5% of an HIV-uninfected control group of Danish railway workers. In the Amsterdam cohort of homosexual/bisexual men, KSHV seroprevalence increased from 21% in 1984 to 36% in 1996; in a parallel cohort of intravenous drug users KSHV infection was much less common, with a small increase in prevalence between 1985 (3.5%) and 1996 (7.5%) (Renwick et al., 1998).

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In Padova, Italy, Calabro etaf. (1998)found 62% of homosexual men with antibodies to either LANA or orf65, compared to 11% in HIV-infected intravenous drug users, and 17% in HIV-infected heterosexuals and 25% in HIV-negative patients with hemophilia. Thus, in this part of Italy, where KSHV is more common in blood donors (19%; see Table I1 and Fig. 2 ) than in the United Kingdom, Denmark, or Holland, the prevalence in homosexual men is again higher than in other risk groups of HIV transmission and in the general population.

C. KSHV Prevalence and Risk for K S in Different Risk Groups for HIV Transmission Thus, the generally higher seroprevalence of KSHV among homosexual/bisexual men, both HIV infected and uninfected, correlates well with the higher incidence of KS in this group of individuals (Fig. 4). In addition, as discussed in more detail in Section VI, detection of KSHV DNA in PBMCs of HIV-infected homosexual/bisexual men precedes (Whitby et al., 1995; Moore et al., 1996b; Lefrkre et af., 1996) and predicts (Whitby et al., 1995) the subsequent appearance of KS lesions. Similarly, antibodies to KSHV predict the subsequent progression to KS (Melbye et af., 1998; Martin et af., 1998; Renwick et al., 1998).

D. KSHV Prevalence in Transplant Recipients KS has long been known to be common in iatrogenically immunosuppressed recipients of organ transplants. The rate of KS in transplant recipients varies widely in different geographic regions, being relatively rare in the United Kingdom, but thought to be the commonest posttransplant tumor in Saudi Arabia (Qunibi et al., 1988). No systematic comparison of KSHV antibody prevalence in transplant recipients from different geographic regions is presently available. Nevertheless, Parravicini et al. (1997a) reported a case-control study of Italian transplant KS patients, in which 10/11 transplant patients with KS, compared to 2/17 control transplant patients, already had antibodies to LANA or the orf65 protein before transplantation, representing an odds ratio of 75 (95'%0 CI 4.7-3500). In this study transplant patients with KS originated mainly from southern Italy, where KSHV is more prevalent (see above). In Saudi Arabia, Qunibi et af. (1998) found a higher rate of KSHV infection in transplant patients (5/18) compared to controls (6144). In a French case-control study of organ transplant recipients, antibodies to KSHV LANA or orf65 protein were found, before or after transplantation, in 22% (9/41)of transplant recipients without KS compared to 89% (16/18) of transplant recipi-

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ents who later developed KS (odds ratio, 48.0; 95% CI, 5.5-2084) (Farge et al., 1998). This case-control study included patients of African and Middle Eastern origin, providing an explanation for the relatively high KSHV seroprevalence in the control group (compared to French blood donors). Transplant KS patients were more likely to originate from Africa and the Middle East than were controls. All five posttransplant KS patients, from whom pretransplant sera were available for testing, had already been infected with KSHV prior to transplantation (Farge et al., 1998). These two studies suggest that, in KSHV endemic countries, most transplant patients who develop KS do so as a result of a reactivation of a preexisting latent KSHV infection. However, transmission of KSHV to the transplant recipient from the organ donor can occur and may play an important role in countries where KSHV is rare, as recently reported from Switzerland (Regameyet al., 1998b). Similarly, Farge et al. (1998) and Parravicini et al. (1997a) reported two cases of KSHV seroconversion after transplantation in their control group.

V. TRANSMISSION OF KSHV A. Sexual Transmission Detailed epidemiological studies on the risk factors associated with AIDS KS in homosexual men predicted that the “KS agent” should be sexually transmitted in this risk group (Beral, 1991; Beral et al., 1990, 1992; Archibald et al., 1992). In accordance with this prediction, KSHV antibodies are found more frequently among sexually transmitted disease (STD) clinic attenders than among blood donors (Simpsonet al., 1996; Kedes et al., 1996). Among homosexual men KSHV is clearly sexually transmitted. Melbye et al. (1998), in a prospective cohort study of Danish homosexual men, found that the presence of antibodies to KSHV LANA or orf65 was linked to promiscuity, duration of homosexual lifestyle, and the frequency of receptive anal intercourse. Individuals who already had antibodies to KSHV in 1981, at the beginning of the AIDS epidemic, as well as those who seroconverted subsequently,were more likely to have had contact with United States homosexual men (Fig. 5 ) . This group also concluded that KSHV had most likely been introduced into the gay community in Denmark in the late 1970s. Interestingly, Zong et al. (1997) reported that a few KSHV strains predominate among United States homosexual men with AIDS KS, thus suggesting a relatively recent infection from a common source. In our experience, isolates from all three major subgroups circulate among United Kingdom HIVinfected homosexual men studied in the past 2 years (Cook et al., 1998). Thus, the situation may now be much more complex than in the early 1980s, when participants in the study reported by Melbye et al. (1998) were en-

Thomas F. Schulz

142

Receptive anal intercourse

Years homosexual OR 0<4

-

3.3 0.97 11.1

I 0 c 19

L I 1.9

0.6 -6.4

3.2

1.2 -8.6

OOL 0

10

C c8

30

20

0

20

30

I

% KSHV seropositive

% KSHV seropositive

I:"F

10

Oral -anal contact

No. of partners

%.4 0.6 -3.7

,

so+

I0

20

20

30

Use of nitrite inhalants

1.7

20

10

% KSHV seropositive

HIV antibody status

10

0.4 -3.8

40

30

% KSHV seropositive

0

0.4-4.2

1.26 3.1 1.04 -9.2 0

0

i.n

1.7 0.6 -4.?

~

0.6-4.8

40

30

z 1

0 Po 20+

p

0

10

20

-

1.3

0.6 -2.9

1.1

0.3 -3.8

30

% KSHV seropositive

% KSHV seropositive

US exposure

us visit 198041

Yes

2.0

0

10

20

30

% KSHV seropositive

0.96 -4.2

40

0

10

20

30

% KSHV seropositive

Fig. 5 Risk factors for sexual transmission of KSHV among homosexual men. The results shown refer to risk for KSHV seropositivity at enrollment in 1981 into a prospective cohort study of Danish homosexual men (Melbye e t a / . 1998).

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rolled. In agreement with the decline of the incidence of AIDS KS among homosexual men during the 1980s (IARC, 1996), the rate of new KSHV infections in this cohort decreased during this period, presumably as a result of a change in lifestyle (“safe sex”) (Melbye et al., 1998). Among United States homosexual/bisexual men, Martin et al. (1998) found a strong correlation between KSHV infection (measured by antibodies to LANA) and homosexual activity, the number of homosexual partners, and a history of other sexually transmitted infections. Thus, there is clear-cut evidence for sexual transmission of KSHV and a link between KSHV transmission and HIV-1 infection among homosexual men. In contrast, there is so far only limited evidence for the sexual transmission of KSHV in the general (HIV-negative) population of KSHV nonendemic countries. For homosexual men, the risk factors for infection with KSHV appear therefore to be largely the same as those initially predicted for the “KS agent” (Beral, 1991; Beral et al., 1990, 1992; Archibald et al., 1992). However, whether oral-anal contact (“rimming”),found by some (Beral et al., 1992), but not by others (Elford et al., 1992; Page-Bodkin et a/., 1992), to increase the risk for AIDS KS, also predisposes to KSHV transmission is still controversial. In the Danish cohort study (Melbye et al., 1998) oral-anal contact did not increase the rate of KSHV infection. In contrast, there is some evidence for oral-anal contact predisposing to KSHV infection in an Australian cohort study (A. Grulich, S. Olsen, P. S. Moore, J. Kaldor, personal communication, 1998). Oral-penile contact was found to be a risk factor for KSHV transmission in the Dutch cohort study (J. Goudsmit, personal communication, 1998). Sexual transmission of KSHV is likely to occur as a consequence of its presence in seminal fluid (Monini et al., 1996a,b; Gupta et al., 1996; Howard et al., 1997).As discussed above, and also reviewed elsewhere (Blackbourn and Levy, 1997), KSHV DNA has been detected in the minority of semen samples from KS patients, as well as from homosexual men without KS (Howard et al., 1997; Gupta et al., 1996), and in prostate specimens of AIDS KS patients by PCR (Corbellino et d., 1996a,b). The fact that KSHV has been detected in saliva by PCR or by culture by some (Vieira et al., 1997; Koelle et al., 1997), although not others (Whitby et al., 1995), could provide a possible explanation for oral-anal, or oral-penile, transmission.

B. Transmission in Childhood Transmission of KSHV before puberty appears to be rare in the United States (Blauvelt et al., 1997), but infection with KSHV does occur in young children of countries where KSHV is more widespread. In Italy, Calabrb et

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al. (1998)found 6/40 Sardinian children to have antibodies to KSHV LANA and/or orf65 protein, and in Zambia, Kasolo et al. (1997) detected KSHV DNA in PBMCs of 8 % of febrile children. One case of a 3-year-old HIV-negative boy with KSHV infection was also reported from Germany (Kusenbach et al., 1997). Mayama et al, (1998) studied the age distribution of antibodies to KSHV LANA and orf65 protein among more than 200 children, adolescents, and young adults in Uganda and found that KSHV seroprevalence reached adult levels well before puberty. Antibodies to KSHV were, however, rare in children before the age of 2 (Mayama et a/., 1998). This pattern of age-dependent seroprevalence is indicative of horizontal transmission of KSHV among young children in Uganda and reminiscent of the pattern known from other herpesviruses, in particular EBV. Furthermore, Mayama et al. (1998) found that the presence of antibodies to two KSHV antigens (LANA, orf65 protein) were independently associated with hepatitis B infection, which is known to be transmitted horizontally among young children in Africa (Edmunds et d., 1996; Kiire, 1996). This observation could suggest that living conditions predisposing to KSHV infection (close contact, crowding) also enhance horizontal KSHV transmission. However, cases of AIDS-associated KS in Ugandan children below the age of 2 have been reported (Ziegler and Katongole-Mbidde, 1996), suggesting that vertical transmission of KSHV may also occur in Uganda. Furthermore, Bourboulia et al. recently reported a link between KSHV seropositivity of mothers and their children, suggesting that mother-child transmission may occur (Bourboulia et al., 1998), and Angeloni et al. found evidence for intrafamilial clustering of KSHV infection in Sardinia (Angeloni et al., 1998). In South Africa, Wilkinson et al. (1998) also found KSHV infection to be common among children below the age of puberty, and in the United Kingdom, Lyall et al. (1998) found antibodies to KSHV LANA and orf65 protein mainly in children of African mothers and in an age distribution similar to that reported by Mayama et al. (1998). As mentioned above, the detection of KSHV in saliva by PCR (Boldough et al., 1996; Koelle et al., 1997) or culture (Vieira et al., 1997) may provide an explanation for horizontal transmission among young children, along the lines suggested for EBV (de ThC et al., 1989). These findings suggest that horizontal or vertical transmission of KSHV is common among young children in Africa. Sexual transmission among highrisk groups in nonendemic countries may represent an exception. Sexual transmission may of course also occur in Africa, as suggested by an age-dependent increase of KSHV prevalence among adults in Africa (Bourboulia et al., 1998; Bestetti et al., 1998). Further detailed studies are obviously required to extend these early observations. However, the presently available data already indicate that one of the early reservations about KSHV being the KS agent, i.e., that, as a herpesvirus, it should not be mainly sexually

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transmitted and would therefore be unlikely to fit the pattern predicted for the KS agent, was probably unnecessary: in an endemic region transmission of KSHV occurs mainly horizontally in childhood, similar to other herpesviruses, and its transmission pattern among homosexual/bisexual men appears to be the exception, rather than the rule, a conclusion that is reminiscent of the epidemiology of HBV infection.

C. Parenteral Transmission There is also some evidence for parenteral transmission of KSHV. Blackbourn et al. (1997) reported a single case of KSHV transmission by blood transfusion. In view of the significant prevalence of KSHV among Italian blood donors (see above; Calabrb et al., 1998; Whitby et al., 1998) this may occur more frequently. However, its clinical relevance is uncertain, AIDS KS being comparatively rare in those who contract HIV through transfused blood (Beral et al., 1990). Several cases of KSHV transmission via an organ transplant (a kidney transplant in one case) have been reported (Parraviciniet al., 1997a; Regamey et al., 1998). Farge et al. (1998) also reported two cases of KSHV serwonversion in their control group, presumably as the result of either transplantation or transfusion. As outlined above, both the reactivation of a pre-existing KSHV infection and acquisition of KSHV from the transplant donor can lead to transplant KS (Parravicini et al., 1997a; Farge et al., 1998; Regamey et al., 1998). Among African children younger than 12 years Mayama et al. (1998) found a weak association between antibodies to orf65, but not LANA, and a history of intravenous injections. Further studies are clearly required before this can be taken to suggest a role for parenteral transmission among children in Africa.

VI. ASSOCIATION OF KSHV WITH DISEASE A. Kaposi’s Sarcoma 1. THE PRESENCE OF KSHV IN KS LESIONS

That KSHV can be detected, by PCR or Southern blot, in all epidemiological forms of Kaposi’s sarcoma, i.e., AIDS KS, classic Mediterranean KS, African endemic KS, and posttransplant KS, and in its different histological stages (fully developed nodular KS, early patch/plaque stage KS), is no longer controversial and has been reviewed elsewhere (Olsen and Moore, 1997;

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Schulz et al., 1998).Detection of KSHV DNA by PCR in paraffin-embedded biopsies is not always successful, but this is thought to be due to the inadequate quality of DNA after formaldehyde fixation. However, even in frozen specimens of fully developed nodular KS the amount of KSHV DNA can be very variable and may sometimes only be detectable by PCR, but not Southern hybridization (Chang et al., 1994). This is likely to reflect the fact that KS lesions contain a mixture of KSHV-infected endothelial tumor (spindle) cells (Boshoff et al., 1995b; Foreman et al., 1997)and KSHV-uninfected cells that may have been recruited into these lesions. Only few KS spindle cells are present in early KS lesions, but these are infected with KSHV, as are some endothelial cells (Foreman et al., 1997). In contrast, a wide variety of other neoplasms has been investigated for the presence of KSHY DNA, and, with the exception of body cavity-based lymphoma, some cases of multicentric Castleman’s disease, and several controversial reports (see below), been found not to be associated with this virus. 2. FORMAL EVALUATION OF T H E ASSOCIATION OF KSHV WITH KS As discussed above, KSHV DNA can occasionally be detected in samples from individuals without KS, in particular among HIV-infected homosexual men, and in KSHV endemic regions. In KS patients, KSHV can also be detected by PCR in non-KS samples, such as uninvolved skin (Chang et al., 1994; Lebbe et al., 1995; Dictor et al., 1996; Corbellino et al., 1996a,b), peripheral blood mononuclear cells (Ambroziak et al., 1995; Whitby et al., 1995; Moore et al., 1996b; Lefrere et al., 1996),semen (Howard et al., 1997; Gupta et al., 1996; Monini et al., 1996a,b), and prostate (Corbellino et al., 1996a,b), albeit at lower levels. Therefore, to evaluate the strength of the association between KSHV infection and the presence of clinical KS formally, the detection of KSHV by PCR in peripheral blood of KS patients has been compared to that in asymptomatic (HIV-infected or HIV-uninfected) individuals in several case control studies and a series of smaller, less formal comparisons (Whitby et al., 1995; Moore et al., 1996b; Lefrkre et al., 1996; Marchioli et al., 1996; Humphrey et al., 1996; Lebbe et al., 1997a; Collandre et al., 1995; Ambroziak et al., 1995; Decker et al., 1996; Heredia et al., 1996; Ariyoshi et al., 1998). With the exception of two small studies reported by Decker et al. (1996) and Collandre et al. (1995), all others, and in particular those that studied larger numbers of patients and controls (Whitby et al., 1995; Moore et al.. 1996b; Marchioli et al., 1996; Humphrey et al., 1996; Lebbi et al., 1997a; Lefrkre et al., 1996; Ariyoshi et al., 1998), found highly significant odds ratios for an association between the detection of KSHV DNA in peripheral blood and the presence of KS. Table I11 shows the odds ratios and 95% confidence in-

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tervals calculated for a comparison of AIDS KS patients with HIV-infected patients without KS on the basis of the findings reported by these five major studies. This strong association between detectable KSHV and KS was seen even in those studies where the control group (HIV-infected patients without KS) also included, or consisted entirely of, homosexual or bisexual men, who are more likely to be infected with KSHV, than other HIV transmission groups (see above). The study by Moore et al. (1996b) allows a comparison between AIDS KS patients and HIV-infected patients with hemophilia (who are only rarely infected with KSHV; see above). Most of these comparisons have been carried out in countries where infection with KSHV appears to be comparatively rare in the general population. Under these conditions it is easier to discern significant associations between KSHV infection and the presence of KS. However, a significant association between KS and the detection of KSHV in peripheral blood was also found in a study from The Gambia, West Africa (Ariyoshi et al., 1998). Although retrospective case control studies of this kind cannot prove causality, a cohort study reported by Whitby et al. (1995) found that detection of KSHV in peripheral blood mononuclear cells of HIV-infected homosexual men predicts subsequent progression to KS. However, the detection of KSHV DNA in peripheral blood of KS patients correlates with tumor burden (Brambilla et al., 1996; Blum et al., 1997; Farge et al., 1998), and the failure to detect KSHV DNA in the control group could conceivably have been due to insufficient assay sensitivity. Formally, the study by Whitby et al. (1995) could also not exclude that the detection of KSHV DNA before KS Table 111 Association of KSHV DNA Detection in Peripheral Blood and the Presence of AIDS KS or Classic KS

KSHV DNA detecteatested KS type

Cases

AIDS KS

24/46

AIDS KS AIDS KS AIDS KS AIDS KS AIDS KS Classic KS Classic KS

11/21 46/99 34/98 10111 48/82 9/15 9/18

Controls HIV+, no KS, 111143 Blood donors, 01134 HIV+, no KS, 3/42 HIV+, no KS, 0164 HI!?+, no KS 12/64 HIV+, no KS, 1/45 HIV+, no KS, 6/43 Matched controls, 0115 Blood donors, 0120

OR (95%CI)= 13 (5.2-34) 146 (21-6053)b 7 (1.6-43) 55 (8.6-2240)b 2.3 (1.0-5.4) 440 (19.9-19,415) 8.7 (3.1-27.6) 21 (1.9-984)b 19.0 (1.9-875)b

Ref. Whitby et al. (1995) Moore et al. (1996b) Marchioli et al. (1996) Humphrey et al. (1996) Lefrkre et al. (1996) Simpson et al. (1996) Simpson et al. (1996) Lebbe et al. (1997a)

aOdds ratios (OR) were calculated from published studies reporting more than 10 cases or controls. bCases in which no KSHV was detected in the control samples; one positive control was assumed in order to permit the calculation of odds ratios.

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diagnosis may, at least to some extent, have reflected the presence of subclinical lesions at the time of testing. Serological studies have, however, led to similar conclusions. Several groups, using different serological assays (see Section III,B,l ), all agree that the presence of antibodies to KSHV shows a strong association with the presence of KS. Understandably, this association appears stronger in countries where KSHV infection is rare, and for studies that use assays of a high specificity, but possibly incomplete sensitivity, such as LANA immunofluorescence or orf65 ELISA/WB (see above). However, significant associations were also found with lytic immunofluorescence assays or “whole virus” ELISAs, and in KSHV endemic countries (see below). The strong association between KSHV antibodies and the presence of KS in HIV-infected patients is not only evident when AIDS KS patients (in nonendemic countries these are mostly homosexual or bisexual men in whom KSHV infection is more common; see above) are compared with HIVinfected individuals belonging to other transmission groups, in whom KSHV infection is much less common. The biggest serological studies comparing HIV-infected homosexual men with and without KS are summarized in Table IV. Comparing the prevalence of antibodies to KSHV LANA, the lytic orf65encoded protein, or a 40-kDa lytic protein in AIDS KS patients versus HIVinfected, asymptomatic homosexual men, the results reported by several groups (Gao et al., 1996a,b; Simpson et al., 1996; Kedes et al., 1996; Miller et al., 1996; Renwick et al., 1998; Martin et al., 1998) amount to odds ratios of higher than 8.5 in all but one study (lower 95% CI at least 2.3; see Table IV Association of KSHV Antibodies with the Presence of AIDS KS in Cross-Sectional Studies of HIV-Infected HornosexuaVBisexual Men

KSHV antigen (assay) LANA (IFA) LANA (IFA) LANA (IFA) LANA (IFA) LANA + orf65/vpl9 LANA (LNA WB) Lytic IFA Lytic IFA Orf65/vpl9 (EIA N B) vp40 (WE)

‘ W R ,Odds ratio.

Cases (HW-infected men with KS) with antibodies

Controls (HIV-infected men without KS) with antibodies

OR (95 % CI)a

Ref.

841103 37/45 35/43 47/91 62/71 32/40 87/91 31/48 46/57 32148

10133 13/37 12/40 16/71 2311528 7/40 64/71 7/54 5/16 7/54

10.2 (5.8-28) 8.5 (2.8-27) 16.3 (4.6-64) 3.7 (1.7-7.8) 8.9 (4.3-18) 18.7 (5.4-70) 2.4 (0.6-11.5) 12.2 (4.2-37.6) 9.2 (2.3-40) 13.4 (4.5-41.6)

Simpson et al. (1996) Kedes et al. (1996) Gao et al. (1996b) Lennette et al. (1996) Renwick et al. (1998) Gao et al. (1996a) Lennette et al. (1996) Miller et al. (1996) Simpson et al. (1996) Miller et al. (1996)

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Kaposi's Sarcoma-Associated Herpesvirus

Table V Association of KSHV Antibodies with the Presence of Classic KS among HIV-Uninfected Individuals in Mediterranean Countries KSHV antigen (assay)

Cases with antibodies

LANA (IFA)

17118

LANA (IFA)

11111

LANA (IFA)

313

orf65lvpl9

17118

orf65lvpl9

313

Controls with antibodies 3126 (matched controls) 41107 (blood donors)b 771404 (blood donors)d 3126 (matchedcontrols) 711404

OR (95% CI)" 130 (10.7-5665)

Ref. Simpson et al. (1996)

257 (23.1-11256)" Gao et al. (1996b) p < 0.01

Calabrb et al. (1998)

130 (10.7-5665)

Simpson et al. (1996)

p < 0.01

Calabro et al. (1998)

aOR, Odds ratio. bKS cases not necessarily from the same geographic region in Italy as the blood donor controls (Milan) (see text). T o calculate odds ratio, one KS case was assumed to be negative for KSHV antibodies. dKS cases and blood donor controls from the Veneto region of Italy.

Table IV). The study by Lennette et al. (1996) also found a significant association with antibodies to LANA (odds ratio, 3.7; 95% CI, 1.7-7.8), but not with antibodies detected by a lytic IFA (odds ratio, 2.4; 95% CI, 0.6-11.5). Similarly, a preliminary analysis (Simpson et al., 1996) of a big case control study on classic (HIV-negative)KS in Greece found a highly significant association of antibodies to LANA and orf65 protein with classic KS (Table V). A more extended analysis of the complete case control study has recently confirmed this strong association (A. Hatzakis, 1998, personal communication). In addition, a comparison of Italian classic KS patients with local blood donor populations showed that these patients virtually always have antibodies to KSHV, in comparison to 5-35% of blood donors from different regions in Italy (see above and Table V). Antibodies to KSHV can usually be detected before the appearance of clinical KS lesions (Gao et al., 1996a; Melbye et al., 1998; Martin et al., 1998; Renwick et al., 1998).However, the strongest single argument for KSHV being the cause of AIDS KS in HIV-infected homosexual men was provided by two prospective cohort studies (Martin et al., 1998; Renwick et al., 1998), which showed that the presence of antibodies to KSHV LANA, or orf73/ LNA and orf65, predicts the subsequent progression to KS. Martin et al. (1998) found that 50% of HIV-infected homosexual men with antibodies to LANA progressed to AIDS KS within 10 years. Renwick et al. (1998) found that the timing of KSHV infection relative to HIV-1 infection may affect the rate of progression to AIDS KS. In their (Amsterdam)

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Thomas E Schulz

cohort, AIDS KS developed more rapidly in those individuals who contracted KSHV infection after HIV-1 (50% of these individuals developed KS 5 years after seroconverting to KSHV), than in those who were already KSHV seropositive when seroconverting to HIV-1 (50% developed KS after 10-12 years; p log rank < 0.04) (Renwick et ul., 1998). This difference was still observed when the two groups were adjusted for differences in CD4 count (which was an independent risk factor for developing KS in this study) and HIV-1 viral load (which was not). A case of KSHV seroconversion in an HIV-infected homosexual man has been described (Oksenhendler et al., 1998). In this patient, infection with KSHV was followed, after 4-6 weeks, by an acute febrile lymphadenopathy and splenomegaly accompanied by a transient decrease in CD4 cells. Histologically, the lymphadenopathy was characterized by a polyclonal B cell proliferation, a pronounced plasma cell infiltration, and angiogenic changes, reminiscent of early KS lesions. Together with the prospective cohort study by Renwick et al. (1998) this case illustrates that infection with KSHV of an already HIV-1-infected individual has the potential to cause rapid-onset severe disease. Thus, KSHV infection in HIV-l-infected homosexual men can be associated with a rapid progression to KS. In contrast, classic KS appears to be a rare event in KSHV-infected individuals (see above), and the rate of AIDS KS among HIV-infected patients with hemophilia, intravenous drug users, and heterosexually infected women from KSHV endemic countries is the same as for their counterparts in countries such as the United Kingdom where KSHV infection in the general population is rare (Casabona et al., 1991; see above). Although immunosuppression clearly promotes the onset of KS in a KSHVinfected individual [the best argument for this is the association of transplant KS and certain forms of immunosuppression (see above and Farge et al., 1998)], it does not fully explain these differences. It is conceivable that sexual transmission of KSHV, common among homosexual men, but probably less important in the general population of endemic countries (see above), may represent a particular hazard. Alternatively, particular routes of sexual transmission (e.g., rectal; see above) may be associated with a particularly high risk of KS. The observation by Renwick et al. (1998), that secondary infection with KSHV of an already HIV-infected homosexual man is associated with a more rapid progression to KS, suggests further possible explanations. Already HIV-infected individuals may mount only an insufficient immune response to curb an acute KSHV infection, whereas immunosuppression of an individual harboring a latent KSHV infection may be less dangerous. It is also conceivable that, in addition to immunosuppression, HIV-1 infection may enhance lytic KSHV replication (during acute KSHV infection) and/or the spread of KSHV to endothelial cells, the likely precursors of KS spindle (endothelial tumor) cells. Several suggestions as to the

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mechanisms involved, which include inflammatory cytokines released during HIV-1 infection and HIV-1 TAT, have been made and recently reviewed elsewhere (Biberfeld et al., 1998). Because endemic (HIV-negative)Kaposi’s sarcoma occurs in East and Central Africa, but not in West Africa, in spite of apparently comparable levels of KSHV seroprevalence (see above), other infections could conceivably act as cofactors in its pathogenesis.

B. Body Cavity-Based Lymphoma/Primary Effusion Lymphoma Body cavity-based lymphoma is a rare B cell non-Hodgkin lymphoma (NHL) that occurs mainly in AIDS patients (Knowles et al., 1998), but has rarely also been seen in HIV-negative individuals (Nador et al., 1995,1996; Hermine et al., 1996).Clinically, BCBL/PEL is usually characterized by malignant effusions in the peritoneal, pleural or pericardial cavities, usually in the absence of a discerible tumor mass. However, lymphomatous infiltration of the pleura or peritoneum can sometimes be found (Komanduri et al., 1996). BCBL/PEL cells lack expression of most lineage-associated B or T lymphocyte differentiation antigens, but usually express the common leukocyte antigen CD45. The consistent rearrangement of the immunoglobulin genes is evidence for their B cell origin. BCBL/PEL cells consistently lack chromosomal changes commonly associated with other mature B cell NHLs, involving the protooncogenes c-myc, bcl-2, bcl-6, n-ras, and k-ras, as well as p53 mutations (Carbone et al., 1996; Nador et al., 1996; Cesarman et al., 1996a,b; Jaffe, 1996).Most BCBL/PEL cells contain EBV, present in the lymphoma cells as multiple latent episomal copies, and all also contain KSHV (Cesarman et al., 1995a,b, 1996a,b; Gessain et al., 1997; Hermine et al., 1996; Pastore et al., 1995; Gaidano et al., 1997; Strauchen et al., 1996). Owing to the rarity of this lymphoma, no formal epidemiological studies have investigated the association between KSHV and BCBL, but from a biological point of view, a causative role of KSHV in the pathogenesis of BCBL appears plausible.

C. Castleman’s Disease and Related Lymphoproliferative Disorders Castleman’s disease (CD) is an atypical lymphoproliferative disorder that may present as generalized lymphadenopathy [multicentric Castleman’s disease (MCD)] or as a solitary enlarged lymph node. It occurs as two histologic variants, the plasma cell variant and the hyaline-vascular variant. The

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plasma cell variant of MCD has been known to be associated with HIV infection and KS in AIDS patients (Soulier et al., 1995; Gessain et al., 1996). KSHV is frequently found in the plasma cell variant of MCD in HIV-infected individuals, but less commonly in the hyaline-vascular variant, and less frequently in MCD of HIV-negative individuals (Soulier et al., 1995; Gessain et al., 1996; Barozzi et al., 1996). It is thought that the virally encoded IL-6 homolog, vIL-6, which has been shown to support the proliferation of plasma cells in uitro and to be expressed in KSHV infected B cells in uiuo, may play a role in the pathogenesis of this disorder (Mooreet al., 1996c; Nicholas et al., 1997). A case of KSHV seroconversion illness in an HIV-infected individual has been described (Oksenhendler et al., 1998). This patient had pronounced lymphadenopathy and splenomegaly, characterized by a polyclonal B cell and plasma cell proliferation, as well as angiogenetic changes, reminiscent of some of the features found in MCD. A similar case, in an HIVl-uninfected transplant recipient in whom KSHV seroconversion at the time of transplantation was followed by Castleman’s disease after 13 months, has been described by Parravicini et al. (1997a), and a lymphoproliferative disorder in KSHV-infected transplant patients has been recognized in which the expression of the viral IL-6 homolog in B cells was documented (Matsushima et al., 1998). It is not known whether other reported cases of MCD, or lymphoproliferative disease with MCD-like features, also occurred in newly KSHV-infected individuals, and whether in some cases MCD could be considered a “seroconversion illness” with this B-lymphotropic herpesvirus (Ambroziak et al., 1995). The presence of KSHV in some cases of angiolymphoid hyperplasia of the skin (Gyulai et al., 1996) and in angioimmunoblastic lymphadenopathy, or lymph node hyperplasia with giant germinal centers (Luppi et al., 1996b), has also been noted.

D. Unconfirmed Links between KSHV and Other Diseases As with many viruses that have been newly discovered over the past two decades, an association of KSHV with a number of diseases has been suggested but not confirmed. Among these is cutaneous sqameous cell carcinoma, frequently found in immunosuppressed transplant recipients (Rady et al., 1995). Several groups have been unable to support this association (Boshoff et a/., 1996; Uthman et al., 1996). Similarly, the reported link between KSHV and sarcoidosis (di Alberti et al., 1997) could not be confirmed by others, is not in keeping with the epidemiology of sarcoidosis, and may have been due to a combination of PCR contamination and the fact that many of the samples came from a KSHV endemic area (Moore, 1998; Regamey et al., 1998; LebbC et al., 1999).

Kaposi'sSarcoma-Associated Herpesvirus

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An involvement of KSHV in multiple myeloma or benign monoclonal gammopathy (Rettig et al., 1997; Said et al., 1997)seemed an attractive suggestion in view of the ability of the virally encoded IL-6 homolog to stimulate plasma cell lines in vitro (Moore et al., 1996c; Nicholas et al., 1997)and its expression in KSHV-infected B cells in vivo (Moore et al., 1996c; Boshoff et al., 1998). However, several serological case control studies, using different assays, could not find any evidence for such an association (MacKenzie et al., 1997; Marcellin et al., 1997; Whitby et al., 1997; Masood et al., 1997; Parravicini et al., 1997b). This was not due to a compromised antibody response in myeloma patients, because antibodies to EBV EBNA-1 (MacKenzie et al., 1997),HCMV, HBV (Parravicini et al., 1997b),HHV-6, and HHV7 ( Masood et al., 1997)were found with expected frequency. However, one group (Gao et al., 1998) did recently report a higher rate of antibodies to orf6.5 protein and LANA, as detected by Western blot, in myeloma cases than in controls. Unlike KS, multiple myeloma is not more common among HIVinfected gay men (Lyter et al., 1994), and its geographical distribution does not match that of KSHV. PCR studies on myeloma bone marrow biopsies (Chang et al., 1994; Parravicini et al., 1997b; Masood et al., 1997) failed to detect KSHV DNA, and several groups have not been able consistently to detect KSHV DNA by PCR in bone marrow dendritic cell cultures (as reported by Rettig et al., 1997), irrespective of whether the culture conditions used favored the development of CD34' bone marrow stromal cells or CD68' macrophage like cells (Masood et al., 1997; Mitterer et al., 1998). However, the same group, and some others, have continued to detect KSHV in bone marrow dendritic cell cultures by PCR and in situ hybridization (Tisdale et al., 1997; Said et al., 1997). A link between KSHV and cutaneous T cell lymphoma (Sander et al., 1996) also could not be confirmed by others (Pawson et al., 1996). Similarly, the presence of KSHV in angiosarcoma (McDonagh et al., 1996)was not observed by others (Tomita et al., 1996; Boshoff et al., 1995a).

VII. CONCLUSION The emerging picture indicates that the epidemiology of KSHV differs in endemic and nonendemic countries. Where it is widespread in the general population, it may follow transmission patterns known from other herpesviruses, whereas in homosexual men of nonendemic countries it appears to be primarily sexually transmitted. Based on three large cohort studies of homosexuaUbisexua1men, risk factors for its transmission are those that had been predicted for the KS agent. In addition to the strong association between KSHV infection and Kaposi's sarcoma, in particular in the context of

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HIV infection, two large prospective cohort studies have now shown that KSHV infection of HIV-positive individuals is followed within a few years by the appearance of AIDS KS lesions. These findings represent extremely strong support for a causative role of KSHV in this disease. However, the available epidemiological evidence also highlights the importance of HIV-1 as an important factor in the pathogenesis of AIDS KS. In HIV-negative, nonimmunosuppressed individuals from KSHV endemic areas, classic KS appears to be a rare consequence of KSHV infection. If this is the case, the existence of KSHV variants or other cofactor(s) may have to be postulated to explain the geographic distribution and clinical features of endemic African KS. Thus, in the same way that careful epidemiological studies had long predicted the existence and transmission pattern of a human tumor virus as a cause for KS, the recent findings on the epidemiology of KSHV have highlighted its interaction with other infections and helped to put into perspective the wealth of available in vitro observations on the cell biology of KS. Some of the findings reviewed above may yet provide further clues for experimental work to unravel the fascinating biology of this virus, which, although probably only of moderate oncogenic potential on its own, appears to be one of the strongest tumorigenic agents in humans in the presence of certain cofactors. such as HIV-1.

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Consensus on Synergism between Cigarette Smoke and Other Environmental Carcinogens in the Causation of Lung Cancer Arnold E. Reif and Timothy Heeren* Mallory Institute of Pathology Boston University School of Medicine and Department of Epidemiology and Biostatistics Boston University School of Public Health Boston, Massachusetts 021 18

I. Introduction A. Biological Interaction B. Synergism C. Statistical Interaction D. Valid Evidence for Presence of Synergism 11. Testing the Significance of a Finding of Synergism A. Linearity of Individual Dose-Response Curves B. Cigarette Smoking Dose-Response C. Asbestos Dose-Response D. Radon Dose-Response E. Interaction between Cigarettes and Asbestos F. Interaction between Cigarettes and Radon G. Interaction between Cigarettes and Alcohol 111. Carcinogenic Synergism and Public Health Iv. Previous Findings on Synergism Involving Cigarette Smoke V. Multistep Carcinogenesis VI. Varying the Time Frame of Data Collection VII. Conclusion References

I. INTRODUCTION Cigarette smoke is by far the most injurious substance in our society today, eventually killing about half of all regular smokers. (Doll et al., 1994). After decades of campaigns against smoking, the public thinks it knows all ‘Address correspondence to Timothy Heeren, Department of Epidemiology and Biostatistics, Boston University School of Public Health, 175 Albany Street, Boston, MA 02118.

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about the health effects of smoking, but is still uninformed about the impact of synergism between smoking and other environmental carcinogens, particularly radon and asbestos. Here, we propose that “synergism” be used as a potent new slogan in the ongoing educational war against smoking in developed countries, where the public is already saturated with information on the medical effects of smoking. Of course, a public education campaign based on synergism must rest on sound evidence that synergism really exists. Thus, it is our intent to examine evidence for statistically verifiable synergism in the interaction of smoking with asbestos, radon, alcohol, and with other environmental carcinogens. Although we note where complexities lie, our focus is not on these, but on whether a simple answer can be given to the question of whether synergism exists in these binary systems. Our objective is to counter an opinion frequently voiced by carcinogenesis workers, i.e., that statistical verification of carcinogenic synergism is so complicated and requires such extensive data that it is not worth undertaking. We begin by defining synergism, autosynergism, antagonism, and biological and statistical interaction. Then we analyze relevant dose-response curves and carcinogenic interactions. Here, we confine ourselves to interactions between two carcinogens.

A. Biological Interaction Because antagonism is defined as negative synergism, it is helpful that the term biological interaction has been coined to encompass both positive and negative synergism. (Rothman, 1986).

B. Synergism Lichens exemplify the expression of synergism in biology. Composed of a fungus in symbiotic union with an alga, their interaction is called synergistic, because they mutually support each other’s growth. The essence of synergism is that two agents together act more effectively than if their effects were merely additive. The problem has been to quantify synergism and determine its statistical significance without introducing artifact. Biologists usually define synergism in such systems along the lines of the United Nations Scientific Committee on the Effects of Atomic Radiations (1982).Synergy is not a function of dose-response, but in the simplest case, the dose-response curves of each of two carcinogens are linear when administered separately, and when applied jointly, they act in synergism if the resultant tumor yield exceeds the sum of the yields from separate administration at the same doses; but if the tumor yield is smaller, the two agents are said to act in antagonism. This definition remains inviolate.

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It has been argued that the interpretation of synergism depends on the model assumed in the analysis (Hogan et al., 1978). In contrast, we believe that instead of allowing the definition of synergism to depend on a particular model, it makes better sense to hold the concept of synergism inviolate, and to structure models to provide results that accord with it. In line with this consideration, the proposal has proved persuasive, that biological interaction be defined as departures from additivity of either incidence rates or risks, whereas statistical interaction be defined with latitude in the choice of a scale of measurement (Rothman et al., 1980). To reduce the possibility of artifact, we suggest a more secure definition of biological interaction. Many authors have taken into consideration the nonlinearity of carcinogen dose-response curves when testing for biological interaction (Thomas 1981; Halpern and Whittemore, 1987; Lubin, 1988; Greenland, 1993). When one or both curves are concave, this is necessary to avoid the possibility that this shape is ascribed to autosynergism. This is a flawed concept that holds that a response above that expected from linearity is due to synergism between an initial and a second dose of the same agent. Acceptance of autosynergism as a valid form of synergism permits one to ignore problems posed by nonlinearity but is untenable, because it would destroy the integrity of the concept of synergism (United Nations, 1982; Steel and Peckham, 1979). For some data, analyzed without taking into consideration the nonlinearity of dose-response curves, it is possible to conclude that stutisticul interaction is present (Wahrendorf and Brown, 1980; Plackett and Hewlett, 1963),although for the same data we could find no evidence for biological interaction. Thus, it is crucial to take into account nonlinearity in evaluating synergism (Kupper and Hogan, 1978; Reif, 1995). Based on the concerns of many of those cited above, we propose a definition of biological interaction that excludes artifacts due to autosynergism: 1. Biological interactions (synergism and antagonism) are special cases of the interactions of two or more agents. 2. Biological interaction is suggested by departures from additivity of measured effects, such as incidence rates or risks. When one or both agents have nonlinear dose-response curves, appropriate consideration of nonlinearity is necessary. 3. Both extent of departure from additivity and degree of statistical significance determine the likelihood that biological interaction is present. 4. The presence or absence of statistical interaction may not denote the presence or absence of biological interaction, unless the dose-response curves of the two agents are linear. 5. Because the extent of biological interaction can vary with the length of the time interval between exposure to the agents and evaluation of their effects, conclusions on synergism should state the time interval of observations on which they are based.

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The above definition does not exclude the possibility that a valid statistical test for synergism can be performed using very different methods-as long as these accord with the essence of synergism and avoid artifacts. For instance, we used the time at which death from tumors occurred to substantiate carcinogenic synergism (Reif and Colten, 1984). But we assumed that the rate of tumor growth was logarithmic, and did not compensate for deviation of the assumed from the actual growth curve. Because this assumption may have introduced artifacts, we now conclude that our method was flawed.

C. Statistical Interaction Synergism, or biological interaction, is distinct from statistical interaction, which is model dependent. For event outcomes, the assumed statistical approaches focus on relative risks (or odds ratios). Because the relative risk scale is based on ratios, statistical interaction in these models is inherently multiplicative. Thus, if the risk ratios of two carcinogens are multiplicative, in statistical terms this indicates independence of the modes of action of the two agents, and thus the absence of statistical interaction. But for biological interaction, a multiplicative situation indicates a high level of excess risk above additive, suggesting synergism (Blot and Day, 1979; Walter and Holford, 1978). In the assessment of statistical interaction, investigators are free to choose any model that they deem fits the data suitably. In the absence of a specific physiological model, the definition of statistical interaction is arbitrary and its choice can depend to some extent on statistical convenience in the analysis of observed data (Walter and Holford, 1978). Even biological interaction between two agents has been defined in terms of mechanism rather than risk: as being present if the qtralitative nature of the mechanism of action of each agent is affected by the presence or absence of the other (Siemiatycki and Thomas, 1981). We agree with the comment that this concept does not lead to an unambiguous definition of independence of effects, and thus does not produce clear analytical implications (Pearce, 1988).There are even public health interactions and decision-making interactions (Rothman et al., 1980; Saracci, 1980).

D. Valid Evidence for Presence of Synergism Tests for synergistic interactions can be complicated and difficult to understand (United Nations, 1982). Because tests of interaction usually have low power (Saracci, 1987), the strength of interaction rather than its statistical significance may be important (Pearce, 1988). However, we believe that the integrity of the scientific approach should not be bent in the face of difficulty (Kyburg, 1990), and that attempts to find better methods of assessment should continue. A statistically significant finding of synergism is sufficiently important to justify the design of studies large enough to make its

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achievement possible. But such a finding depends not only on the magnitude of the synergism and sample size, but also on the statistical analysis chosen and how it accommodates to the presence or absence of linearity in dose-response curves. Data that seem to show a high magnitude of synergism often are not extensive enough to produce statistical significance (Reif, 1984). To deal with this problem, we proposed a classification of the degree of carcinogenic synergism based on answers to several questions (Reif, 1995). Now, we replace the requirement that data collection be continued to the end of the life of all subjects with the condition that the time for which data was collected be stated alongside any claim of synergism, qualifying its validity. Several groups have used our classification in reporting a finding of carcinogenic synergism. Thus, “apparent synergism” was used to summarize work on the interaction between radiation and 2-dimethylhydrazine in the induction of colonic tumors in rats (Sharp and Crouse, 1989), and to report the effect of treatment with a combination of 10 heterocyclic amines on the development of hepatic foci in rats (Hasegawa et al., 1994). The concept of “potential synergism,” synonymous to “apparent synergism,” has been used to specify the interaction between five heterocyclic amines in producing liver cell foci (It0 etal., 1991), and for the interaction between wheat bran and psyllium in enhancing the inhibition of colon cancer (Alabaster et al., 1993). Should such a classification seem problematic, an alternative approach to summarize the validity of a finding of synergism is to attach to it a brief description of the evidence on which it is based, by answering the following three questions: 1. What was the magnitude of the synergism that was found? 2. Was the level of synergism significant when tested by a method that included proper consideration of dose-response data? 3. What was the time period for which subjects were observed? Here, we test evidence for the linearity of the dose-response curves of each of three respiratory carcinogens, namely, cigarette smoke, radon, and asbestos. Having established linearity for each of these carcinogens, then we can validly apply Eq. (2) (given in Section 11) to data on their interaction. For situations wherein the dose-response curves are nonlinear, we have already attempted to substantiate synergism by a conceptually simple method (Reif, 1995).

11. TESTING THE SIGNIFICANCE OF A FINDING OF SYNERGISM Because we have previously illustrated statistical testing for synergism when the two carcinogens have linear dose-response curves (Reif, 1985, 1995), we merely summarize the method here. Synergism may be present if

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yAB

-

>

-

+ (yB - yo),

Or

’

yA

+ yB

-

(l)

where A and B denote two carcinogens A and B applied separately at doses of respectively, A and B, or else together at the same doses (AB) used for single application. The unexposed control group is designated 0. The values Y denote the responses observed (e.g., cancer incidence or death) in the four relevant groups A, B, AB, and 0. Group designations (such as A) represent both the group and the dose at which it is applied. Values of Y can represent either the number of individuals who experienced the effect out of the total number in that group, or the corresponding rates or percentages of response. When r denotes death from tumor rather than tumor incidence, yAB cannot exceed 1.0, for Y = 1.0 means that all subjects have died. To leave the definition of synergism uncomplicated, time-related effects (Pearce, 1989) are not considered, even though relevant. For instance, in the extreme case where the end point of Eq. (1) is death and one waits long enough, all persons will die regardless of exposure status; then the inequality does not hold and the two exposures do not exhibit synergy. However, none of the studies quoted included sufficient data on the time dependence of the two exposures to permit an analysis that utilizes time dependence in testing for synergism. Synergism may be present if Eq. (1)gives a positive result, and a modified z-test can be used to test its significance:

z

= (yAB - yA - yB

+ yo)/[yAB(1

- yAB)/nAB

+ r A ( l - rA)/nA

+ yB( 1 - rB)/nB+ yo( 1 - ~ , ) / n , ] ” ~ ,

(2)

where yAB = xAB/nAB, yA = xA/nA,yB = xB/nB,and yo = xo/no,and x is the number of occurrences (e.g., cancer) among a subgroup of n individuals. The value of z is entered into a two-tailed table oft at infinite degrees of freedom, to obtain the probability P that the difference between expected and observed effects could be fortuitous rather than due to the presence of synergism.

A. Linearity of Individual Dose-Response Curves The first question is whether dose-response data for inhalation of three types of human carcinogens (cigarette smoke, radon gas, and asbestos fibers) are compatible with linearity. If true, then the statistical test for synergism (see above) is valid. To show that dose-response curves for separate exposure to these three carcinogens are linear, we shall fit a linear model to data on lung cancer incidence over a variety of exposure levels. Then we shall compare the fit of this model to the observed data. In the studies utilized below, the risk of cancer mortality at various levels of exposure is described not by rates but by standardized mortality ratios

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(SMRs), which are the ratios of the response rate in a target group relative to the expected rate, if the age-specific cancer rates from the general population were to apply to the age distribution of the target group. If increased risk due to exposure is proportional across all age categories, then linearity of SMRs is equivalent to linearity of underlying response rates. If the effect of exposure differs by age, and the age distribution differs by exposure level, then a linear response may not be reflected by linear SMRs. Because available data are in the form of SMRs, our analysis requires the assumption that age-specific dose-response curves are parallel. We use a weighted leastsquares regression routine to acknowledge different standard errors for estimated responses at different exposure levels. Because people with no exposure should have cancer rates equivalent to the general population, we forced these models to have an SMR of 1.0 for zero dose. This assumption was tested for each of the examples presented here and was found to hold.

B. Cigarette Smoking Dose-Response Dose-response data for causation of lung cancer by cigarette smoking have been reviewed in detail (U.S. Department of Health and Human Services, 1982), and a roughly linear dose-response curve was found in several studies. Here we present data from an American Cancer Society study (Fig. 1) involving a million men and women, which remains by far the most extensive study to date (Hammond, 1966).A linear model fits these SMRs very well, with an R-square of 0.98. Although the data involve cigarettes smoked per day, this exposure rate is directly related to cumulative exposure, unless

SMR

Cigarettes per Day

Fig. 1 Relationship between the number of cigarettes smoked per day and lung cancer mortality ratios for United States males. Data from the American Cancer Society study by Hammond (1966).

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duration of smoking is a function of cigarettes per day. This seems false, in that a 1996 case-control study found a near-linear relationship between CUmulative exposure to cigarette tar and the relative risk of Kreyberg type I and of Kreyberg type I1 lung cancers (Zang and Wynder, 1992).Further, the content of arsenic per gram of lung correlates linearly with the incidence of lung cancer in cigarette smokers (Liu and Chen, 1996). Here, arsenic functions not as a carcinogen but as a marker for deposition of other carcinogens, and reflects total exposure.

C. Asbestos Dose-Response Inhalation of asbestos fibers can produce mesothelioma, lung cancer, cancers of the gastrointestinal tract and larynx, asbestosis, and benign pleural disorders. Although approximately 80% of all diffuse malignant mesotheliomas occur in men exposed to asbestos in the workplace, and sometimes in their family members or in people who live near asbestos mines, smoking seems not to enhance the prevalence of this neoplasm. In contrast, smoking enhances the incidence of lung cancer in asbestos-exposed people. However, the carcinogenicity of asbestos fibers depends on their type and size distribution. Amphiboles are more pathogenic than chrysotile asbestos because of differences in deposition and clearance of the fibers. The straighter amphibole fibers penetrate deeper into the lung; the larger chrysotile fibers tend to occur in bundles and to be deposited at bifurcations of the airway, and they are also more soluble (Mossman and Gee, 1989). Further, the risk of lung cancer in the mining and milling industry is 10- to SO-fold lower than in industries that process and use asbestos, such as textile manufacture and insulation. In the latter, bundles of fibers are broken up into shorter, thinner fibers that are readily inhaled and retained in the alveoli (Landrigan, 1998). Thus it is important that within each of the studies discussed below, the type of asbestos and its size distribution are constant. For lung cancer mortality following asbestos exposure, three studies (Dement et af., 1982; McDonald et al., 1983; Thomas et al., 1985) provide dose-response information. The first two present cancer risk associated with varying levels of asbestos exposure in terms of SMRs rather than of simple response rates. The dose-response data from these studies are shown in Fig. 2. With the outcome measured in terms of SMR, the SMR at 0 dose must be 1. So we fit a weighted linear regression model with intercept 1 to the above data. At each dose, the weight is the reciprocal of the variance of the SMR, which we estimate using the Poisson distribution to describe the variance in the observed number of cancers. For both sets of data, the linear models fit very well. Analysis of the Dement et al. (1982) data yields an R-square of

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I69

SMR

15

10

--

0

20

40

60

80

100

120

140

1 60

CumulativeAsbestos Exposure

Fig. 2 Relationship between cumulative exposure to asbestos fibers in males and lung cancer mortality ratios. Data on dose of fibers measured as ~ r n -day ~ by Dement et al. (1982),and data on dose of fibers measured as million particles per cubic foot-years by McDonald et al. (1983).

0.97, and analysis of the data of McDonald et al. (1983) data gives an Rsquare of 0.98. The results of the study of Thomas et al. (1985)are also consistent with a linear dose-response.

D. Radon Dose-Response Data obtained prior to 1988 on radon exposure and lung cancer have been summarized in detail (Committee on the Biological Effects of Ionizing Radiations, 1988). Here, also in terms of the SMR, we present data from two of the best studies of uranium miners (Fig. 3). A weighted linear regression model with an intercept of 1 fits well for both sets of the SMR data. Analysis yields an R-square of 0.91 for the Ontario data (Muller et al., 1985),and an R-square of 0.95 for the Eldorado data (Howe et al., 1986).Although the dose-response is linear, the magnitude of the trend is strongly dependent on age. But strong support for linearity comes from a recent pooled analysis of 11 cohorts of miners exposed to radon, at exposure levels only three to six times higher than lifetime exposure in an average house: little evidence for departure from a linear excess relative risk model was found (Lubin et al., 1997). Several studies have found no convincing evidence that domestic inhalation of radon causes lung cancer (Lees et al., 1987; Blot et al., 1990, Auvinen et al., 1996), but negative results can be expected because the risk of lung cancer from indoor radon is much lower than from smoking, and because of population mobility and exposure errors (Stidley and Samet, 1994; Lubin et al., 1994, 1995).

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16

SMR

12 i

4

” ’

2 00

50

100

150

200

Muller

250

300

Cumulative Radon Exposure (WLM)

Fig. 3 Relationship between cumulative exposure to radon gas in uranium miners and lung cancer mortality ratios. Cumulative working-level months (WLM) was used to measure exposure. Data of Muller et a/. (1985) and of Howe et al. (1986).

E. Interaction between Cigarettes and Asbestos The above analysis shows that all three of the pulmonary carcinogens have dose-response curves that are closely fitted by straight lines. Therefore, next we may employ Eq. (2)to test whether data on their interaction accords with a statistically significant degree of synergism. What follows is a further verification of previous reports of synergism in this system (Saracci, 1977,1987; Pearce, 1988). Regarding the biologic interaction between cigarette smoking (A) and asbestos (B) in the production of lung cancer, the data of Hammond et al. (1979) are suitable for testing with Eqs. (1)and (2). Lung cancer incidence in the four relevant groups was as follows: 276/6841 for the group exposed to both carcinogens (rhB in our notation); 303/37,000 (r,) for cigarette smokers; 4/891 (rB) for asbestos workers who were nonsmokers; and 28/ 37,000 (yo) for the control group. First, these data are positive for synergism when inserted in Eq. (1).Second, this finding of synergism is significant (P < 0.01) when tested by Eq. (2). Third, the data demonstrate multiplicative risk for the group exposed to both carcinogens. These data provide strong evidence for the presence of synergism. In terms of our classification system, the data are consistent with strict synergism. This conclusion is based on data gathered over a period that did not extend to the end of the life span of all subjects; according to the United Nations report of 1982, a study must extend over this time period to constitute “long-term” data gathering.

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F. Interaction between Cigarettes and Radon BEIR VI (Committee on the Biological Effects of Ionizing Radiations, 1998) has presented convincing evidence that the interaction between cigarette smoking and radon is synergistic. Here we illustrate how the admittedly circumscribed data of a recent report by Pershagen et al. (1994) can be utilized to demonstrate synergism between cigarette smoking (A) and residential radon (B) in producing lung cancer. The data of Pershagen et al. are unique in involving the low levels of radon inhalation actually encountered in the most relevant situation, the home. In this study, the proportions of lung cancer incidence for cases and controls were as follows: 16/20 (rAB)for the group exposed to both carcinogens; 347/583 (r,) for cigarette smokers; 5/36 (rB)for the group exposed to high residential radon levels; and 135/1070 (ro)for the control group. First, these data are positive for synergism when inserted in Eq. (1).Second, the finding of synergism is of borderline statistical significance (0.05 < P < 0.10) when tested by Eq. (2).Third, the data deviate only 18% from a perfect fit of multiplicative risk in the doubly exposed group. These data provide strong but not convincing evidence for synergism, and therefore fall just short of providing evidence consistent with the presence of strict synergism. But when Pershagen et al. tested statistical interaction in a model that permitted the input of all their data, they concluded that they had documented the presence of carcinogenic synergism (P = 0.02). The above conclusion is supported by an earlier study on the effect of inhaling cigarette smoke and radon on the production of lung cancer in uranium miners (Lundin et al., 1969).The data translate into the following values in our notation: r,, = 60, rA = 15.5, rB = 6, and ro = 1.5. First, these data are positive for synergism when inserted in Eq. (1).Second, this finding of synergism is significant ( P < 0.01) when tested by Eq. (2).Third, the data closely fit multiplication of risk in the group exposed doubly. Hence the above results are in accord with strict synergism during the period for which subjects were monitored, which did not extend to the end of the life span of all subjects.

G. Interaction between Cigarettes and Alcohol Saracci (1987) has reviewed in detail 15 papers that either provide or analyze data on the effect of the combined action of smoking and alcohol on the risk of cancer of the mouth, pharynx, larynx, and esophagus. For none of these cancers were sufficient data available for the action of alcohol alone, such that we could establish the shape of the dose-response curve for production of a particular type of cancer with sufficient certainty. Because none

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Table I Interaction between Inhalation of Radon and of Cigarette Smoke in the Generation of Lung Cancer Lung cancer cases/(cases + controls)b

Smoking habit Never smoked Heavy smokerse

Low-level residential High-level residential radon inhalationc radon inhalationd 13511070 3471583

Relative risk due to smoking Heavy smokerslnonsmokers 4.72

Relative risk due to radon (high leveYlow level)

5/36 16/20

1.10 1.34

5.76

6.34

‘%ased on data on residential radon exposure, smoking, and lung cancer in Sweden. Data from the study of Pershagen et al. (1994). bControls were frequency matched for age and calendar year of residence with the case group. A slight difference in numbers for the two groups resulted from exclusion of immigrants. ‘Data were pooled for the groups exposed to up to 50, 50 to 80, and 80 to 140 Bq/m3 (corresponds to 2.7 pCi radon per liter). In the United States, the recommended level beyond which action should be taken to reduce the radon concentration is 4 pCi per liter (150Bq/m3). dGroups exposed to more than 140 Bq/m3. T h e data are for smokers of 10 or more cigarettestday.

of the studies incorporated consideration of dose-response into the statistical demonstration of interaction, and because we cannot substantiate that the dose-response curve is linear, the available data fail to meet our criteria for statistical substantiation of synergism. Nevertheless, when the interaction between smoking and alcohol in the production of a particular cancer is multiplicative or more than multiplicative, then the data permit the conclusion that synergism is probable (see Table I) (Reif, 1995). This is the case for laryngeal cancer, for which a number of different studies have substantiated a multiplicative interaction (Saracci, 1987). Synergism also is probable for cancer of the tongue, for which a study of 200 cases and 200 controls indicates a more than multiplicative interaction between the two carcinogens (Herity et al., 1981). According to Saracci (1987), for esophageal cancer the data are less convincing: although the data of Wynder and Bross (1961) suggest a multiplicative interaction, some of the data of Tuyns et al. (1977) yielded less than a multiplicative value when analyzed by Day and Munoz (1982).

Ill. CARCINOGENIC SYNERGISM AND PUBLIC HEALTH A strong reason for studying the interaction between tobacco and radon or asbestos is that hundreds of millions of the world’s population are exposed

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not only to tobacco, but also to radon and/or to asbestos. But safeguarding people from exposure is a very different problem than solving the scientific problems posed by synergism (Reid, 1996).Can synergism be used as a new catchword to spark public awareness of smoking’s additional risks? Worldwide, smoking is the most important cause of cancer, causing three million deaths per year. In developed countries, it causes, respectively, 24 and 7% of all male and female deaths, but in former socialist countries those figures rise to, respectively, 40 and 17% (U.S. Department of Health and Human Services, 1982; U.S. Environmental Protection Agency, 1993; Peto et al., 1996). In those workplaces where occupational diseases pose hazards, “smoking control and reduction in exposure to hazardous agents are effective, compatible and occasionally synergistic approaches to the reduction of disease risk for the individual worker” (U.S. Department of Health and Human Services, 1985). Because the mortality from smoking represents preventable deaths, scientists should not be content merely with proving and explaining this carnage. The real challenge-far harder-is to devise strategies to discourage smoking, and to become involved in their implementation in society. Here, based on studies with follow-ups shorter than to the end of the life span of all participants, we report a consensus that the interaction between cigarette smoke and either asbestos or radon is synergistic. How can this finding be used for prevention? It can be argued that proof of synergism is irrelevant, because it is sufficient merely to state the relevant risk ratios. But use of that single word synergism to express a complex idea is thoroughly embedded in modern science (Kaiser, 1997) and society. In contrast, risk ratios are merely numbers that express mechanistic nuts and bolts. Synergism is a sophisticated concept that forces one to try to recall what it means. Thus it has acquired a mystique reserved for concepts only dimly understood. But it could be popularized simply as a red flag signifying high risk. If placed in that light, it could have a psychological impact that no mere statement of figures can make. For this reason, a focus on synergism between smoking and other pulmonary carcinogens such as radon and asbestos, to which all of us living in a modern society have been exposed to a greater or lesser extent, could be a highly potent new publicity weapon in the fight against smoking-but not in third-world countries, where the concept would be too sophisticated for the education that most of the populations have received. For them, we urgently need a worldwide campaign against smoking that focuses on the ironclad data on the horrendous effects of cigarette smoke: cardiovascular disease, cancer, and emphysema. Ideal leaders for initiating such a campaign would be experienced antismoking campaigners such as former Surgeon General Koop, the present Surgeon General, or former Food and Drug Association Commissioner Kessler. Better yet, all three could collaborate to enroll their counterparts in countries throughout the world, perhaps working through the United Nations.

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In contrast, in developed countries, the public is saturated with all too familiar messages on the health effects of smoking. The new slant can be that synergism can multiply the risk from smoking. Thus, smokers must not only worry about damage from cigarettes, but now will have to steer clear of living in homes or working in places or occupations where they will encounter high levels of radon or asbestos. This constitutes yet another “hassle” for smokers, who already feel much besieged. In the case of radon, the Environmental Protection Agency (EPA)estimates that approximately 14,000 lung cancer deaths are caused annually in the United States. The EPA has spearheaded prevention efforts (Brenner, 1989). It has advocated nationwide testing of homes and schools, lowering radon levels in the five million homes believed to have levels above 4 pCi/liter, and constructing radon-resistant housing; however, these guidelines have been hotly contested (Horgan, 1994). It is always relevant to question extrapolation from high dose levels (as in uranium mines) to relatively low dose levels (as in homes). However, the data of Pershagen et al. (1994) concern only residential exposure (Table I). Further, the BEIR VI committee reworked the latest evidence on residential radon exposure, and concluded that in 1995, radon-related lung cancer deaths could have been as low as 3000 or as high as 32,000, but most likely were between 15,400 and 21,800 (Committee on the Biological Effects of Ionizing Radiations, 1998). These estimates are remarkably close to those independently derived by the EPA. The EPA’s estimates have been sufficiently persuasive such that 25 states already have real estate laws that mandate sellers to state the radon level in their home (Grillo, 1996). Now, the EPA could launch a campaign based on synergism. Because cigarette smoke is a far more powerful carcinogen than radon (Table I), the EPA could focus both on abating radon and on the need to quit smoking for people living in a home with high levels of radon (Fabrikant, 1990; Chaffey and Bowie, 1994),because the risk of lung cancer from radon exposure is substantially decreased by quitting smoking (Finkelstein, 1996). An added incentive to quit would be to safeguard their nonsmoking family members, who already are passive smokers with an increased risk of lung cancer (U.S. Environmental Protection Agency, 1993), from an even higher risk due to synergism with radon. Similar strategies also could be used to campaign against occupational exposure to asbestos (Gardner et al., 1988; Mossman and Gee, 1989). Regarding occupational exposure to pulmonary carcinogens, there is strong evidence for synergism in the development of lung cancer in occupationally exposed people who also possess the genetic trait for high metabolism of debrisoquine (Carporaso et al., 1989,1990). The risk of lung cancer is already so high for those homozygous for a second lung cancer susceptibility gene (Sellers et al., 1990), and for carriers of yet a third such gene

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(Makachi et al., 1991), that few who knew they had a high susceptibility would consent to further occupational exposure (Reif, 1986).Knowledge of genetic susceptibility to lung cancer provided by the genetic testing of people before they decide (or continue) to work where they will receive exposure to pulmonary carcinogens would seem a vital benefit. Although genetic predisposition affects lung cancer incidence, to conclude that therefore one may smoke (Fisher, 1959; Burch, 1982) is wrong (Reif, 1981b). Irrespective of genetic susceptibility, whether (or how early) lung cancer develops depends largely on tobacco use (Reif, 1991; Carporaso, 1991; Sellers et al., 1991). Still, highly complex issues surround the ethics of genetic testing for occupational purposes (Li, 1996; Kahn, 1996).To protect individuals, testing should be done only in situations of strictly guaranteed privacy, where test subjects alone have access to the information. This will permit them to make an informed choice on whether to begin (or to continue) occupational exposure. Strict privacy for test results would even help employers, by protecting them from knowingly employing high-risk people, for which they could eventually be sued. Most persons in the one-third of the United States population who smoke cigarettes have tried to quit and failed. Strategies such as screening high-risk smokers with periodic chest X-rays and sputum cytology fail to reduce lung cancer incidence (Coultas and Samet, 1992).But the knowledge that one carries a lung cancer susceptibility gene would be a very strong incentive to quit. Still, if all smokers were to be screened for such a gene, the danger would be that the large majority, who would be found not to carry a susceptibility gene, might misinterpret that finding to mean that they could continue to smoke-when in fact their risk of developing lung cancer would still be almost as high as for all smokers, and their risk of death from heart disease and emphysema would not have been considered. Therefore, what is necessary for all smokers is not genetic testing, but quitting. Then, even after decades of smoking, the risk of lung cancer gradually decreases greatly (Reif, 198la). Stopping exposure produces more benefit for a predominantly latestage carcinogen such as tobacco than for the early-stage carcinogen radon (Brown and Chu, 1987). Synergism can also occur between chemical carcinogens and oncogenic viruses. Herpes simplex virus and tobacco-specific N'-nitrosamines act synergistically in cell transformation (Park et al., 1991).We do not know as yet whether this means that smoking is very dangerous for those infected with herpes. However, woodchuck hepatitis virus acts synergisticallywith dietary aflatoxin B, to produce liver cancer in woodchucks. Hence, it seems very likely that synergism also explains the 100-fold increase in the risk of developing hepatocellular carcinomas in Southeast Asia, where human hepatitis B virus (HBV) interacts with high dietary levels of aflatoxin B, (Bannasch et al., 1995).

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Exposure to one carcinogen is bad enough, but when smokers are exposed to other carcinogens that act synergistically, the result can be even worse.

The public already thinks that it knows what the medical effects of tobacco smoke are, yet 25% of American adults continue to smoke cigarettes (Shopland et al., 1995). Now the public can be alerted to a potent new concept that underlines the urgency of quitting (Reif, 1979): the thought that synergism with other environmental carcinogens makes smoking even more dangerous than it is already by itself. Several questions remain. Do our findings on synergism accord with previous work? Can analysis of synergism give insight into the carcinogenic mechanism? How can two interacting carcinogens be synergistic in the short term, yet additive in the long term?

IV. PREVIOUS FINDINGS ON SYNERGISM INVOLVING CIGARETTE SMOKE Present results show that all three carcinogens (tobacco smoke, radon, and asbestos) possess linear dose-response curves, in agreement with previous work. These findings mean that for these carcinogens, statistical interaction and biological interaction are identical. Thus, previous findings of statistical interaction are now validated as denoting synergism. Early work on synergism was concerned mainly with drug interactions (Hewlett and Plackett, 1959; Ashford and Corby, 1974). Later work on carcinogenic synergism has raised awareness that the events summarized by this blanket term are complex and involve many relevant intricacies, only some of which have been taken into account. For tobacco, these include dose rate (number of cigarettes smoked per day and over time) and type of tobacco; for those who quit, years since cessation; and for the subjects involved, age at first exposure, sex, diet, and genetic predisposition (Committee on the Biological Effects of Ionizing Radiations, 1988). Further, time-related factors can complicate the assessment of cancer epidemiology (Rothman, 1976). Despite these problems, a consensus has emerged that the production of lung cancer from the joint effects of cigarettes and asbestos is closer to multiplicative than to additive (Saracci, 1977,1987; Frank, 1986; Thomas et al., 1987; de Klerk et af., 1991; Cheng and Konh, 1992; Zhu and Wang, 1993), a result that agrees with our findings. As to the mechanism of synergism between tobacco smoke and asbestos, Eastman etal. (1983)showed that when benzo(a)pyrene is added to cultures of hamster epithelial cells, the amount of this carcinogen retained by the cells after an 8-hr incubation period was eight times higher if it was first adsorbed onto asbestos fibers, than when added without asbestos. The authors con-

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cluded that synergism could result because asbestos binds tobacco carcinogens, and additional experimental evidence for this was provided by Harvey et al. (1984). Other explanations for synergism are that asbestos acts as a promoter that produces cell proliferation (Mossman, 1994), or else that cigarettes and asbestos interact in producing *OHradicals, thereby increasing DNA damage (Jackson et al., 1987; Valavanidis et al., 1996). As for gene changes, heavy lifetime exposure to tobacco smoke causes mutations of p 5 3 and perhaps also of K-ras, and asbestos exposure increases their frequency (Wang et al., 1995; Husgafvel-Pursiainen et al., 1993). There also exists a consensus that interaction of cigarettes and radon produce risks close to or at multiplicative levels (Whittemore and McMillan, 1983; Halpern and Whittemore, 1987; Lubin, 1988; Saracci, 1987; Committee on the Biological Effects of Ionizing Radiations, 1988; L'Abbe et al., 1991; Moolgavkar et al., 1993; Yao et al., 1994; Hornung, 1995; Lubin and Steindorf, 1995). Our analysis yields the same result. Most important, the latest version of BEIR (VI) published by the National Academy of Science (Committee on the Biological Effects of Ionizing Radiations, 1998) concluded that there was a synergistic interaction, which was somewhat short of multiplicative. For the interaction between smoking and radiation in atom-bomb survivors, the relative risks fit a multiplicative or additive model equally well (Committee on the Biological Effects of Ionizing Radiations, 1988). These results differ from those for smoking and radon, because atom-bomb survivors received total-body rather than only pulmonary irradiation. Because tobacco carcinogens cause more than 50% additional cases of cancer at sites other than lung, namely, mouth, larynx, esophagus, bladder, pancreas, and kidneys (Reif, 1976), and because this percentage could increase with radiation exposure, deaths from such cancers might be expected to diminish the numbers at risk for lung cancer. Synergism also exists for the production of lung cancer from exposure to cigarettes plus arsenic. This holds true whether arsenic exposure is occupational (Hertz-Picciotto et al., 1992) or from drinking contaminated water (Tsuda et al., 1995). Because smoking causes cancer in many body organs (Reif, 1979; Doll, 1996), it may be fruitful to look for synergism with other carcinogens that target the same organs as cigarettes.

V. MULTISTEP CARCINOGENESIS Klein (1998) has summarized the evidence that most forms of cancer develop through multiple steps, as long ago suggested by Foulds (1958,1969, 1975). Indeed, the distinct steps of initiation, promotion, development, and

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progression are well understood (Potter, 1980; Weisburger and Williams, 1989). But our understanding of the mechanism of carcinogenesis was changed radically by discovery of oncogenes. Initially, the oncogene process seemed simple, depending either on direct activation of oncogenes or else on inactivation of suppressor genes. We still believe that the driving force in the evolution from a normal to a malignant cell is accumulation of genetic damage in the form of activated protooncogenes and inactivated tumor-suppressor genes (Anderson et al., 1992). But now we recognize that inactivation of DNA repair genes and of genes that influence programmed cell death can also be vital factors in the process of carcinogenesis, and that the molecular mechanism is generally far more complex (Klein, 1998) than first conceived. Germ cell mutations are heritable, thus mutations that affect the abovenamed genes are vital factors in determining genetic susceptibility to cancer (Evans and Prosser, 1992). Further, epigenetic factors also may have to be perturbed for cancer to emerge (Weinberger, 1990; Harris, 1993), and genetic polymorphisms may affect cancer risk (Gonzalez, 1995). Also, normal immune response genes regulate the immune competence of the host, and thus susceptibility to immunogenic tumors (Herberman, 1982). Further, there are many different causes of cancer (Doll, 1978; Reif, 1981a; Weisburger and Horn, 1991; Colditz et al., 1996), and their disparate nature makes it very unlikely that all processes are identical at a molecular level. Once cancer cells have emerged, the host has defense mechanisms against genetically aberrant cells, but these succeed only if the cancer cell bears antigens recognized by the host’s surveillance cells as distinctly aberrant (Reif, 1997). Although epidemiologists have attempted to elucidate the mechanism of carcinogenesis from a study of interactions, this is a difficult task (Siemiatycki and Thomas, 1981: Saracci, 1987). Nevertheless, fitting the Armitage-Doll model of carcinogenesis to data on lung cancer mortality in asbestos workers has led to the conclusion that radiation and asbestos act mostly on early stages of carcinogenesis (Day and Brown, 1980; Brown and Chu, 1987; Pearce, 1988).In contrast, cigarette carcinogens seem to be more effective in late events rather than in initiating events (Doll and Peto, 1978; Brown and Chu, 1987, 1989). However, in the context of their two-stage clonal expansion model, Moolgavkar et af. (1993) show that smoking and radon both affect the first-stage transition rate and the rate of clonal expansion, and do not affect the transition rate of initiated cells. Even when the mechanism of carcinogenesis involves multiple progressive changes, it is likely that one particular step is rate limiting for a particular carcinogen. Indeed, the above findings support the thesis that synergism occurs when the main rate-limiting step in the production of a given type of tumor differs for two carcinogens, such that one agent relieves the main ratelimiting bottleneck for the other (Reif, 1984). Thus, the conclusions drawn

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by Doll and Peto (1978), Day and Brown (1980), Brown and Chu (1987), Pearce (1988), and Brown and Chu (1989) suggest that early-stage carcinogenesis by radiation or asbestos supplies a population of initiated cells that the powerful late-stage actions of tobacco carcinogens then promote to overt cancer. This mutually synergistic interaction entails potentiation of a relatively weak initiator (tobacco smoke) by either one of two potent initiators (radiation or asbestos).Such a mechanism closely resembles two of those first proposed by Hitching (1973) for drug synergism, and later espoused by Hlatky and Whittemore (1991), that synergistic drugs act independently at different sites-either within a complex pathway or within a branched pathway. In contrast, when two carcinogens affect the same stage of carcinogenesis, then the relative risks are additive, and there is no synergism (Brown and Chu, 1989). Strong support for a "bottleneck relief" mechanism comes from the finding that two different oncogenes can fail to produce cancer when injected separately into newborn hamsters, yet when injected together produce a 73% incidence of malignant sarcomas (Corallini et al., 1987). Similarly, ras and myc oncogenes have different effects on cells, yet cooperate to produce neoplastic transformations (Stern et al., 1986).In line with our thesis, these results show that two synergistic oncogenes must act on a different portion of an oncogenic process that has two main rate-limiting steps. Carcinogenesis progresses to its completion only when the suppression of both rate-limiting steps is removed. But if the first rate-limiting step is initiation, then the second step can be the promotion of the initiated cells, requiring both proliferative and developmental changes. This was demonstrated incisively by Sukumar and co-workers, who found in animal models that the ras oncogene can remain latent within the mammary gland without producing breast cancer, until exposure to a hormonal promoter (estrogen) occurs, producing both cell proliferation and development (Kumar et al., 1990; Sukumar et al., 1995). Experimental studies have produced evidence for much of what the above investigators deduced from epidemiological studies in human beings. Cigarette smoke contains a complex mixture of complete carcinogens and promoters (Wynder and Hoffman, 1979). It functions both as an initiator and as a potent promoter in animal and in human systems (Van Duuran et al., 1971). In molecular terms, exposure to cigarette smoke is correlated with GC + TA transversions of the tumor suppressor gene p53, as found in lung carcinomas of smokers. This suggests that these specific mutations are caused by cigarette smoke and that they reduce or destroy the ability of p53 to suppress lung tumors (Takeshima et al., 1993). In this connection, other tumor promoters such as phorbol esters not only bind to specific cell surface receptors, but also can damage DNA sufficiently to activate or amplify cellular oncogenes, which then permanently encode the transformed genotype (Marx, 1982).

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Regarding genetic susceptibility to lung cancer, the three genes so far discovered already have been alluded to. Further details are that 90% of the United States population carries the gene for extensive metabolism of debrisoquine, which increases the risk of lung cancer, with only 10% of our population being resistant to it (Carporaso et al., 1990); this low percentage of resistance will give scant reassurance to smokers. Further, a gene carried in homozygous form by 0.3% of the population has been found to confer on its carriers a lung cancer risk manifold higher than for the 9.9% who carry it in heterozygous form (Zellers et al., 1990). This gene causes 27% of the lung cancer prior to age 50, when lung cancer is still rare. But for all ages, only 10.4% of lung cancer is attributable to this gene (Reif, 1991).

VI. VARYING THE TIME FRAME OF DATA COLLECTION A recent study on the interaction of cigarette smoke and radon in the production of lung cancer suggests that our “bottleneck relief” model for the mechanism of carcinogenic synergism fails to cover all eventualities. Archer has found that these carcinogens interact multiplicatively if the follow-up time is relatively brief, additively if long (Archer, 1988); another additive result has been reported also (Radford, 1981). Similarly, for the interaction of cigarette smoke and asbestos fibers, weaker evidence for interaction was found at the end of an extended follow-up period (Berry et al., 1985). Here we present a model that explains Archer’s surprising conclusion (1988),that either synergism or addition can result when one of the two carcinogens is not a sufficiently potent promoter to bring the promotional phase of carcinogenesis to completion. Our model simplifies the carcinogenic process by assuming that it is a twostep procedure, and by combining all initiation steps into one category (I), and all promotion steps into another (P). We suppose that three identical populations X, Y, and Z are exposed to these carcinogens. Exposure is either separate, so that X is exposed only to carcinogen A and Y to B, or else joint, so that Z is exposed to both A and B. If we assume that initiation by carcinogens A and B is additive without overlap, then the respective fraction of the populations X, Y, and Z in which cancer cells are initiated can be designated as I,, I,, and I, + I,. Let us assume with Pitot that promotion cannot take place until initiation has occurred (Pitot, 1981), and that the likelihood that at least one of the initiated cells is promoted to a growing cancer cell is PA for carcinogen A and P, for B. If A and B promote independently, then when acting jointly

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their promotional effect will be (PA + P, - PAP,), where PAPBcorrects for initiated cells for which the actions of the two carcinogens overlap. For separate exposure to each of the two carcinogens, the fractions of the populations in which initiated lung cells are promoted to cancer cells will be I,P, for X and I,P, for Y, for a total additive response of I,P, + IBPB. But for the population 2 with joint exposure to both carcinogens, the fraction developing cancer will be (I, + IB)(PA+ P, - PAP,). When 100% of initiated cells are promoted to cancer, then PA, PB, and (PA + P, - PAP,) all equal 1.00, and there is no difference between joint or separate exposure, because in both cases the fraction developing cancer is Iq + I,. But for values of PA and P, below 1.00, development of cancer on joint exposure in population Z exceeds the additive result for separate exposure of population X to A and Y to B. For instance, suppose that after a relatively short follow-up period, the values or I,, I,, PA, and P, all equal 0.2. Then the fraction of the population developing lung cancer as a result of separate exposure will be 0.2 X 0.2 or 0.04 (4%)for each of the two carcinogens, and 0.08 (8%) as the additive response. But for joint exposure, we obtain (0.2 + 0.2) (0.2 + 0.2 - 0.2 X 0.2) or 0.144 (14.4%).Thus, joint exposure produces a response that does not quite reach the multiplicative effect of separate exposures, 4% X 4% = 16.0%. But after a long-term follow-up period, promotion of initiated cells to cancer could reach loo%, and PA, P,, and PA + P, each could attain their maximum value of 1. Then, cancer incidence for joint exposure will be (IA + I,) X 1. This is the same result as is obtained by adding the responses for separate exposure of X and Y. Hence for this case there is no synergism. The above model is only one of many that could be chosen to analyze the effect of the length of the follow-up period on the presence of synergism. For simplicity’s sake, we did not choose to analyze a more realistic multistep model. Despite this limitation, our model illustrates the possibility that a short-term follow-up may indicate synergism, whereas a long-term one may not do so. Also, our model illustrates why synergism might not quite reach a multiplicative level even for a short-term follow-up. Another way of looking at the same problem is to posit that synergism be defined in terms of time-specific measures of risk, for instance, the hazard rate at specific times. Further, the joint effects of smoking and radon may differ, depending on the time sequence in which exposure to the two carcinogens occurs. But even if joint exposure to cigarette smoke and radon is merely additive in the long term, in the short term smokers will lose irreplaceable years of life because of synergism. Although they may have contracted cancer later, its early incidence robbed them of the intervening years. Thus, synergism has a potent deleterious effect even when it does not extend to the end of life.

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V11. CONCLUSION The concept that smokers face a serious risk from the synergism of cigarette smoke with other common respiratory carcinogens, particularly radon and asbestos, so far has not been exploited in antismoking campaigns in developed nations, where the public is already well-informed on the hazards of smoking. But use of synergism for publicity is valid only if a consensus exists on the evidence for synergism. We begin by defining carcinogenic synergism and antagonism, and illustrate how synergism can be substantiated statistically when the dose-response curves of two interacting carcinogens are linear; if nonlinear, this requires consideration if artifacts are to be avoided. From an examination of the dose-response curves for lung cancer causation by each of the three carcinogens, we conclude that all are linear. Our results validate those previous studies that reported synergism for these binary interactions but without consideration of dose-response curves. The evidence we cite indicates that a consensus exists on the presence of carcinogenic synergism in these systems, and is based on valid statistical analyses of the available data. Although this is the overall finding that applies to the most commonly encountered situations, there are complexities and exceptions. For instance, we illustrate how results for short- or medium-term follow-ups can indicate synergism, whereas those for long-term follow-ups may not do so. We conclude that a consensus exists, that the causation of lung cancer by cigarette smoke interacting either with asbestos or with radon is synergistic. This finding can now be used in a new publicity campaign against smoking, focused on the previously unappreciated additional dangers of smoking that stem from its synergism with other airborne carcinogens.

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Stern, D. E, Roberts, A. B., Roche, N. S., Sporn, M. B., and Weinberg, R. A. (1986).Mol. Cell. Riol. 6, 870-877. Stidley, C. A., and Samet, J. M.(1994).Am. /. Epidemiol. 139,312-322. Sukumar, S., McKenzie, K., and Chen, Y. (1995).Mutat. Res. 333,37-44. Takeshima, Y., Seyama, T., Bennett, W. P., Akiyama, M., Tokuoka, S., h i , K., Mabuchi, K., Land, C. E., and Harris, C. C. (1993).Lancet 342, 1520-1521. Thomas, D. C. (1981).Biometrics 37, 673-686. Thomas, D. C., McNeill, K. G., and Dougberty, C. (1985).Health Pbys. 49, 825-845. Thomas, D. C., Brown, C . C., Chu, K. C., Goldsmith, D. F., and Saracci, R. (1987).J . Chron. DlS. 40 (SUPPI. 2), V-VI. Tsuda, T., Babazono, A., Yamamoto, E., Kurumatani, N., Mino, Y., Ogawa, T., Kishi, Y., and Aoyama, H. (1995).Am. /. Epidemiol. 141, 198-209. Tuyns, A. J., Pequignot, G., and Jensen, 0. M. (1977).Bull. Cancer 64,45-60. United Nations Scientific Committee on the Effects of Atomic Radiations (1982).In “Ionizing Radiation: Sources and Biological Effects,” pp. 727-773; summary of main conclusions, paragraph S3:ll. United Nations, New York. U.S. Department of Health and Human Services (1982). “The Health Consequences of Smoking. Cancer. A Report of the Surgeon General.” Office on Smoking and Health, Rockville, Maryland. U.S. Department of Health and Human Services (1985).“The Health Consequences of Smoking. Cancer and Chronic Lung Disease in the Workplace. A report of the Surgeon General.” Office o n Smoking and Health, Rockville, Maryland. U.S. Environmental Protection Agency ( 1993). “Respiratory Health Effects of Passive Smoking: Lung Cancer and Other Disorders.” Monograph No. 4, NIH Publ. No. 93-3605. Vaiavanidis, A., Balomenou, H., Macropoulou, I., and Zarodimos, I. (1996).Free Rad. Biol. Med. 20, 853-858. Van Duuran, B. L., Sivak, A., Katz, C., and Melchionne, S. (1971).J. Natl. Cancer Inst. 47, 235-240. Wahrendorf, J., and Brown, C. C. (1980).Biometrics 36, 653-657. Walter, S. D., and Holford, T. R. (1978).Int. J. Epidemiol. 108, 341-346. Wang, X., Christiani, D. C., Wiencke, J. K., Fischbein, M., Xu, X., Cheng, T. J., Mark, E., Wain, J. C., Kelsey, K. T. (1995).Cancer Epidemiol. Biomaikers Prevent. 4,543-548. Weinberger, R. A. (1990).“Oncogenes and the Molecular Origins of Cancer.” Monograph 18. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Weisburger, J. H., and Horn, C. I-. (1991).In “American Cancer Society Textbook of Clinical Oncology.” (A. I. Holleb, D. J. Fink, and G. P. Murphy, eds.), pp. 80-98. American Cancer Society, Atlanta, Georgia. Weisburger, J. H., and Williams, G. M. (1989).Cell Biol. Toxicol. 5, 377-391. Whittemore, A. S., and iMcMillan, A. (1983).1. Natl. Cancer Inst. 71, 489-499. Wynder, E. L., and Bross, I. J. (1961).Cancer 14, 389-413. Wynder, E. L., and Hoffman, D. (1979).N.Engl. /. Med. 300, 894-903. Yao, S. X., Lubin, J. H., Qiao, Y. L., Boice, Jr., J. D., Li, J. Y., Cai, S. K., Zhang, F. M., and Blot, W. J. (1994).Radzai. Res. 138, 326-336. Zang, E. A., and Wynder, E. L. (1992).Cancer 70,69-72. Zhu, H., and Wang, Z. (1993).BY.J. Indust. Med. 59,271-278.

Carcinogenesis and Natural Selection: A New Perspective to the Genetics and Epigenetics of Colorectal Cancer Jarle Breivik and Gustav Gaudernack Section for Immunotherapy The Norwegian Radium Hospital N-0310 Oslo, Norway

I. Introduction 11. Evolution and Cancer A. Natural Selection and Multicellularity B. Cancer Evolution 111. The Microsatellite Instability Pathway A. Microsatellite Instability, Mismatch Repair, and BAX B. Mismatch Repair and Methylation Tolerance C. Bile Acids, Methylation, and Proximal Location IV. The Chromosomal Instability Pathway A. Chromosomal Instability and Cell Cycle Control B. Bulky-Adduct-Forming Carcinogens and Nucleotide Excision Repair C. p53 Inactivation D. Environmental Factors and Distal Location V. MIN versus CIN A. Point Mutations and Carcinogens B. Pathways and Prognosis VI. DNA Methylation and the Epigenetics of Cancer A. DNA Methylation Balance B. Epigenetic Evolution C. Methylation and Cancer D. Hypermethylation and the MIN Pathway VII. Location-Related Carcinogenic Environments VIII. Conclusions and Perspectives References

I. INTRODUCTION Evolution through natural selection is a scientific concept, which in a unique manner grasps the fundamental features of biology (Darwin, 1859; Dawkins, 1989; Alberts et al., 1994).Neoplastic transformation is also increasingly understood in terms of evolutionary mechanisms, and it is now Advances in CANCER RESEARCH 0065-2301099 $30.00

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generally agreed that cancer development is a multistep process involving natural selection of somatic mutations (Vogelstein and Kinzler, 1993; Bodmer et al., 1994; Klein, 1998). In close agreement with this view, a growing amount of data indicates that tumor-specific genetic alterations are not random, but closely related to different tissues and specific classes of carcinogens (Lawley, 1994; Levine et al., 1995). A number of reports also reveal distinct patterns of genetic alterations in different tumors, thus implying that the carcinogenic process may proceed through alternative genetic pathways (reviewed by Shackney and Shankey, 1997). A number of studies on colorectal cancer have found significant associations between specific genetic alterations and different clinicopathological variables. We clearly demonstrated some of these differences in a study of colorectal carcinomas from 282 different patients, and found different genetic alterations to be dependent on age and gender of patient (Breivik ct al., 1997). The most striking finding in this, and a number of other studies, was, however, the marked differences between cancers in the proximal and distal segments of the bowel (Fig. 1; see color plate). Different genetic pathways to colorectal cancer thus appear to be closely related to specific segments of the colorectum. Determined to explore this phenomenon, we searched the literature for other differences related to proximal and distal colorectal cancer. This resulted in a substantial amount of data from very different fields of cancer research, with few apparent connections. However, based on the fact that all the data represented different perspectives of the same biological process, we continued to search for unexplored links between the various pieces of information. Considering neoplastic transformation as an evolutionary process, we also assumed that this fundamental aspect of biology was the key to organizing the data. In this work we therefore present a model of the carcinogenic process based on the principles of molecular evolution. We then apply this model as a tool for organizing data related to colorectal cancer, and suggest some unexplored connections between genetic and biochemical as well as epidemiological data related to cancer development. We also describe how DNA methylation and the epigenetics of cancer may be understood in terms of natural selection, and present an integrated hypothesis on the molecular etiology of colorectal cancer.

11. EVOLUTION AND CANCER

A. Natural Selection and Multicellularity The foundation underlying all of biology is self-replication of genetic information in the form of DNA sequences or genes (here used as synonyms)

Fig. 1 Distribution of K-RAS mutations, TP53 mutations, and microsatellite instability (MIN) in colorectal carcinomas in relation to mean age (69 years) and gender of patient, and location of tumor. Genetic information is presented as sectors of circles representing tumors. Colored sectors illustrate the presence of genetic alterations: blue, K-RAS mutation; red, MIN; green, TP53 mutation. Notice the marked differences between tumors in the proximal and distal segments of the colorectum. See Breivik et al. (1997) for further details. Reproduced from Breivik et al., Int. J. Cancer. Copyright 01997 John Wiley & Sons, Inc.

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(Dawkins, 1989; Alberts et al., 1994). Collections of different genes cooperate to their own reproduction by building complex “copying machines” known as organisms. Natural selection occurs when one version of a gene (an allele) contributes to the building of organisms that are more efficient copiers than are the organisms made from other versions of that gene. This better adjusted allele will thus multiply at the expense of others. Which alleles are favored in the selection process is, however, totally dependent on the surrounding milieu, and different environments may promote genes and organisms with completely different properties. Multicellular organisms represent clonal expansions of single cells, and may thus be viewed as colonies of closely related genetic entities working together toward reproduction. The genes that build multicellular organisms have, among other factors, been selected because of their ability to coordinate the different cells of the organisms. It is therefore not surprising that the genomes of multicellular organisms encode a large number of proteins involved with the transmission of signals between the different cells of the body. The genes, which encode these communication and control proteins, ensure that each cell fulfills its chores and promotes the primary goal of the organism, i.e., reproduction. The genes of multicellular organisms thus have a composition, and exist in an environment that promotes cellular cooperation. However, this does not imply that genes in somatic cells are exceptions to the general rule of natural selection. On the contrary, recent discoveries have shown that somatic evolutionary processes are important mechanisms in human biology, and that this may be both a curse and a blessing to an organism. The adaptive immune system is probably the best known example of natural selection occurring at the somatic level. Immunological research has revealed that lymphocytes, by genetic rearrangements and a consecutive series of selection steps, evolve antigen-specific receptors with a crucial role in the immunodefense system (Robey and Fowlkes, 1994; Posnett, 1995). These somatically evolved features of the lymphocytes enable the organism to recognize foreign elements and are the key to immunological memory. DNA has, however, no sense of team spirit or future expectations, and is reproduced only because of its biochemical interactions with the surrounding environment (Dawkins, 1989). Somatic mutations are therefore selected solely based on their ability to improve the reproductive capacity of a somatic clone, and this may not always agree with the objectives of the organism. On the contrary, natural selection will favor cells that escape the organism’s control mechanisms and that proliferate even if to do so contradicts the goal of reproduction of the organism. The somatic evolutionary process will thus inevitably drive replicating cells toward neoplastic transformation, and cancer might be viewed as the price an aging organism pays for the advantages of multicellularity.

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B. Cancer Evolution Evolution through natural selection depends on two essential elements, selection pressure and availability of genetic variation (Dawkins, 1989; Alberts et al., 1994). Because neoplastic transformation represents evolution at the somatic level, it should also be viewed in this context. As already described, a selection pressure that favors escape from the organism’s growth control mechanisms is an inevitable consequence of multicellularity. In other words, a somatic mutation will have a larger potential to reproduce, if it involves an escape from the suppressive signals brought on by the other genes and cells of the organism. Based on the same logic, any factor that increases the reproductive ability of a mutation will necessarily promote a somatic evolutionary process. If, e.g., a dietary component inhibits proliferation of the colorectal mucosae through an effect on a hormone receptor, natural selection will favor mutants that disrupt this signaling pathway. Such mutated cells and their progenitors will also lack the ability to receive other signals transmitted through this pathway. Consequently, the organism has lost important channels for controlling the growth of these mutants, and the cells have moved one step in the direction of neoplastic transformation. Any environmental factor or endogenous biochemical disturbance that favors escape from cellular control mechanisms must therefore also be expected to accelerate the carcinogenic process. Availability of genetic variation is the second essential factor in an evolutionary process. Without variation there can be no evolution. Preservation of genetic homogeneity is therefore essential to prevent neoplastic transformation. However, DNA replication and repair are not perfect and accumulation of genetic variants is an inevitable consequence of aging (Hurst, 1995; Simpson, 1997). Additionally, any factor, intrinsic or extrinsic, that increases the somatic mutation rate must be expected to accelerate the transformation process. An evolutionary view of neoplastic transformation is therefore in good agreement with the fact that most potent carcinogens are also mutagenic. The organism’s homeostatic processes involve a number of mechanisms, including antioxidation and detoxification, which prevent mutagens from affecting the integrity of the genome. In addition, there are a number of cellular mechanisms that stop established DNA damage from spreading in the somatic environment. The coordination of cell cycle arrest and enzymatic DNA repair is the organism’s first line of defense against established DNA damage (Murnane, 1995; Sanchez and Elledge, 1995).These mechanisms prevent DNA damage from spreading in the somatic environment, by halting proliferation until genomic integrity has been reestablished. A DNA-damaging environment will

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Fig. 2 A general evolution-based model of neoplastic transformation. (1)An organ or a group of cells is exposed to a DNA-damaging environment. ( 2 )DNA damage promotes genetic variation and induces growth arrest and DNA repair. (3) Variants that escape growth arrest and compromise DNA repair are favored through natural selection. (4) Repair deficiency results in a genome-wide accumulation of DNA damage and genetic alterations, i.e., a general genomic instability. (5)The excessive DNA damage provides more genetic variation and induces apoptosis. (6) Apoptosis-deficient variants survive. (7)The cells, which have escaped cell cycle control, compromised DNA repair, acquired a general genomic instability and escaped apoptosis, continue to proliferate and evolve irrespective of the organism’s “intention” to survive and reproduce. In addition and parallel to these key steps, any genetic variants that constitute a growth advantage will promote the somatic evolution process, and contribute to the neoplastic process.

thus involve a growth-inhibiting signal to normal cells, and consequently constitutes a selection pressure that favors escape of this control mechanism. The resulting breakdown of the arresthepair mechanism will therefore involve a breakdown of the cell’s protection against DNA damages, and thus results in a general increase in mutation rate. Apoptosis, also known as cellular suicide or programmed cell death, is a mechanism that deletes cells from multicellular organisms. Excessive DNA damage (Gotz and Montenarh, 1996)and immunorecognition (Berke, 1997)

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are among the factors that trigger apoptosis, and programmed cell death is central to the preservation of genomic integrity. Cells that escape apoptotic death will obviously have a growth advantage in the somatic environment, and an environment that induces apoptosis will consequently favor such mutants. Accumulation of DNA damage due to mutagenic environments and compromised repair mechanisms must therefore be expected to favor defects in the apoptotic pathways. From this it may be concluded that DNA-damaging environments contribute to somatic evolutionary processes by increasing the genetic variation as well as constituting a selection pressure that favors breakdown of the organism’s control mechanisms. Figure 2 illustrates this evolution-based scenario of neoplastic transformation. In addition and parallel to these key steps, any genetic variants that constitute a growth advantage will promote the somatic evolutionary process, and contribute to neoplastic transformation. Neoplastic transformation may thus be viewed as a continuous evolutionary process whereby every generation of cells is evaluated by the inevitable law of natural selection. The concept that carcinogenesis is a progressive process driven by evolutionary mechanisms is not a new idea in the scientific community (Klein, 1998; Foulds, 1958). The era of molecular biology has, however, provided the theoretical and technical tools that allow specific testing of this hypothesis, and the evidence that supports a multistep model to malignancy is already overwhelming. A growing amount of data indicate that cancers develop by different molecular pathways recognized as common patterns of genetic alterations (reviewed by Shackney and Shankey, 1997). Such patterns divide colorectal cancer into two distinct categories, and these malignancies appear to develop by molecular pathways related to two different types of genomic instability (Lengauer et al., 1997b).

Ill. THE MICROSATELLITE INSTABILITY PATHWAY

A. Microsatellite Instability, Mismatch Repair, and BAX iMicrosatellite instability (MIN), also referred to as replication errors or the mutator phenotype, is recognized as genome-wide alterations in repetitive DNA sequences (reviewed by Lothe, 1997).Such mutation patterns have been found in almost all analyzed malignant tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC), and in about 15% of the sporadic (noninherited) cancers. MIN is caused by defects in the nucleotide mismatch repair (MMR) machinery, which, in addition to correcting mismatched nucleotides, also repairs incision/deletion mutations in re-

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Fig. 3 An evolution-based model of colorectalcarcinogenesis.(A)The MIN pathway. (B)The CIN pathway. (C) Hypermethylation and its relationship to the MIN pathway. (D) Carcinogenic environments in the colorectum in relation to location and carcinogenetic pathways. NS, Natural selection; MGMT, 06-methylguanine-DNA methyltransferase; MMR, mismatch repair; BAF, bulky adduct forming; NER, nucleotide excision repair; MCP, mitosis checkpoint. See text for further details.

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peated DNA sequences. Germ-line mutations in the MMR genes hMSH2, hMLHl, hPMS1, and hPMS2 have been identified in HNPCC patients, and somatic MMR alterations are found in tumors from hereditary as well as sporadic tumors with MIN. Defects in the MMR pathway represent a general increase in mutation rate, and MIN has been specifically related to alterations in cancer genes harboring repetitive DNA sequences. One such gene, B A X , has a central role in the promotion of the apoptosis process, and frameshift mutations in B A X have been identified in more than 50% of tumors expressing MIN (Rampino et al., 1997). MMR deficiency generates frameshift mutations in numerous encoding sequences, resulting in potentially immunogeneic proteins (Parsons et al., 1995; Rampino et al., 1997). As previously mentioned, induction of apoptosis is a central immunological mechanism, and immunorecognition may be a mechanism by which MMR deficiency promotes apoptosis. In fact, MIN tumors display a profound lymphocyte infiltration that may be related to this immunological phenomenon (Kim et al., 1994). The relationships between MMR deficiency, MIN, apoptosis, and B A X mutations fit very well with steps 3 to 6 (Fig. 2) of the general evolutionbased model of neoplastic transformation. Compromise of DNA repair results in an increased mutation rate that induces apoptosis and promotes escape from the apoptotic pathway. In Fig. 3A these key events in the MIN pathway are incorporated in the model.

B. Mismatch Repair a n d Methylation Tolerance MMR deficiency and MIN have been related to cellular tolerance to the methylating agents N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanine (MNNG). Different studies suggest that such methylation tolerance arises through the acquisition of defects in the MMR pathway (Branch et al., 1995; Karran and Hampson, 1996; Kawate et al., 1998), and a mechanism that explains this relationship has been proposed (Karran and Bignami, 1994): MNU and MNNG induce methylation, preferentially in the 0" position in guanine (G) residues in the DNA molecule. The enzyme 06-methylguanine-DNA methyltransferase (MGMT), which transfers the methyl group to a cysteine residue within its own structure, reverses such DNA damages (Pieper, 1997). However, MGMT is susceptible to epigenetic silencing through hypermethylation (Qian and Brent, 1997), and expression in the colorectal mucosae is low compared to other tissues. The resulting low level of MGMT thus promotes accumulation of 06-methylguanine ( 06-mG)

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residues in the colorectum. 06-mG codes ambiguously, and promotes formation of 06-mG/T hybrids that are recognized by the MMR machinery. MMR is, however, targeted to the newly synthesized DNA strand, and is apparently unable to correct the 06-mG in the old strand (Modrich, 1997). The cell may thus be caught in a futile cycle of unsuccessful repair attempts, causing G, arrest and persistent strand breaks that eventually lead to cell death (Karran and Bignami, 1994; Hawn et al., 1995). Methylating agents thus impose a selection pressure that favors cells that avoid these “deathprone” repair attempts, and that promote mutation, loss, or silencing of genes involved in the MMR pathway. This relationship between MMR deficiency and methylation tolerance fits very well with steps 1 to 3 (Fig. 2) of the evolution-based model to neoplastic transformation. A mutagen causes DNA damage that induces growth arrest and DNA repair, and constitutes a selection pressure that favors disruption of this control mechanism (Fig. 3A).

C. Bile Acids, Methylation, and Proximal Location A number of animal studies have found bile acids to enhance colorectal carcinogenesis (Cohen and Raicht, 1981; McSherry et al., 1989), whereas others have found an inhibiting effect on the neoplastic process (Magnuson and Bird, 1993; Bird, 1995; Shirtliff and Bird, 1996). The effect of bile acids on tumor development thus appears to be strongly dependent on the model system, and these apparently contradicting results support the hypothesis that bile acids are related to specific molecular pathways to colorectal cancer. Different studies also implicate bile acids in human colorectal carcinogenesis (Nagengast et al., 1995), and epidemiologically this association is most evident in the proximal segments of the colon (McMichael and Potter, 1985). It is therefore interesting that about 90% of sporadic and 70% of familial cancers expressing the MIN phenotype are localized to these segments (Lothe et al., 1993; Young et al., 1993; Breivik et al., 1997). The bile acid conjugates taurocholic acid and glycocholic acid can undergo N-nitrosation to form compounds analogous to the methylating agents (Puju N-methyl-N-nitrosourea and N-methyl-N’-nitro-N-nitrosoguanidine et al., 1982; Busby et al., 1985). Considering the data that link MMR deficiency and methylating agents, this suggests that bile acid conjugates constitute the selection pressure that favors MIN in the proximal colon. Methylating bile components are therefore good candidates as a promoter of step 1 in an evolution-based model of the MIN pathway (Fig. 3A). Epidemiological, biochemical, and genetic data related to MIN and col-

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orectal cancers thus appear to form a logical connection when viewed in the context of our general evolution-based model of neoplastic transformation.

IV. THE CHROMOSOMAL INSTABILITY PATHWAY A. Chromosomal Instability and Cell Cycle Control In contrast to MIN tumors, whereby mutations are predominantly localized to repetitive DNA sequences, the majority of colorectal tumors have numerous alterations at the chromosomal level. These alterations range from different types of chromosomal rearrangements, to loss and gain of large fragments of the genome, including entire chromosomes (Meuth, 1996), and may be measured as abnormal numbers of chromosomes or shifts in the total nuclear DNA content (Hiddemann et al., 1984; Thompson and Thompson, 1998). Tumors expressing such features are classified as aneuploid, in contrast to the diploid properties of normal cells and most tumors with MIN. Aneuploidy has been associated with a persistent defect in chromosomal segregation (Lengauer etal., 1997b),and as a parallel phenomenon to MIN, this phenotypic property of cancer cells has been designated chromosomal instability (CIN). Furthermore, recent findings have shown that CIN is related to the loss of function of a mitotic checkpoint involving the hBUBl gene (Chahill et al., 1998).Breakdown of such control mechanisms may thus cause CIN in a manner similar to that in which defects in the MMR pathway cause MIN. These findings indicate that CIN is a direct consequence of defects in the cell cycle control machinery, and fit well with step 4 of our evolution-based model of neoplastic transformation. In Fig. 3B this key event in the CIN pathway has been incorporated in the model.

B. Bulky-Adduct-Forming Carcinogens and Nucleotide Excision Repair Carcinogens can be divided into methylating and bulky-adduct-forming (BAF) agents depending on the DNA lesions and the repair mechanisms that they induce (Mitra et al., 1989; reviewed by Lawley, 1994). In contrast to methylating carcinogens, BAF carcinogens, including UV radiation, free oxygen species, and a miscellaneous group of chemicals, induce relatively large alterations in the three-dimensional structure of the DNA molecule. Such le-

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sions induce growth arrest, and are exclusively repaired by nucleotide excision repair (NER) (reviewed by Sancar, 1995). NER is the most important of the DNA repair pathways, and also appears to function as a backup for other repair mechanisms. The NER process involves removal and resynthesis of large fragments of DNA, and includes an intermediate stage with single-stranded gaps in the DNA molecule. These NER-induced incisions, as well as the bulky adducts themselves, prompt chromosomal rearrangement (reviewed by Bohr, 1995). Furthermore, unbalanced chromosomal rearrangement comprising centomere regions must be expected to distort spindle formation and chromosome segregation. BAF carcinogens may thus inhibit cellular growth by activating the mitotic checkpoint, and consequently constitute a selection pressure that favors breakdown of this control mechanism. In other words, BAF carcinogens may promote CIN in a manner similar to that whereby methylating agents promote the MIN phenotype. This model thus links CIN directly to a carcinogenic environment, and provides an explanation to the relationship between CIN and chromosomal rearrangements. It is also in close agreement with steps 1 to 4 in our model and have been incorporated in Fig. 3B.

C. p53 Inactivation TP.53 is the single gene most frequently altered in human malignancies, and such alterations are found in about 70% of colorectal cancers (Hollstein et al., 1991; Goh et al., 1994). The protein (p53) has a key role in the cellular defense against DNA damage, and appears to function as a “guardian of the genome” (Lane, 1992).p53 is stabilized by bulky DNA adducts and single-stranded DNA (Lee et al., 1995), and the resulting accumulation of p53 promotes cell cycle arrest or apoptosis (reviewed by Gotz and Montenarh, 1996). Both BAF carcinogens and NER-induced strand-breaks will thus promote p53-dependent growth arrest or apoptosis. Either way, such agents must be expected to constitute a selection pressure that favors inactivation of p53. The observed relationship between p53 inactivation and resistance to numerous carcinogenic and chemotherapeutic agents (reviewed by Hickman, 1996) is thus in close agreement with the evolutionary view of neoplastic transformation. Different studies have found an association between p53 inactivation and CIN, and a causal relationship between the two has been proposed (Shackney and Shankey, 1997). However, additional studies strongly indicate that CIN is not caused by p53 inactivation (Lengauer et al., 1997b), and as previously described CIN has now been related to another molecular pathway

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(Chahill et al., 1998). In this context it is therefore interesting that our evolution-based model offers an alternative explanation to the association between p53 inactivation and CIN. Because both molecular phenomena may be understood as adaptations to an environment of BAF carcinogens, we propose that the observed association between p53 inactivation and CIN reflects their relationship to common etiological factors. This does not, however, exclude that p53 inactivation may enhance the CIN phenotype, nor the possibility that CIN promotes p53 inactivation. The dual role of p53, with relevance to cell cycle control as well as apoptosis, suggests that it may be involved in different events during tumor progression. p53 inactivation appears, for instance, to be a very early event in ultraviolet radiation-related skin cancer (Nakazawa et al., 1994), whereas this is not the case for colorectal cancer. TP53 alterations are actually very rare in the early stages of colorectal carcinogenesis, and p53 inactivation appears to be involved in the malignant conversions of advanced adenomas (Fearon and Vogelstein, 1990). Although results are contradictory (Shackney and Shankey, 1997), this suggests that p53 inactivation succeeds CIN in colorectal carcinogenesis. We thus propose p53-related apoptosis and p53 inactivation as candidates for step 5 and 6 of our evolution-based model of the CIN pathway (Fig. 3B).

D. Environmental Factors and Distal Location Although cancers in the proximal colon are associated with intrinsic factors such as bile acids and sex hormones, distal cancers are more closely related to environmental factors such as diet, cigarette smoking, and alcohol consumption (Wynder et al., 1992; Heineman et al., 1994; Giovannucci and Willett, 1994; Giovannucci and Martinez, 1996). Of dietary components, heterocyclic amines found in fried fish and meat are the only known animal colon carcinogens that humans consume on a regular basis (Sugimura, 1992). Diets containing high levels of such compounds are associated with increased risk of colorectal cancer (Roberts-Thomson et al., 1996). Heterocyclic amines as well as a variety of compounds found in cigarette smoke are BAF carcinogens, and have been related to NER (Marwood et al., 1995). These environmental factors may thus contribute to an environment that promotes colorectal cancer by the CIN pathway, and are good candidates for step 1 of our model (Fig. 3B). Aneuploidy and TP53 alterations have consistently been related to distal tumor location (Meling et al., 1993; Breivik et al., 1997; Changchien et al., 1997). We have described how these genetic alterations as well as the epidemiological factors related to distal colorectal cancer may be linked to BAF carcinogens. Viewed in the context of our model, there thus appears to be a

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logical connection between CIN and epidemiological and genetic data related to distally occurring colorectal cancer.

V. MIN VERSUS CIN A. Point Mutations and Carcinogens Methylating and BAF carcinogens are related to distinct patterns of point mutations (reviewed by Lawley, 1994). BAF carcinogens have been related to a variety of mutations and promote transversions as well as transitions. Conversely, methylating mutagens such as MNNG and MNU almost exclusively induce GC -,AT transitions, predominantly in the second base of GpG sites (not to be confused with CpG sites; see later) (Lukash et al., 1991; Zhang and Jenssen, 1991; Bishop et al., 1996). Our model implies a relationship between the two types of carcinogenic agents and the MIN and CIN pathways, respectively. Based on this relationship our model therefore predicts an association between the spectrum of point mutations and type of genomic instability. In general, tumors with MIN are predicted to have a high frequency of methylation-related GC -,AT transitions, whereas CIN should be associated with a high frequency of transversions. Not many molecular studies on colorectal cancers have been large enough to investigate reliably the relationship between specific types of point mutations and genomic instability, and to our knowledge there are no reports of any significant associations between the two phenomena. However, Giaretti et al. (1995) found a positive correlation between aneuploidy and transversions in the K-RAS oncogene in colorectal adenomas, thus suggesting a link between transversions and CIN. To further explore these relationships we therefore reinvestigated previously published data from a large number of studies of colorectal carcinomas (Breivik et al., 1994, 1997). Tumors with MIN from four different patients also displayed K-RAS mutations. All were methylation-related transitions. One of the four patients had three additional synchronous primary carcinomas also analyzed for KRAS mutations. Two of these carried methylation-related transitions; the third had no detectable mutation. We also looked for differences in K-RAS mutation patterns in relation to the presence of TPS3 mutations ( n = 75), and as predicted methylation-related transitions were most frequent in tumors without TP53 mutations (62 vs. 39%; p = 0.05). Transversion in both K-RAS and TPS3 was also related to distal tumor location (Breivik et al., 1994; Bsrresen-Dale et al., 1998). Point mutation spectra thus support our hypothesis by substantiating the

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links between the MIN and CIN pathways, and methylating and BAF carcinogens, respectively. Larger collaborative studies and multivariate analyzes are, however, needed to confirm these associations.

B. Pathways and Prognosis Distally occurring colorectal cancers tend to be more infiltrating, recur more often, and are associated with a poorer clinical outcome compared to proximal lesions (Bottger et al., 1993; Tang et al., 1995). Furthermore, genetic alterations related to the CIN pathway, including aneuploidy, TP.53 alterations, and transversions have all been related to an unfavorable prognosis and distal tumor location (Meling et al., 1993; Moerkerk et al., 1994; Breivik et al., 1994, 1997; Yamashita et al., 1995; Changchien et al., 1997; Bsrresen-Dale et al., 1998). Conversely, carcinomas of the proximal colon tend to grow as polypoid fungating masses, and to be associated with a somewhat favorable prognosis (Cotran et al., 1989). Furthermore, MIN has also been associated with a favorable prognosis and is found almost exclusively in proximal tumors (Lothe et al., 1993; Thibodeau et al., 1993; Breivik et al., 1997). There thus appears to be a connection between tumor behavior, anatomic location, and different genetic pathways to colorectal cancer. Considering the evidence, which suggests that the MIN and CIN pathways are promoted by different classes of mutagens, this also suggests a relationship between patient prognosis and etiologic factors.

VI. DNA METHYLATION AND THE EPIGENETICS OF CANCER A. DNA Methylation Balance Methylation of cytosine residues (5-methylcytosine) at CpG sites is found throughout the human genome, characteristically clustered to so-called CpG islands in gene promoter regions. Hypo- and hypermethylation of these regions are related to activation and inhibition of transcription, respectively, and this type of gene regulation is essential to cell differentiation and embryological development (reviewed by Monk, 1995). DNA methylation patterns are maintained during cell division by 5-cytosine DNA methyltransferase (DNA-MTase) (reviewed by Ramsahoye et al., 1996). This enzyme preferentially methylates hemimethylated CpG sites, thus copying established methylation patterns to the newly synthesized DNA

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strands. This epigenetic replication process ensures that information related to gene expression and chromatin structure is maintained through cell division. Different tissues and stages of development are related to specific methylation patterns, and a growing number of data have related cancer development to epigenetic alterations (reviewed by Baylin et al., 1998). Furthermore, different biochemical agents have been shown to modify gene expression through their effects on DNA methylation. Despite a relatively high-fidelity maintenance, DNA methylation thus represents a highly dynamic system that responds to endogenous as well as exogenous factors (Zingg and Jones, 1997; Slattery et al., 1997). A large number of factors have been found to affect the general level of DNA methylation (reviewed by Zingg and Jones, 1997). Enzyme activities related to methyl group metabolism and the DNA methylation process are evidently of importance. Great interest has been focused on the regulation of DNA-MTase, and activity of this key enzyme has been related to differentiation and cell cycle control as well as neoplastic transformation (Chuang et al., 1997; Baylin et al., 1998). An intricate relationship between different genetic factors, including somatic mutations, must therefore be expected to affect DNA methylation (Baylin, 1997). The genetic code thus influences DNA methylation patterns through two different mechanisms-by structurally determining possible methylation sites and by encoding proteins involved in the methylation process. Conversely, expression of the genetic code is extensively regulated by methylation, and methylation may directly affect the nucleotide sequence by promoting mutagenesis (Zingg and Jones, 1997). Combined, this establishes an intricate relationship of regulation and dependence between the genetic and the epigenetic code (Fig. 4). Different studies have suggested DNA methylation as a key factor in the aging process, and increasing age has been related to a general hypermethylation of CpG islands, in different tissues (Issa et al., 1994, 1996). Accumu-

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Fig. 4 The genetic-epigenetic interaction. Although DNA methylation is structurally dependent on CpG sites in the DNA molecule, its replication and regulation are directly dependent on expressed nucleotide sequences, particularly the DNA-MTase gene. Concurrently, gene expression is extensively regulated by methylation, and methylation may directly affect the genetic code by promoting mutagenesis. Combined, this establishes an intricate relationship of regulation and dependence between the genetic and the epigenetic code.

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lation of methyl groups at CpG sites thus appears to be a normal phenomenon during the life of an organism. S-Adenosylmethionine ( AdoMet) is the substrate of DNA-MTase, essential to CpG methylation, and the prime methyl donor in the body (Zingg and Jones, 1997).The dietary factors choline, methionine, folate, and vitamin B,, are key components in AdoMet synthesis, and insufficient supply of these metabolites may thus promote hypomethylation. Conversely, ethanol and a number of experimental and environmental carcinogens inhibit AdoMet synthesis, and are thus also promoters of hypomethylation (Anonymous, 1994). Different chemotherapeutic agents also interfere with the DNA methylation process. The nucleotide analog, 5-azacytidine (5-Aza-C), is the most studied inhibitor of DNA-MTase and has been used to treat solid tumors and myelogenous leukemia. Similarly, methotrexate interferes with methionine metabolism, and may thus also inhibit the methylation process. Conversely, cisplatinum and 5-fluorouracil promote hypermethylation (Zingg and Jones, 1997). From this it may be concluded that a combination of different genetic, environmental, and therapeutic factors inhibit as well as promote DNA methylation. There thus appears to be a balance between hypo- and hypermethy-

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Fig. 5 The DNA methylation balance. Disturbances in the DNA methylation process appear to be a central phenomenon in cancer development. Both hyper- and hypomethylation have been related to the neoplastic phenotypes, and different intrinsic and extrinsic factors affect the general level of DNA methylation. We therefore propose a balanced methylation level to represent an important homeostatic mechanism. This figure illustrates how a selection of factors may influence and disturb this balance. The genetic constitution, including somatic mutations, may contribute on either side of the balance. Specific factors may have different effects in different segments of the genome. AH, Aromatic hydrocarbons.

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lation related to the genetic composition and the microenvironment of a particular cell. Figure 5 illustrates how a selection of different factors may affect this DNA methylation balance.

B. Epigenetic Evolution The conventional way of illustrating CpG sites and DNA methylation patterns is illustrated in Fig. 6 (Qian and Brent, 1997).These patterns represent a molecular coding system that is both replicated and mutated in the somatic environment (Baylin et al., 1998). Like somatic mutations, replication of methylation patterns depends on cellular proliferation. A methylation pattern that involves a cellular growth advantage will thus increase in number, relative to other patterns. Current data thus imply that methylation patterns evolve and adapt through natural selection. Evolving DNA methylation patterns undoubtedly add a new level of complexity to our understanding of biology, and thus also to the neoplastic transformation process. Natural selection is, however, a universal concept, not limited to the DNA molecule. The principles of molecular evolution are therefore the key also to incorporating DNA methylation into a model of the carcinogenic process. Evolution of methylation patterns is a normal and essential mechanism in the human organism, not confined to neoplastic cells. The epigenetic evolution of methylation patterns may thus be viewed as a phenomenon analogous to the rearrangement and selection of antigen receptor genes. The evolution of the immune system and the epigenetic evolution of methylation patterns both represent evolution of molecular information in a closely regulated somatic environment, and are employed as adaptive tools for propagating germ-line DNA to the next generation of organisms. However, like somatic mutations, methylation patterns are reproduced solely based on their biochemical ability to interact with their environment. u

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Fig. 6 The epigenetic code. Schematic representation of a CpG island. Vertical lines represent CpG sites. Filled and open dots indicate the presence and absence of methylation, respectively. P1 and P2 illustrate alternative methylation patterns, representing hyper- and hypomethylation, respectively. This clearly shows that DNA methylation is a binomial coding system, similar to a computer language. The code is replicated and mutated in the somatic environment, and thus is subject to the general laws of natural selection. Evolution of this epigenetic code is essential to embryologic development and cell differentiation, and appears also to have a central role in the carcinogenic process.

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An epigenetic mutation that involves a growth advantage will thus proliferate even if it contradicts the organism’s intention to survive and reproduce. The epigenetic evolution process will consequently promote breakdown of mechanisms such as cell cycle control, apoptotic pathways, and immunorecognition. Evolving methylation patterns, in concert with evolving genetic variants, will thus inevitably drive cells toward neoplastic transformation and malignancy. As previously described, variation is, in addition to selection pressure, the key to evolution. Mutagens and repair deficiencies promote genetic variation. Similarly, exogenous and endogenous factors that disturb methylation fidelity and the DNA methylation balance (Fig. 5 ) will promote epigenetic variation. Exogenous factors such as ethanol and 5-aza-C may thus be classified as “epigenetic mutagens” (Klein and Costa, 1997), whereas endogenous defects in the methylation process result in different types of epigenetic instability (Baylin et al., 1998). The question of whether genetics or epigenetics is the primary cause of neoplastic transformation is controversial (Baylin et al., 1998). The reciprocal relationship between the two molecular structures (Fig. 4) suggests, however, that this is an impossible question to answer. The somatic evolution of DNA sequences and methylation patterns takes place in an environment determined by the dynamic interaction between genetics and epigenetics. These processes can consequently not be viewed independently, but are linked together so that the behavior of one system always depends on the state of the other. Both systems do, however, represent molecular coding systems that replicate and mutate in the somatic environment. Neoplastic transformation may thus be viewed as somatic evolution of molecular information, regardless of whether this information is DNA sequences or methylation patterns.

C. Methylation a n d Cancer Many cancers display global hypomethylation compared with normal tissues, and this has been proposed to represent the central phenomenon in the neoplastic process (Counts and Goodman, 1995). Demethylation has been related to overexpression of known oncogenes, and 5-aza-C promotes demethylation and cancer in animal model systems (Wainfan and Poirier, 1992). In both animals and humans methyl-deficient diets are related to increased risk of liver and colorectal cancer (Giovannucci et al., 1993), and feeding methyl-deprived rats with AdoMet leads to remethylation of DNA and reversal of the tumorigenic state (Christman et al., 1993). Conversely, many tumors also display regional hypermethylation associated with the inactivation of tumor suppressor genes, and increased mutation rate at CpG sites (Zingg and Jones, 1997). Furthermore, mice geneti-

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cally deficient in DNA-MTase exhibit resistance to colorectal tumorigenesis initiated by mutation of the APC tumor suppressor gene, and further demethylation by 5-aza-C treatment enhances this resistance (Laird et al., 1995). The terms hypo- and hypermethylation should be used with caution, because we have limited knowledge regarding the methylation status in the normal progenitors of neoplastic cells. The term normal cell is also difficult to define, considering that neoplastic cells evolve in a stepwise process, and might use decades to evolve recognizable phenotypes. However, current data strongly imply that both hypo- and hypermethylation contribute to the carcinogenic process. Tilting of the proposed DNA methylation balance (Fig. 4) in either direction thus appears to promote disturbances in cellular control mechanisms and promote neoplastic transformation.

D. Hypermethylation and the MIN Pathway Recent studies have revealed a striking correlation between genetic instability and DNA methylation in colorectal cancer. Colorectal cancer cell lines expressing the MIN phenotype were found to have increased capacity for silencing introduced retroviral genes by de novo methylation when compared to cell lines with CIN (Lengauer et al., 1997a). Others have found MIN to be strongly associated with tumor-specific hypermethylation of promotor region CpG islands (Ahuja et al., 1997). There is thus a close but yet undetermined relationship between MIN and this “hypermethylator phenotype.” Alterations in DNA methylation occur very early in the neoplastic process, and transfection studies have shown that the increased methylation capacity is not caused by absence of MMR activity (Lengauer et al., 1997a).The opposite relationship, i.e., the MIN pathway promoted by hypermethylation, is, however, supported by a number of observations. First of all, hypermethylation contributes to the accumulation of G/T mismatches through silencing of MGMT (Qian and Brent, 1997), and directly by promoting spontaneous deamination at CpG sites (Zingg and Jones, 1997).Second, epigenetic silencing of p16 and hMLH2, respectively related to breakdown of cell cycle control and MMR deficiency, is found at high frequency in sporadic MIN tumors (Ahuja et al., 1997; Kane et al., 1997).And third, methylation-related down-regulation of Fas may promote escape from immunologically induced apoptosis in colorectal cancer (Moller et al., 1994; Wang et al., 1997; Houghton et al., 1997). Hypermethylation may thus contribute to the selection pressure, as well as the epigenetic variation that promotes a number of steps in the MIN pathway (Fig. 3C). MIN tumors are predominantly localized to the proximal segments, whereas age-dependent hypermethylation increases toward the distal seg-

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ments of the colorectum (Issa et al., 1994). If hypermethylation promotes colorectal carcinogenesis, there must therefore be a location-specific factor that directs the process onto the MIN pathway. We have already suggested that bile components represent this factor, and have discussed the relationship between bile metabolites, proximal carcinogenesis, and methylation tolerance (tolerance to carcinogen induced 06-methylguanine, not to be confused with the CpG methylation). Methylating bile derivatives and age-related hypermethylation may thus be the combination that promotes the MMR deficiency. With reference to the reciprocal relationship between genetics and epigenetics (Fig. 4), these factors may have synergistic effects at different levels, and in combination provide both the selection pressure and the biochemical variation underlying the MIN phenotype (Fig. 3 , A and C).

VII. LOCATION-RELATED CARCINOGENIC ENVIRONMENTS As previously described, proximal and distal colorectal carcinogenesis are related to different genetic pathways. We have shown that these differences may be traced to specific classes of carcinogens. The biological basis for these apparent differences in carcinogenic environments is, however, unclear, although a number of mechanisms have been suggested. Different lines of evidence have implemented bile acids in colorectal carcinogenesis, and a number of factors related to increased risk of colorectal cancer may act through their effect on bile metabolism (reviewed by Nagengast et al., 1995).Each day approximately 20% of the bile acid pool escapes reabsorption and the enterohepatic circulation. In the colon, bile acids are metabolized by the anaerobic bacterial flora. First, deconjugation takes place and the amino acid molecule on the carboxyl group is removed. Second, the primary bile acids, cholic acid and chenodeoxycholic acid, are dehydroxylated into the secondary bile acids, deoxycholic acid and lithocholic acid, respectively. Deoxycholic acid is partly reabsorbed in the colon, while the further bacterial degradation in the bowel produces the tertiary bile acids. In combination this implies an alteration in the composition of bile acids throughout the length of the colorectum, and this gradient may thus contribute to different carcinogenic environments in the proximal and distal segments (McMichael and Potter, 1985). Interestingly, it is N-nitrosation of conjugated bile acids entering the proximal colon that produces analogs to the niethylating carcinogens MNNG and MNU (Puju et al., 1982; Busby et d., 198s). This is thus in good agreement with our model, and may explain the proximal location of tumors with MIN.

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Gastric cancer reflects a strikingly similar relationship between the MIN phenotype and tumor location. MIN has been consistently related to the distal stomach (the antral location), and negatively associated with TP.53 mutations, which are most frequent in the proximal locations (Strickler et al., 1994; Seruca et al., 1995; Renault et al., 1996; Gleeson et al., 1996; Ottini et al., 1997; Wu et al., 1998).Considering that the distal stomach is anatomically most susceptible to duodenal reflux, this adds another circumstantial link between the MIN phenotype, bile acids, and tumor location. Reduced transit time and increased fecal concentrations toward the rectum have also been related to the observed differences between proximal and distal cancers (McMichael and Potter, 1983, 1985). Much of the direct-acting mutagenic activity in human feces can be correlated to fecapentanes (reviewed by Povey et al., 1991). Different studies indicate that these compounds are BAF carcinogens that induce NER and single-strand breaks in DNA (Plummer et al., 1986; Zarkovic et al., 1993). Increasing concentrations of fecapentanes toward the distal colorectum may thus be related to the high frequency of CIN in this segment. Phenotypic differences between cells in the proximal and distal mucosae have also been suggested to promote different carcinogenetic pathways. The current view is that exogenous carcinogens such as heterocyclic and aromatic amines are activated by different metabolic processes and reach the colorectal mucosa by its blood supply (Minchin et al., 1993; Lang et al., 1994). Cytochrome P-4501A2 in liver, and N-acetyltransferase in liver and colon, catalyze this activation process, and “rapid” phenotypes of these enzymes have been related to an increased risk of colorectal cancer (Minchin et al., 1993; Lang et al., 1994; Roberts-Thomson et al., 1996). Elevated activity of Nacetyltransferase in the distal colorectum might thus contribute to an overload of activated BAF carcinogens in this segment. However, animal studies suggest a decrease in N-acetyltransferase activity toward the rectum (Minchin et al., 1993). Similarly, age-related hypermethylation increases toward the rectum (Issa et al., 1994).This is thus also opposite from what might be expected if these phenotypic differences in the mucosae were the cause of different susceptibility to carcinogenetic pathways. However, it is exactly what might be expected if the organism and the mucosae have adapted to different carcinogenic environments in the proximal and distal colorectum. These phenotypic differences between the proximal and distal mucosae are thus more likely consequences of carcinogenic environments rather than causes of cancer susceptibility. In conclusion, we therefore propose a model whereby the proximal and distal mucosae are exposed to different carcinogenic environments set up by a combination of bile acid metabolites, fecal mutagens, and circulating carcinogens (Fig. 3D).

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VIII. CONCLUSIONS AND PERSPECTIVES In this article we have presented a general model of neoplastic transformation based on evolutionary principles and established molecular mechanisms related to the carcinogenic process. We then applied this model as a tool for organizing a large amount of data related to colorectal cancer. Somewhat surprisingly we found that this approach resulted in a pattern wherein lines may be drawn from epidemiological risk factors through clinicopathological variables, and further to molecular biology. In conclusion, we found that different genetic alterations related to the MIN and CIN pathways, respectively, may be traced to different carcinogenic environments. A number of studies in the field of DNA methylation have emphasized the epigenetic dimension of neoplastic transformation. Here we have described how this phenomenon may also be viewed in the light of natural selection, and have presented a model that attempts to integrate the genetics and epigenetics related to the MIN pathway. Besides addressing the location differences related to colorectai carcinogenesis, this model provides a possible explanation for the association between MIN and hypermethylation. In conclusion, we propose the MIN phenotype to be promoted by a combination of hypermethylation of DNA and methylating carcinogens. This hypothesis is currently being tested in an animal model system. When organizing data in this manner there is an unavoidable bias toward data that fit the predetermined hypothesis, and a number of contradicting results are not referred to in this work. We are, however, confident that our model is in agreement with the major trends in the data, and believe that this evolutionary view to carcinogenesis may serve as a framework for integrating other data related to the neoplastic process. In cancer-related research there is growing awareness that neoplastic transformation must be viewed as an evolutionary process (Klein, 1998). We have addressed this issue and present a model that integrates evolution and carcinogenesis at the molecular level. This model offers new perspectives to colorectal carcinogenesis and relates the neoplastic process to the basic evolutionary concept of biology.

ACKNOWLEDGMENTS Supported by the Norwegian Research Council for Science and the Humanities (JB). We express our gratitude to Paula DeAngelis and Ole Petter F. Clausen for their critical reading of the manuscript.

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McSherry, C. K., Cohen, B. I., Bokkenheuser, V. D., Mosbach, E. H., Winter, J., Matoba, N., and Scholes, J. (1989).Human Genet. 49,6039-6043. Meling, G. I., Lothe, R. A., Berresen, A. L., Graue, C., Hauge, S., Clausen, 0. P., and Rognum, T. 0. (1993).Br. J. Cancer 67, 93-98. Meuth, M. (1996). Cancer Surv. 28,33-46. Minchin, R. F., Kadlubar, F. F., and Ilett, K. F. (1993).Mutat. Res. 290, 35-42. Mitra, G., Pauly, G. T., Kumar, R., Pei, G. K., Hughes, S. H., Moschel, R. C., and Barbacid, M. (1989). Curcinogenesis 86, 8650-8654. Modrich, P. (1997).J. Biol. Cbem. 272,24727-24730. Moerkerk, P., Arends, J. W., van Driel, M., de Bruine, A., de Goeij, A., ten Kate, and J. (1994). Human Genet. 54,3376-3378. Moller, P., Koretz, K., Leithauser, F., Bruderlein, S., Henne, C., Quentmeier, A., and Krammer, P. H. (1994).Int. J. Cancer 57,371-377. Monk, M. (1995).Dev. Genet. 17, 188-197. Murnane, J. P. (1995). Cancer Metastasis Rev. 14, 17-29. Nagengast, F. M., Grubben, M. J., and van Munster, I. P. (1995). Eur. J. Cancer 31A, 1067-1070. Nakazawa, H., English, D., Randell, P. L., Nakazawa, K., Martel, N., Armstrong, BK, and Yamasaki, H. (1994).Proc. Natl. Acud. Sci. U.S.A. B91, 360-364. Ottini, L., Palli, D., Falchetti, M., D’Amico, C., Amorosi, A., Saieva, C., Calzolari, A., Cimoli, F., Tatarelli, C., De Marchis, L., Masala, G., Mariani-Costantini, R., Cama, A., Renault, B., Calistri, D., Buonsanti, G., Nanni, O., Amadori, D., Ranzani, G. N., Gleeson, C. M., Sloan, J. M., McGuigan, J. A., Ritchie, A. J., Weber, J. L., Russell, S. E., Seruca, R., Santos, N. R., David, L., Constancia, M., Barroca, H., et al. (1997).Human Genet. 57,4523-4529. Parsons, R., Myeroff, L. L., Liu, B., Willson, J. K. V., Markowitz, S. D., Kinzler, K. W., and Vogelstein, B. (1995). Cancer Res. 55, 5548-5550. Pieper, R. 0. (1997).Pbarmacol. Tber. 74,285-297. Plummer, S. M., Grafstrom, R. C., Yang, L. L., Curren, R. D., Linnainmaa, K., and Harris, C. C. (1986). Curcinogenesis 7,1607-1609. Posnett, D. N. (1995).Ann. N.Y Acad. Sci. 756,71-80. Povey, A. C., Schiffman, M., Taffe, B. G., and Harris, C. C. (1991).Mutat. Res. 259,387-397. Puju, S., Shuker, D. E., Bishop, W. W., Falchuk, K. R., Tannenbaum, S. R., and Thilly, W. G. (1982). Cancer Res. 42,2601-2604. Qian, X. C., and Brent, T. P. (1997).Human Genet. 57,3672-3677. Rampino, N., Yamamoto, H., Ionov, Y., Li, Y., Sawai, H., Reed, J. C., and Perucho, M. (1997). Science 275,967-969. Ramsahoye, B. H., Davies, C. S., and Mills, K. I. (1996).Blood Rev. 10, 249-261. Renault, B., Calistri, D., Buonsanti, G., Nanni, O., Amadori, D., and Ranzani, G. N. (1996). Human Genet. 98,601-607. Roberts-Thomson, I. C., Ryan, P., Khoo, K. K., Hart, W. J., McMichael, A. J., and Butler, R. N. (1996).Lancet 347,1372-1374. Robey, E., and Fowlkes, B. J. (1994).Annu. Rev. Immunol. 12,675-705. Sancar, A. (1995).J. Biol. Cbem. 270,15915-15918. Sanchez, Y., and Elledge, S. J. (1995).BioEssays 17, 545-548. Seruca, R., Santos, N. R., David, L., Constancia, M., Barroca, H., Carneiro, F., Seixas, M., Peltomaki, P., Lothe, R., and Sobrinho-Simoes, M. (1995).Int. J. Cancer 64, 32-36. Shackney, S. E., and Shankey, T. V. (1997). Cytometry 29, 1-27. Shirtliff, N., and Bird, R. P. (1996). Carcinogen. 17,2093-2096. Simpson, A. J. (1997).Adv. Cancer Res. 71,209-240. Slattery, M. L., Schaffer, D., Edwards, S. L., Ma, K. N., and Potter, J. D. (1997).Nutr. Cancer 28,52-62.

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Antitumor Immunity at Work in a Melanoma Patient Pierre G. Coulie, Hideyukl Ikeda, Jean-FranqoisBaurain, and Rita Chiari Catholic University of Louvain Cellular Genetics Unit B-1200 Brussels, Belgium

I. Introduction 11. Melanoma Patient LB33 and Melanoma Cell Lines 111. Autologous CTLs against MEL.A Cells A. Frequency of CTLs against MEL.A B. Anti-MEL.A CTL Clones C. Presence of Numerous Antigens on MEL.A IV. Identification of Antigens Recognized by CTLs on MEL.A Cells A. Antigen LB33-B B. Antigen LB33-A C. Antigens LB33-K and LB33-L V. The MEL.B Cells A. In Vivo Immunoselection? B. Anti-MEL.B CTL Clones C. Identification of the Antigen Recognized by CTL 17 VI. A New Class of Antitumor CTL A. Presence of NK Inhibitory Receptors on CTL 17 B. A Role in Antitumor Defense for CTLs Expressing NK Inhibitory Receptors? VII. Conclusions References

This review covers the results obtained so far with a chronological analysis of the antitumor cytolytic T lymphocyte (CTL) cell response of a melanoma patient who enjoys an unusually favorable evolution. Two melanoma cell lines, MEL.A and MEL.B, were derived from metastases removed from the patient in 1988 and 1993, respectively. The patient developed a very strong CTL response against the MEL.A cells. Several antigens on these cells, presented by various HLA class I molecules, result from point mutations present in the genome of the tumor. The MEL.B cells, on the other hand, resist lysis by these CTLs because they have lost expression of most HLA class I molecules, suggesting that they were selected in vivo by the anti-MEL.A CTLs. New CTLs recognize MEL.B cells specifically, however. Analysis of their specificity indicates that they carry inhibitory receptors similar to those present on natural killer (NK) cells. These results illustrate the relationship between a tumor and the immune system in vivo over a period of several years. They are discussed in the context of the recent identification of many human

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Copyright 0 1999 by Academic Press. All rights of reproductionin any form reserved.

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tumor antigens recognized by CTLs, and the perspectives of specific immunotherapy opened up by these discoveries. 0 1999 Academic Press.

I. INTRODUCTION The identification of antigens recognized by T lymphocytes on cancer cells has prompted renewed interest in tumor immunology. The pioneering work of Boon and colleagues with the murine mastocytoma P815 cells led to the characterization of the first nonviral antigens that were recognized by cytolytic T lymphocytes (CTLs) mediating rejection responses (Boon, 1992). These studies made use of a genetic approach to the identification of antigens targeted by CTLs, a strategy that could then be transposed to human tumors. CTL clones that show specificity for autologous tumor cells can be derived from the blood or tumor-infiltrating lymphocytes of melanoma patients (Boon et al., 1994). They can be maintained in culture for long periods of time and represent tools that are crucial to the identification of tumor antigens. Dozens of antigens recognized by such antitumor CTL clones have now been identified, and the list continues to grow. Based on the pattern of expression of the parent protein, the tumor antigens can be divided into five groups (Van den Eynde and van der Bruggen, 1997) (Fig. 1): 1. Antigens encoded by genes that are silent in most normal tissues but are activated in different types of tumors. Prototype antigens of this group are those encoded by the MAGE genes. 2 . Differentiation antigens present on melanomas and encoded by genes, such as tyrosinase, that are expressed only in melanocytes and in melanomas. 3 . Antigenic peptides derived from regions of ubiquitous proteins that are mutated in the tumor cells. Because they have been found in several independent tumors and have a demonstrated effect on the activity of the encoded proteins, some of these mutations may be involved in oncogenesis. 4. Antigens encoded by nonmutated genes expressed in normal tissues and overexpressed in many tumors. 5. Viral antigens, such as those encoded by human papilloma viruses. Although it seems easier to derive autologous tumor-specific CTLs against melanomas than against other tumors, antigenicity is not restricted to melanomas: several genes encoding tumor-specific antigens have been found to be expressed by many types of tumors. It is becoming increasingly clear, therefore, that most if not all human tumors carry antigens. One may then

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ask why the immune system does not eliminate these antigenic tumor cells. Many explanations have been proposed, pertaining either to an absence of stimulation of the immune system (tolerance, ignorance, no access of lymphocytes to the tumor cells, presence of suppressive soluble factors, etc.), or to tumor cells escaping immune attack (loss of antigen expression, Fas-mediated killing of T cells, etc.). We surmise that the relationships between tumors and the immune system, when analyzed and understood, will display great diversity. It is likely, however, that within this diversity, several mechanisms whereby tumors escape immune surveillance will be of a more general interest than others. A valuable approach to identify such mechanisms is to carry out longitudinal analyses of antitumor immune responses in the very few patients who enjoy a favorable clinical course despite recurrent metastases, with the aim of understanding which role has been played by the immune system in determining their clinical evolution.

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11. MELANOMA PATIENT LB33 AND MELANOMA CELL LINES A female patient presented in 1985, at the age of 40, with a superficial, spreading, primary melanoma of the left leg (Fig. 2). No other tumoral localization was found and the tumor was excised (Clark III-IV, Breslow 1.3 mm). In 1987 an ipsilateral inguinal lymphadenopathy was observed. The treatment involved surgery (two out of three inguinal lymph nodes proved to be invaded by tumor cells) and chemotherapy [seven courses of vincristine, actinomycin D, dacarbazine, and l-(2-chloroethyl)-3-cyclohexyll-nitrosourea (CCNU)]. A subcutaneous achromic nodule (3.4 mm) appeared in January 1988 in the left paravertebral area. The patient received three courses of IL-2 (3.5 X lo6 U/day for 5 days) and IFN-y (16.8 X lo6 U/day for 5 days). After this treatment, the nodule had increased in size and showed inflammatory signs; it was excised. In February 1989, a left supraclavicular metastasis and a right axillary lymph node metastasis were observed. The patient received four courses of IL-2 alone (3.5 x lo6 U/day for 5 days). The first adenopathy regressed completely, whereas the second continued to grow and was excised. The patient then had no sign of disease for 4 years, during which period she was vaccinated repeatedly with autologous melanoma cells, and received no other treatment. Each vaccine consisted of two to five intradermal inoculations of lethally irradiated autologous tumor cell clones isolated from cell line MEL.A, derived from the metastasis removed in 1988. These clones were derived from tumor cells that had received a mutagenic treatment with N-methyl-N’-nitro-Nnitrosoguanidine. The rationale for this form of autologous vaccine came from the work on P815 cells: immunogenic tumor variants, derived from P815 by mutagen treatment, proved capable of eliciting a specific immune protection against the original P815 cells (Van Pel et al., 1983).N o evidence was obtained, though, that in the case of patient LB33 the mutagenized tumor cells were more antigenic than the parental MEL.A cells. In 1993 the patient suffered from anemia and a tumoral localization was found in the wall of the small bowel, a not unusual localization of melanoma metastases (Elsayed et al., 1996; Klaase and Kroon, 1990). The tumor was resected. Four months later, a left subclavicular metastasis appeared and was excised, although not completely. The patient has been disease free since then. Since 1994, the autologous vaccines have consisted of a mixture of mutagenized MEL.A cells, as before, and MEL.B cells, derived from the intestinal metastasis. A notable clinical characteristic of melanoma is its unpredictable clinical course. However, the median survival time for patients with two distant metastases, the clinical status of patient LB33 in 1989, is only 4-6 months

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(Balch et al., 1992), and less than 5 % of patients with a disease corresponding to that of patient LB33 in 1993-1994 are expected to be alive after 5 years (Buzaid et al., 1997). Altogether, the clinical evolution of patient LB33 seems to be exceptionally favorable. But this type of evolution is not unique: there are several descriptions of patients with long-term survival despite recurrent metastases treated by surgery (Brand et al., 1997; Huffman and Sterin, 1973; Wong et al., 1993). Three cell lines could be derived from tumor material excised from patient LB33. Cell line MEL.A was derived from the subcutaneous metastasis removed in 1988, grafted into nude mice, and subsequently adapted to culture. Cell lines MEL.B and MEL.C were obtained by adapting directly to culture the intestinal and cutaneous metastases removed in 1993 and 1994, respectively. MEL.A cells are adherent and grow with a doubling time of lt40 hr, whereas 1MEL.B and MEL.C cells barely adhere to tissue culture flasks and grow more slowly than MEL.A. We could not exclude a priori that MEL.A, MEL.B, and MEL.C were derived from independent tumors resulting from distinct transformation events occurring at several years’ distance. We observed, however, that a mutation in the ubiquitously expressed gene MUM1, which is responsible for the expression of an antigen on the tumor (see below), was found in DNA extracted from all three cell lines. This mutation is not present in the normal cells of the patient; nor, we infer, is it frequently found in melanomas, because none of the 300 tumor samples that we tested carried it. We conclude, therefore, that MEL.A, MEL.B, and MEL.C derive from the same tumor.

111. AUTOLOGOUS CTLs AGAINST MEL.A CELLS

A. Frequency of CTLs against MEL.A A first indication of the presence of a strong antitumor immunity in patient LB33 came from a frequency analysis of antitumor CTLs. We have described a method to evaluate the frequency of precursors of antitumor CTLs (CTLp) in the blood of melanoma patients (Coulie et al., 1992). These frequencies varied greatly from patient to patient, but among 15 cancer patients, patient LB33 clearly stood out with an antitumor CTLp frequency of approximately 1/1000 blood mononuclear cells, a value comparable to those obtained with control stimulations of blood lymphocytes with allogenic tumor cells. The first blood sample with this frequency of CTLp was collected in 1990, before the injections of irradiated autologous tumor cells, suggesting that a strong CTL response had been induced by the tumor. Blood samples collected in 1994 and 1996 also contained this high number of tumor-

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specific CTLs. The persistence of this strong antitumor immunity may seem a little surprising, especially long after periods of clinically detectable disease. It is possible that the CTLs were periodically restimulated in vivo either by tumor cells of micrometastases, even during “disease-free” intervals, or by the irradiated autologous tumor cells used as a vaccine from 1990 on.

B. Anti-MEL.A CTL Clones To further our understanding of the antimelanoma immune response, we decided to derive tumor-specific CTL clones, making use of a standard protocol of autologous mixed lymphocyte-tumor cell culture (MLTC). Blood peripheral mononuclear cells (PBMCs) were stimulated each week with irradiated MEL.A cells and IL-2, and around day 20 the responder cell population was able to lyse specifically MEL.A cells. CTL clones, derived from these populations of responder cells by limiting dilution, were restimulated each week by the addition of IL-2, irradiated MEL.A cells, and irradiated allogeneic EBV-transformed B cells as feeder cells. More than 100 stable CTL clones were derived from MLTC experiments performed with several batches of blood lymphocytes collected between 1990 and 1992. All these CTL clones expressed the CD8 molecule and showed specificity for MEL.A cells inasmuch as they lysed these cells but did not recognize autologous EBVtransformed B cells, autologous T lymphocytes activated with phytohemagglutinin A and IL-2, or the prototype NK target, K562.

C. Presence of Numerous Antigens on MEL.A To characterize the antigens of MEL.A, antigen-loss variants were selected in vitro with CTL clones and recognition of these variants by the complete panel of CTLs was examined. The procedure of immunoselection is straightforward: approximately 3 X lo7 tumor cells are mixed with a similar number of cells of a given CTL clone. A few hours are required for most of the tumor cells to be killed. They are then gently washed off the culture flasks together with the CTLs and the surviving adherent tumor cells are amplified for the next round of selection. Usually, after three such rounds, the population of melanoma cells is completely resistant to lysis by the CTLs, and it can be cloned by limiting dilution. On the basis of the pattern of recognition of the various antigen-loss variants by the panel of CTL clones, four antigens could initially be defined and named (LB33-A, LB33-B, etc.). An obvious mechanism whereby tumor cells become resistant to lysis by CTLs is the loss of expression of HLA molecules. Patient LB33 was typed by serology as HLA-A24, A28, B13, B44, Cw6, and Cw7. cDNA clones encoding

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these six HLA class I alleles were cloned from mRNA extracted from MEL.A cells. We could then set up reverse transcriptase-polymerase chain reaction (RT-PCR) assays for each class I allele to measure their expression semiquantitatively on RNA extracted from each antigen-loss variant. The results were confirmed by labeling the cells with monoclonal antibodies recognizing the A24, A28, and B13 alleles. Out of the five antigen-loss variants derived from MEL.A, three proved to have lost expression of HLA class I genes (Fig. 3 ) . The A-B-D- variant had lost expression of one complete haplotype (A28, B44, Cw7) and the C- variant had lost expression of the other haplotype (A24, B13, Cw6). Analysis of genomic DNA indicated that gene deletions were responsible for these losses of expression of HLA haplotypes. The HLA molecules that presented each of these antigens were identified by transfecting into the HLA haplotype-loss variants cDNA clones encoding each of the missing alleles. In the course of these experiments we realized that the panel of anti-C CTLs actually consisted of two groups of clones, restricted by either HLA-B13 or HLA-Cw6. Former antigen C was therefore split into Ca and Cb (Lehmann et al., 1995). Later on, other MLTC experiments were performed and new antigenic specificities were defined, leading to the characterization of at least nine antigens recognized by autologous CTLs on MEL.A (Fig. 4). Four different HLA class I molecules present those antigens, whereas no tumor-specific CTLs could be found that recognized antigens presented by either HLA-A24 or Cw7 molecules. The efficiency of obtention of antigen-loss variants in vitro indicates that in the starting population of MEL.A cells the frequency of cells that resist a given CTL is approximately 1OW’. In addition, because these immunoselections were performed on tumor cell clones derived from MEL.A by limiting dilution and amplified for only 10-20 passages before the selection, this value of is an underestimation of the proportion of the same cells in vivo. Nevertheless, it should be borne in mind that, in vivo, an escape variant has to lose expression of several antigens, recognized by different CTLs. Is it surprising to find that at least nine antigens are targeted by CTLs on the autologous melanoma cells? First, one could argue that these CTLs were raised through primary in vitro stimulations against MEL.A and do not result from an in vivo immunization against the tumor. When the experimental conditions of primary in vitro stimulation of CTLs were set up for defined antigenic peptides encoded by MAGE genes, the obtention of CTL was found to require IL-6 and 1L-12, and the estimated frequency of precursors was 1 in 5.106 CDS cells (Herman et al., 1996; van der Bruggen et al., 1994a). In view of these results, it seems unlikely that the classical MLTC experiments, starting with 1 to 2.106 CD8 lymphocytes and IL-2 alone, allowed the stimulation of naive antitumor CTLs. Thus, the strong in vitro CTL response against MEL.A probably reflects an immunization against the tumor in vivo. Second, there are few extensive analyses of antitumor CTL

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responses that can be compared with the anti-MEL.A response. Two other melanoma lines, MZ2-MEL and SK29-MEL, have been studied in detail with a n experimental approach similar to that described here (Van den Eynde et al., 1989; Wolfel et al., 1993). Autologous CTLs were found to recognize at least seven distinct antigens on MZ2-MEL and at least five on SK29MEL. It may not be a coincidence that, like patient LB33, patients M Z 2 and SK29 also enjoy an exceptionally favorable clinical course, being disease free more than 10 years after a metastatic melanoma. It is noteworthy that these two patients also received inoculations of autologous irradiated melanoma cells. Finally, we have to make a clear distinction between the number of antigens present on tumor cells and the number of different CTLs against these antigens. As many of the antigens recognized by CTL on melanomas were identified, the corresponding genes were found to be expressed by a large proportion of these tumors. Thus, most melanomas probably present many antigens to the immune system, and we have so far no reasons to believe that MEL.A cells bear more antigens than d o the other melanomas. The very strong antitumor CTL response of patient LB33 appears therefore to be a consequence, not so much of the presence of an unusually high number of different antigens on the tumor, but rather of the efficient stimulation of the immune system against several of these antigens. In other words, the immunogenicity of MEL.A cells is more remarkable than its antigenicity.

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IV. IDENTIFICATION OF ANTIGENS RECOGNIZED BY CTLs ON MEL.A CELLS A. Antigen LB33-B The first antigen identified on MEL.A cells was antigen LB33-B, presented by HLA-B44 molecules (Coulie et al., 1995).We used a cDNA transfection approach that had been used previously to identify several other tumor antigens (Coulie et al., 1994). Briefly, a cDNA library prepared with mRNA from MEL.A cells was cloned into an expression vector containing the SV40 origin of replication. Such plasmids multiply to a very high copy number in cells such as COS that express the SV40 gene product, a DNA helicase that binds to the SV40 origin of replication. The library was divided into 700 pools of approximately 100 bacteria, and DNA from each pool was cotransfected with that of a cloned HLA-B44 cDNA into microcultures of COS cells. Twenty-four hours later, the transfected cells were tested for the expression of antigen LB33-B by adding the anti-B CTL clone to each microculture, and measuring the production of tumor necrosis factor by the CTL. One pool of cDNA proved positive. It was subcloned, and a cDNA clone was isolated that transferred the expression of antigen LB33-B. The sequence of this cDNA showed no significant homology to sequences recorded in data banks. The corresponding gene was found to be expressed ubiquitously. The antigenic peptide was localized by digesting the cDNA with an exonuclease and transfecting the truncated cDNA clones together with the HLA-B44 cDNA into COS cells. One peptide of nine amino acids was found that sensitized HLA-B44 cells to lysis by the anti-B CTL. Because the gene coding for this antigenic peptide was expressed ubiquitously, including autologous Epstein-Barr virus (EBV)-transformed B cells that were not lysed by the CTL, we considered the possibility that the tumor might carry a point mutation in the gene encoding the antigen. We compared the sequence of the gene in DNA extracted from MEL.A cells or from blood mononuclear cells of patient LB33. The sequence found in the tumor cells corresponded to that of the cDNA clone encoding the antigen, whereas the sequence found in normal cells differed by a single nucleotide, replacing a serine with an isoleucine at position 5 of the antigenic peptide. The normal peptide was not at all recognized by the anti-B CTL clone. Competition experiments indicated that this normal peptide could bind to HLA-B44 molecules with an affinity similar to that of the mutated peptide, suggesting that the mutated amino acid in the antigenic peptide is an essential part of the epitope recognized by the antitumor CTLs. The gene encoding antigen LB33-B was provisionally named MUM-1 (melanoma ubiquitous mutated). The mutated MUM-1 al-

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leie could not be found in DNA extracted from the B- antigen-loss variant, suggesting that these cells resist lysis by the anti-B CTLs because they carry a deletion involving the mutated allele. Antigen LB33-B was the first human tumor antigen encoded by a mutated gene to be identified. Subsequently, point mutations in the genes encoding CDK4 (Wolfel et al., 1995), p-catenin (Robbins et al., 1996), HLA-A2 (Brandle et al., 1996), CASP-8 (Mandruzzato et al., 1997), and KIAA0205 (GuCguen et al., 1998) were also found to be responsible for the expression of tumor antigens recognized by autologous CTLs. The mutations in the CDK4 and p-catenin genes are most probably involved in oncogenesis, because they were found in several independent tumors and they modify the function of the corresponding protein. On the other hand, none of approximately 300 samples of tumors, including melanomas and other types of tumors, was found to contain the same MUM-1 mutation as MEL.A cells, making it unlikely that this mutation played a role in the transformation of the tumor. When we analyzed the sequence of other MUM-1 cDNA clones that hybridized with the cDNA encoding the antigen, we realized that the latter cDNA contained intronic sequences (Fig. 5). It appears that in all cells a fraction of the MUM-1 mRNA is incompletely spliced and still contains intron 2. The sequence coding for the antigenic peptide actually straddles the junc-

...

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Fig. 5 Structure of the 5' end of gene MUM-1 encoding antigen LB33-B. Exons and introns are represented as gray and white boxes, respectively. Exonic or intronic sequences are in uppercase and lowercase type, respectively. The sequence of the antigenic peptide recognized by the anti-B CTL clone is boxed.

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tion between an exon and this following intron, which is kept in the mRNA. The point mutation that generates the antigen is located in position 6 of the intron, and is followed immediately by a stop codon, suggesting that the antigenic peptide derives from the C terminus of a shortened version of the MUM-1 protein. We have no indication as to the function of protein MUM1, and we do not know whether the truncated version of the protein is functional. Because the MUM-1 mRNA species containing intron 2 is ubiquitous, the lack of splicing of this intron is independent of the point mutation. Therefore, the specificity of the antigen for the tumor is caused only by the mutation and not by a splicing difference.

B. Antigen LB33-A We mentioned that a high frequency of anti-MEL.A CTLp was found in the blood of patient LB33 and that CTL clones derived from anti-MEL.A MLTC recognized at least nine different antigens. To examine the distribution of these various specificities in the population of CTLs, we stimulated limited numbers of blood lymphocytes with irradiated MEL.A cells, in multiple mini-MLTCs, and tested the lytic activities of the responding populations against the panel of antigen-loss variants. We found that more than 95% of the anti-MEL.A CTLs recognize antigen LB33-A, indicating that antigen A is immunodominant in vivo. This preponderance of the anti-A CTL response was found in several blood samples collected between 1990 and 1997. To analyze the diversity of the anti-A CTL response we isolated cDNA clones encoding the T cell receptor (TCR) a and p chains of a dozen anti-A CTL clones derived from several MLTC experiments. The sequences of these TCRs proved to be identical, indicating that the very strong anti-A CTL response in patient LB33 results from the amplification of a single CTL clone. It is possible that this is a consequence of the injections of IL-2 in 1988 and 1989: an anti-A CTL activated by its antigen could have been sent through many more cycles of division than in normal conditions by a very high systemic concentration of IL-2. It may seem surprising that the dominance of one antigen was maintained over several years, despite repeated immunizations with tumor cells that present several other antigens, which also stimulated a CTL response. A possible interpretation is that once a dominance is installed it is maintained in the following immunizations (Brichard et al., 1995).The increased proportion of anti-A CTLs will make it very likely that, at each vaccination, anti-A CTLs encounter the tumor cells and start to proliferate before the other CTLs. If all CTLs proliferate at the same rate until the tumor cells are eliminated, the bias is maintained. Interestingly, in vitro, the anti-A CTL clones proliferate significantly more than the other anti-MEL.A CTLs.

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We recently identified antigen LB33-A, presented by HLA-A28 molecules (Baurain, 1998). It is encoded by a new gene that is expressed ubiquitously. This gene appears to belong to a family of genes that can encode helicases containing a DExH motif, which seems important for ATP hydrolysis (Subramanya et al., 1996). One copy of the gene is mutated in MEL.A cells, and the point mutation changes one amino acid in the antigenic peptide. We do not know yet whether mutations in this new gene are found in other tumors. It is worth noting that an antigen recognized by CTLs on a murine UV-induced tumor was found to be encoded by a mutated form of the RNA-helicase p68 (Dubey et al., 1997). Because several helicases are involved in the process of DNA repair (Friedberg, 1996), it is possible that mutations in those genes contribute to an increased accumulation of mutations in tumor cells, some of which are oncogenic or immunogenic.

C. Antigens LB33-K and LB33-L The two antigens, LB33-K and LB33-L, presented by HLA-B44 and Cw6 molecules, respectively, have been identified by Chiari ( 1998). Both antigens proved to be encoded by the same cDNA clone, which was found in a library prepared with RNA from MEL.A cells. This cDNA corresponds to a new gene that is also expressed ubiquitously. Again, a point mutation is responsible for the expression of the antigen. The antigenic peptides presented to CTLs by HLA-B44 or. by HLA-Cw6 molecules are different, but their sequences overlap and contain the same mutated amino acid. It is remarkable that a single amino acid difference leads to the expression of two new immunogenic peptides at the surface of the tumor cells. Up to now, four antigens recognized by autologous CTLs on MEL.A cells have been identified and all of them result from point mutations. It is somewhat surprising that MEL.A cells, which seem quite immunogenic, did not trigger a significant CTL response against antigens of the other categories mentioned in Section I. MEL.A cells express genes MAGE-2, -3, and -6, however, and a peptide encoded by gene MAGE-3 and presented by HLAB44 molecules is efficiently processed and presented at the cell surface because an anti-MAGE-3.B44 CTL clone lyses MEL.A cells (Herman et d., 1996). In contrast, MEL.A cells do not express any of the genes encoding melanocyte differentiation antigens. We will try to find out whether the predominant CTL response against mutated antigens on MEL.A is the result of a higher number of mutations in the genome of these cells compared to other melanoma cells. It is interesting to compare the profile of antigens recognized by CTLs on the autologous melanoma cells of two patients with an exceptionally favorable clinical course: LB33 and MZ2. Contrary to the LB33-MEL antigens,

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seven of eight antigens identified on MZ2-MEL cells are encoded by nonmutated genes that are specifically expressed in tumors: MAGE-1, MAGE3, BAGE, and GAGE (Boel et al., 1995; Gaugler et al., 1994; Traversari et al., 1992; Van den Eynde et al., 1995; van der Bruggen et al., 1994b). The eighth antigen is encoded by the tyrosinase gene (Brichard et al., 1996). It is possible that genes MAGE-3, BAGE, and GAGE are expressed in tumors as the result of a global demethylation of the genome, as shown for gene MAGE-1 (De Smet et ul.., 1996). One may speculate that in patient MZ2 the DNA of a fraction of the tumor became heavily demethylated, leading to a strong expression of genes MAGE, BAGE, and GAGE and hence to the stimulation of the corresponding CTL. Even though conclusions cannot be drawn from only two cases, these observations suggest that different tumors may display a highly immunogenic phenotype through very different mechanisms, possibly controlled by different genes.

V. THE MEL.B CELLS A.

In Vivo Immunoselection?

A salient feature of the clinical course of patient LB33 is the disease-free interval between 1989 and 1993, which was associated with a very strong antitumor CTL response detected in blood samples collected between 1990 and 1993. The patient then relapsed with an intestinal metastasis that was completely excised and from which a second cell line could be derived: MEL.B. We reasoned that if the anti-MEL.A CTL response had contributed to this favorable clinical evolution, new metastases would probably not present to CTLs the antigens carried by MEL.A. We tested the sensitivity of MEL.B to lysis by a panel of anti-MEL.A CTL clones. The results were strikingly clear: MEL.B cells were completely resistant to the lytic activity of all these CTL clones (Lehmann et ul., 1995) (Fig. 6). Analysis of HLA expression by RT-PCR and with monoclonal antibodies indicated that MEL.B cells had lost expression of all HLA molecules with the exception of HLA-A24. The mechanism of this HLA loss is only partially understood: MEL.B cells have a gene deletion involving the complete A28-B44-Cw7 haplotype. For B13 and Cw6, a faint signal is present with RT-PCR, and we assume that expression at the cell surface is very low. Interestingly, HLA-A24, the only class I allele that remains expressed at a high level on MEL.B cells, was, with Cw7, the only class I molecule against which no CTLs could be derived with MEL.A cells. The loss of all the HLA molecules that presented antigens to CTLs, and the persistence of an HLA molecule that did not present antigens, suggest that MEL.B cells were selected irt vivo by the anti-MEL.A CTL response.

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CTL clones (HLA restriction)

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The loss of expression of HLA molecules is often observed in tumors (Garrid0 et al., 1995; Momburg et al., 1989). Complete losses are frequently found by labeling tissue sections with monoclonal antibodies recognizing nonpolymorphic HLA class I determinants. Losses of haplotypes or alleles are more difficult to document, but analyses with anti-HLA-A2 monoclonal antibodies, for example, have indicated that partial HLA losses are frequent (Cabrera etal., 1996; Kageshita etal., 1993; Natali et al., 1989).These losses of HLA expression are usually thought to be a consequence of immune selection, and the identification of numerous tumor antigens targeted by CTLs validates this concept. The high proportion of tumors with partial or complete HLA losses suggests in turn that the immune system is much more active against human tumors than is often thought. However, the frequent occurrence of HLA-loss variants raises a cautionary flag about obtaining long-term remissions by stimulating tumor-specific CTLs in vivo. In this respect it is interesting to note that complete HLA losses are far from being the rule, and that neither the MEL.B nor the HLA-loss variants that we derived in vitro from MEL.A lost expression of all their HLA class I alleles, a phenotype usually associated with a P,-microglobulin or TAP defect (Bicknell et al., 1994; Cromme et al., 1994; D’Urso et al., 1991).

B. Anti-MEL.B CTL Clones The absence of anti-MEL.B CTL clones in the group of CTLs derived from anti-MEL.A MLTC could be due to the absence of antigens on MEL.B, to the presence on MEL.B of antigens that were not present on MEL.A, or to a bias in the anti-MEL.A MLTC, as the vast number of precursors of CTLs against the other antigens could have prevented the growth of a small proportion of A24-restricted CTLs. We set up new MLTC experiments in which MEL.B cells were used instead of MEL.A to stimulate blood lymphocytes collected either in 1990 or in 1994, after the resection of the metastasis from which MEL.B was derived (Lehmann et al., 1995). Essentially no lytic activity against MEL.B could be detected in the responder cells derived from the 1990 lymphocytes, indicating that no antigen presented on HLA-A24 molecules stimulated a significant CTL response before 1990 (Fig. 7 ) .On the contrary, the lymphocytes from 1994 differentiated into a population of responder cells that lysed MEL.B cells very efficiently, indicating that an antiMEL.B CTL response developed in vivo between 1990 and 1994. Surprisingly, this CTL population did not recognize MEL.A cells. Several CTL clones were derived that showed specificity for MEL.B. CTL clone 17 was one of these CTLs.

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Stimulator cells:

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Fig. 7 Lyric activities of MLTC responder cells. Blood mononuclear cells collected from patient I.R.33 in 1990 or 1994 were srimulated each week by the addition of irradiated MEL.A or MELB cells in the presence of IL-2 and IL-4. On day 21 the lytic activities of the responder lymphocytes were measured against MEL.A o r MEL.B cells in the presence of a 50-fold excess of unlabeled K.562 to inhibit the lysis by NK-like effectors.

C. Identification of the Antigen Recognized by CTL 17 CTL clone 17 lysed MEL.B but did not recognize autologous EBV-transformed B cells or K562 cells. It expressed CD8 and, as expected, it was restricted by HI,A-A24 molecules (Fig. 8). The antigen recognized by CTL 17 was named LB33-E. Using the approach mentioned before, a cDNA clone was found that could transfer the expression of antigen E into COS cells on cotransfection of an HLA-A24 cDNA. This cDNA corresponded to a new gene that we named PRAME (for preferentially expressed antigen of melanoma). PRAME mRNA encodes a putative protein of 509 amino acids that shows no significant homology with known proteins. Northern blots indicated that gene PRAME was expressed by several tumor cell lines, but not by normal adult tissues except testis. The expression of the PRAME gene in normal tissues was also studied by RT-PCR amplification. High levels of PRAME expression were found in testis. Endometrium samples expressed the PRAME gene at levels corresponding to 5-30% of that found in the LB33 melanoma cells. Samples of ovary and adrenals expressed 2-5% of that level. Lower levels of expression were detected in some other normal tissues, including kidney, brain, or skin (Ikeda et al., 1997). A wide variety of tumor samples expressed gene PRAME at significant levels, that is, at least 3% of the level found in the LB33 melanoma cells. The highest proportions lung squamous cell of positive tumors were found among melanomas (91YO), carcinomas (78%)or adenocarcinomas (46%), renal carcinomas (41YO),sar-

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Fig. 8 Lytic activity of CTL clone 17. Target cells: (1)MEL.B cells alone or in the presence of an anti-HLA-A24 monoclonal antibody; (2)autologous EBV-transformed B cells; (3)K562, the prototype of target N K cells; (4) melanoma cell lines derived from patients LG2 and LB34; both lines carry HLA-A24 molecules and express gene PRAME; (5) MEL.A cells (A24B13-Cw6-A28-B44-Cw7); (6) the A-B-D- antigen-loss variant, which expresses only A24-Bl3-Cw6; (7)the C- antigen-loss variant, which expresses only A28-B44-Cw7; (8) Ccells expressing HLA-A24 molecules after transfection with an HLA-A24 cDNA clone; (9) MEL.A cells; (10) MEL.A cells in the presence of monoclonal antibody B1.23.2, which recognizes HLA-B and HLA-C molecules; (11) the variant A-B-D-; (12) A-B-D- cells expressing HLA-Cw7 molecules after transfection with an HLA-Cw7 cDNA clone.

comas (39%), head and neck squamous cell carcinomas (39%),and acute leukemias (33%). A high level of gene expression, comparable with that found in the LB33 melanoma cells, was found in most melanoma samples,

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in 24% of lung squamous cell carcinomas, 35% of sarcomas, and 2 % of renal carcinomas. The expression of the PRAME gene shares several characteristics with that of genes of the MAGE, BAGE, and GAGE families, which are expressed in tumors of many different histological types. However, PRAME is expressed by a higher proportion of samples than any of the other genes, and it is also expressed by acute myeloid leukemias, which never express the MAGE, BAGE, or GAGE genes (Chambost et al., 1993; Shichijo et al., 1996). A second similarity among all these genes is their high level of expression in testis. The mouse gene family SMAGE, which is homologous to the human MAGE gene family, is expressed in testis by germinal cells (Chomez et al., 1995), and the MAGE-1 and MAGE-4 proteins were detected in human spermatogonia and spermatocytes (Takahashi et al., 1995). It is possible that the expression of PRAME in testis will also be restricted to germinal cells, which do not bear HLA molecules and should therefore not suffer from an antiPRAME T cell response. The activation of the MAGE-1 gene in tumor cells appears to be due to the demethylation of the promoter, and this was correlated with a genome-wide demethylation process (De Smet et al., 1996). We observed that the treatment of primary fibroblasts or phytohemagglutinin Astimulated blood mononuclear cells with the demethylating agent S-aza-2’deoxycytidine activated the MAGE-1 gene and also the PRAME gene. This effect of DNA demethylation may explain why PRAME is expressed in testis, because male germ-line cells undergo a genomewide demethylation. In contrast with the MAGE genes, PRAME is expressed in some normal tissues other than testis. Except for endometrium, which expresses up to 30% of the level found in the LB33 melanoma cells, the levels of expression in normal tissues corresponded to less than 3-5% of that found in melanoma cells. It was shown that anti-MAGE-1 CTLs were unable to recognize tumor cell lines that expressed MAGE-1 at levels corresponding to less than 5% of that found in the melanoma cells that were efficiently lysed (Lethi et al., 1997). It is therefore possible that the expression of PRAME in most normal tissues is unsufficient for CTL recognition.

V1. A NEW CLASS OF ANTITUMOR CTL A. Presence of NK Inhibitory Receptors on CTL I 7 We observed that, surprisingly, MEL.A cells were not lysed by CTL 17, although they expressed PRAME and HLA-A24 at the same high level as MEL.B. In addition, when we tested the lytic activity of CTL 17 on a panel of HLA-A24 melanoma cell lines expressing the PRAME gene at a high lev-

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el, we observed that some of these cell lines were sensitive to lysis by the CTLs, whereas others were totally resistant (Fig. 8). A first clue to the specificity of CTL 17 was provided by the sensitivity to lysis of the antigen-loss variant A-B-D- that was derived from MEL.A and had lost expression of the A28-B44-Cw7 haplotype (Fig. 8). On the contrary, the C- variant that had lost expression of the A24-Bl3-Cw6 haplotype was not recognized after transfection of a cDNA encoding HLA-A24. These results suggested that there was an inverse correlation between the expression of the A28-B44Cw7 haplotype and the sensitivity to lysis by CTL 17. We tested the lytic activity of CTL 17 against MEL.A cells in the presence of a monoclonal antibody recognizing HLA-B and HLA-C molecules. In these conditions, MEL.A cells were very efficiently lysed by the CTLs (Fig. 8). We concluded that the PRAME antigenic peptide was expressed by both the MEL.A and MEL.B cells, and that HLA-B or HLA-C molecules protected MEL.A cells from lysis by CTL 17. These results were reminiscent of the inhibition of the cytolytic activity of NK cells, and of a fraction of T cells, by MHC class I molecules expressed on the target cells (Ljunggren and Karre, 1985; Moretta et al., 1992; Phillips et al., 1995).To identify the inhibitory HLA-B or HLA-C molecules, we tested the lytic activity of CTL 17 on the HLA haplotype-loss variant A-B-Dtransfected with cDNA clones encoding HLA-B44 or HLA-Cw7. No significant inhibition of lysis was observed after transfection with the B44 construct, whereas transfection with the HLA-Cw7 cDNA protected the cells from lysis by the CTLs (Fig. 8). Therefore, an inhibitory receptor binding to HLA-Cw7 appeared to prevent CTL 17 from lysing MEL.A cells. Several HLA class I-specific NK cell inhibitory receptors have been identified (Moretta et al., 1996). The inhibitory receptors specific for HLA-C molecules are represented by two members of the p58 molecular family, which belongs to the immunoglobulin superfamily (Colonna and Samaridis, 1995; Moretta et al., 1996; Wagtmann et al., 1995). The p58.1 receptors are recognized by the EB6 monoclonal antibody and interact with several HLA-C molecules, including Cw2,4, and 6 (Moretta et al., 1990).The p58.2 receptors are glycoproteins of 58 kDa recognized by the GL183 antibody. They show specificity for a subset of HLA-C molecules that contain serine and asparagine at positions 77 and 80, respectively, such as HLA-Cwl, 3, 7, and 8 (Biassoni etal., 1995; Colonna, 1996; Colonna etal., 1993; Moretta et al., 1993).CTL 17 was labeled with monoclonal antibody GL183, indicating that it carried the p58.2 receptor. The lytic activity of CTL 17 was tested against MEL.A cells in the presence of antibody GL183, aimed at blocking the interaction between p58.2 and HLA-Cw7 molecules (Fig. 9). Addition of increasing concentrations of the antibody restored lysis of the MEL.A cells. These results indicated that blocking the p58.2 receptors on the CTLs suppressed the inhibition of lysis of the MEL.A cells. Taken together, the results indicate that

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% Specific lysis

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Fig. 9 Lytic activity of CTL 17 against 1MEL.A cells in the presence of an anti-p58.2 monoclonal antibody. M E L A cells are not lysed by CTL 17, even at higher effector/target ratios than those shotvn here. CTL 17 was incubated first with the indicated concentrations of the antip58.2 antibody GL183, and then used at an ef'fector/target ratio of 5 in a lysis assay against A1F.L.A cells.

1MEL.A cells express the PRAME-encoded antigen, but that they are not lysed by the anti-PRAME CTL 17 because they bear HLA-Cw7 molecules, which inhibit the lytic activity of the CTL on engagement of the NK inhibitory receptor p58.2. The antigenic peptide recognized by CTL 1 7 was localized by transfecting truncated cDNA clones into COS cells and testing the recognition of the transfectants by the CTLs. Candidate peptides were synthesized and incubated with EBV-transformed B cells derived from patient LB33. CTL 17 was added, together with the anti-HLA-B/C monoclonal antibody to block the inhibition by HLA-Cw7 molecules. Nonapeptide LYVDSLFFL sensitized the cells to lysis with a half-maximal effect at 100 nM. In the absence of the antiHLA-B/C antibody, however, EBV-B cells from patient LB33 could not be lysed at all by CTL 17, even when high concentrations of the antigenic peptide were used. These results clearly show that the inhibitory receptors dominate the activation machinery of CTL 1 7 and that when they are properly engaged by their ligand, even very high densities of antigen on the target cell are unable to stimulate the CTLs. We observed that inhibition of the activation of CTL 17 extends beyond the lytic activity. When the CTLs are incubated with cells that present the PRAME peptide and that also express Cw7 molecules, not only do the CTLs not lyse the target cells, but they neither produce lymphokines such as TNF or IFN-y nor proliferate. Therefore, we think that in uiuo CTLs such as CTL 17 were not stimulated by MEL.A cells, but only by tumor cells with a partial loss of HLA expression, such as MEL.B. This probably explains why we

Antitumor Immunity in Melanoma

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I

- - - - - - -**

'--

Fig. 10 Lytic activity of the anti-PRAME CTL clone 17 against LB33-MEL cells.

did not detect an anti-MEL.B CTL response ivt vitro in the MLTC performed with PBMCs of 1990 (Fig. 7), because MEL.B-like tumor cells were probably not yet present at that time. The results provide an explanation for the unusual specificity of CTL 17. Both MEL.A and MEL.B cells present the PRAME peptide on HLA-A24 molecules. MEL.A cells are not lysed because they express HLA-Cw7 molecules, which engage the p58.2 inhibitory receptors on the CTLs. MEL.B cells can be lysed by CTL 17 because they have lost expression of HLA-Cw7 molecules (Fig. 10). The pattern of recognition of allogenic A24'PRAME' melanomas is also explained by the presence of p58.2 receptors on the CTLs: the melanoma lines that express ligands of p58.2 (HLA-Cwl, 3,7, or 8) are not lysed by CTL 17. It is worth mentioning that CTL 17 expresses an ctp TCR, that its lytic activity is inhibited by an anti-CD3 antibody, and that it does not lyse K562 cells, which do not express HLA class I molecules (Fig. 8). Thus, CTL 17 is a bona fide CTL and, although it bears an inhibitory receptor present on NK cells, it does not exert the MHC-unrestricted lytic activity of these cells.

B. A Role in Antitumor Defense for CTLs Expressing NK Inhibitory Receptors? CTL 17 is not the only anti-tumor CTL derived from patient LB33 that bears functional inhibitory receptors. We derived other anti-MEL.B CTLs that recognize antigens that are not encoded by gene PRAME. These CTLs also express p58.2 inhibitory receptors and appear to be regulated in a similar way to CTL 17. We have screened the LB33 anti-MEL.A CTL clones and autologous antimelanoma CTLs from other patients with a panel of monoclonal antibodies recognizing different families of NK receptors, and found no antitumor CTLs with these receptors. This is probably not very surprising because the stimulator tumor cell lines used to derive all these CTLs did not have major defects in MHC class I expression.

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A substantial proportion of T cells bear NK receptors (Mingari et al., 1996; Phillips et al., 1995). The numbers vary greatly from one receptor to another, but 1-30% of blood T cells in normal individuals appear to express one or several of these receptors. Because thymocytes and cord blood T lymphocytes are consistently negative, expression of NKR by some T cells seems to be acquired during or after stimulation by antigens. The role of those receptors on T cells is not clear. They could participate in the physiologic downregulation of T cell responses. In the case of the anti-PRAME CTL clone 17, there are two explanations for the presence of inhibitory receptors. A first possibility is that, stimulated by MEL.A or MEL.B, an anti-PRAME CTL response developed first, involving classical CTLs without inhibitory receptors. This implies that the expression of gene PRAME in some normal tissues does not lead to the expression at the cell surface of an amount of antigen that is sufficient to trigger lysis, otherwise we would expect to have seen autoimmune disorders involving, for example, the adrenals, which we did not find in patient LB33. Some of the anti-PRAME T cells may then have expressed NK inhibitory receptors as part of a normal mechanism to down-regulate their activation. The concomitant presence of MEL.B cells, capable of stimulating the CTLs and unable to inhibit them through Cw7, would have amplified this response further. A second possibility is that anti-PRAME T cells are normally not stimulated, because they recognize a self antigen. This antigen is not ubiquitously expressed and there would be no clonal deletion of the anti-PRAME precursors during thymic development, but peripheral tolerance would prevent the activation of these autoreactive T cells. If some of these cells then express NK inhibitory receptors, they would no longer be harmful for normal cells, but could target cells such as MEL.B. In the first hypothesis PRAME is a genuine tumor antigen, whereas in the second it 1s to be considered more as an autoantigen, and, of course, this modifies its usefulness as a source of antigens for tumor vaccines. We stimulated LB33 blood lymphocytes with the PRAME peptide that binds to HLA-A24, trying to find the “classical” anti-PRAME CTL predicted by the first hypothesis, and could not derive such CTLs. Of course this does not prove that they do not exist-they simply could be very rare. Using the data obtained with patient LB33 as a model of what may happen in other patients, it is interesting to try to find a role in antitumor defense for CTis bearing NK inhibitory receptors.’Antitumor lytic immune effector cells are the CTL and the NK cells. CTLs recognize antigenic peptides presented by HLA molecules on the tumor cells but not on normal cells. Lymphocytes recognizing MAGE-derived peptides, or peptides encoded by genes mutated in the tumor cells, clearly belong to this category. They are the effector arm of the immune system against those tumor cells that have normal HLA class I expression, whereas NK cells, whose function was clarified by

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the identification of the inhibitory receptors, require a loss of expression of HLA class I molecules to lyse target cells. The lytic activity of the NK cells is triggered through engagement of receptors such as the FcyRIII receptor and other receptors whose function is not yet fully delineated (Bottino et al., 1996; Moretta and Moretta, 1997; Sivori et al., 1997). It is possible that some of them bind to ubiquitous ligands, and in that case the specificity of the Iytic activity of the NK resides entirely in the absence on the target cells of those HLA class I molecules that trigger the inhibitory receptors. Because most NK cells appear to coexpress several types of inhibitory receptors, recognizing HLA-A, HLA-Byor HLA-C molecules, a complete loss of HLA class I will render tumor cells very sensitive to NK lysis. An appropriate way of dealing with tumors having completely lost expression of HLA class I molecules would be to activate NK cells in vim. Because one of the effects of the infusions of IL-2 is to increase the proportion of blood mononuclear cells bearing NK markers (Vlasveld et al., 1993), it is possible that some of the clinical successes of IL-2 in cancer patients are correlated with a previous loss of HLA class I molecules by the tumor. It may be informative to study retrospectively the expression of HLA molecules on tumor samples from those patients who responded to such treatment. HLAloss seems to be a major mechanism of escape from immune attack, thus the stimulation of NK cell lytic activity in vim by IL-2 or by more specific activators may prove to be a crucial complement to immunizations with tumorspecific antigens. It is very likely that transformed cells initially express HLA class I molecules and that, later on, they are selected for the loss of HLA expression by antitumor T lymphocytes. In many instances an intermediary step has to be a partial HLA loss (Fig. 11). CTLs expressing NK inhibitory receptors may play a role in antitumor immunity by eliminating specifically this type of tumor cell. They recognize an antigen presented by one of the remaining HLA alleles, and bear NK inhibitory receptors engaged by those HLA molecules whose expression is lost by the tumor cells. It is noteworthy that the antigen ought not to be tumor specific: it may even be an ubiquitously expressed antigen, because normal cells are protected by their expression of the inhibitory HLA molecules. We have actually identified several cDNA clones that appeared to encode antigens recognized by CTLs on melanomas other than LB33-MELYand that proved to be nonmutated sequences from ubiquitously expressed genes. It is not easy to reconcile such results with the specificity of the corresponding CTLs for the autologous tumor cells. Either the antigen encoded by the cDNA is homologous to the real tumor antigen, and molecular mimicry explains the observation, or the cDNA really encodes the antigenic peptide that is recognized by the TCRs of the CTLs and we have to explain why these CTLs do not lyse other cells bearing this ubiquitous antigen. A possibility is the presence of inhibitory receptors on the CTLs, rec-

Pierre C. Coulie et al.

Tumor

+u Tumor

Fig. 1 I .A model of the interaction of different types of lytic effectors with tumors at different stages. Tumor-specific CTLs are stimulated to lyse and proliferate after recognizing an antigenic peptide that is not presented o n normal cells. CT1.s bearing NK inhibitory receptors recognize antigens that are not necessarily tumor specific, and lyse tumor cells that have lost expression of the HIA that interacts with the inhibitory receptors. NK cells are activated by structures that are as yet poorll- defined, and Iyse cells that have lost expression of those HLA molecules that engage their inhibitory receptors.

ognizing a ligand that is present on normal cells but lost from tumor cells. We could not label such CTLs with a panel of antibodies recognizing some of the known NK inhibitory receptors. But as the number of such receptors continues to grow, and several of them display an allelic polymorphism, the hypothesis cannot be ruled out.

VII. CONCLUSIONS Burnet proposed in his theory of immune surveillance that the immune system was able to detect and destroy tumor cells. Today we can appreciate how much these ideas, formulated when essentially no evidence of an immunological control of cancer was obtained, were farsighted. Rigorous genetic approaches have allowed us to obtain what could only be a dream for Burnet, namely, a growing number of perfectly specific tumor antigens that can be recognized by T lymphocytes. These results fulfill the first part of the immune surveillance theory: detection. Using all these antigens in a variety of formulations designed to increase their immunogenicity is the logical next step. Numerous clinical studies are underway, with encouraging preliminary results (Marchand et al., 1995), aiming to achieve the second part of Burnet’s

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theory: destruction. Because the now demonstrated antigenicity of most tumors implies that mechanisms of escape from immune destruction participate in the development of most cancers, the forthcoming challenge of tumor immunology will be to understand and counter these escape strategies. Patient LB33 represents an interesting example of this fight of the immune system against a tumor. The tumor bears many antigens. For a still unknown reason a strong immune response was triggered. The tumor escapes through a loss of expression of most HLA class I molecules. The immune system fights back with a new wave of CTLs, apparently tailor-made for this type of tumor cell. The results underscore the plasticity of the tumor cells, but also the versatile power of the immune system. It is our hope that the understanding of the modalities of this fight will provide us with new tools for cancer therapy. Detailed analyses of carefully selected patients and of their tumor material are the operating instructions for us to decipher.

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Index

A Actin, 9,67, 70,93-94 Actin binding protein, 68 Acute myeloid leukemia, 67,232 Adhesion, see also Focal adhesions; RGD cell attachment sequence cell-cell, 5, 102 cell-extracellular matrix, 2-4, 100 of colon carcinoma, 84 and cortactin, 100 and FAK, 99 Age factor and colon cancer, 188 and KSHV, 133,143-145 and lung cancer, 180 and methylation, 201,207 and mutations, 189, 190 Alcohol, 171-172,198 Aneuploidy, 196, 198, 199,200 Angiogenesis c-Src effect, 98, 102 FGF receptor role, 69 HGFISF role, 65,66 protein expression, 15-16 Anoikis, 5-6, 7, 8 Antagonism, 162 Antibodies, to KSHV, 126-135, 138-141, 145,149 Antigens and carcinogenesis, 178 EBV viral capsid, 128 expression types, 214-215 KSHV, 126-135,138-141 in melanomas, 219-227,230-232 middle-T, 102 Anti-smoking campaigns, 161-162, 173175,182 Apoptosis as cancer control mechanism, 191-192, 194 and c-Myb oncogene, 31-32,49

due to displacement from extracellular matrix, 5-6, 7, 8 Arsenic, 168, 177 Asbestos dose-response, 168-169 and smoking, 170,176-177,178-179, 180-181 Astrocytic tumors, 69 Autocrine loop, 67-68 Avian leukemia virus E26, 23 Avian myeloblastosis virus, 22-23,49 5-azacytidine, 202,203

B BAGE genes, 227 Basement membranes, 10-11, 12 Basic fibroblast growth factor, 88 BAX gene, 194 B cells, 88,223 Bcl-2 gene, 31-32 Bcl-2 protein, 6, 7, 12 Bile acids, 195-196,206 Biological interaction, 163-164 Bladder cancer, 5,69, 73,177 Blood, 12-13,30-31 Blood vessels, 12, 15-16, 66 Bone metastases, 14 Brain cancer, 65 Breast cancer and breast tumor kinase, 89 and cortactin, 100 and CSF-1,67 and c-Src, 84-85,95,103 and EGFR, 72,73,74, 75,77-78, 95-96 and estrogen receptor, 76, 78 and FGF receptor, 69 Met tyrosine kinase expression, 66 and Myb oncogene, 30 and PDGF receptors, 71

243

244 Breast Cancer (continued) progesterone receptor negative, 85 sFN effect, 13 BS69 protein, 46 Bulky-adduct-forming agents, 196-1 97, 198,199,207 Burkitt’s lymphoma, 88

C C. elegans, and Myb oncogenes, 35 Carcinogenesis, see also Methylation epigenetic explanations, 203-204,208 molecular pathways chromosomal instability, 196-200 microsatellite instability, 192-196, 199-200,205-206 and natural selection, 188-192, 205 of smoking synergies, 177-180 CAS, see pl30Cas Castleman‘s disease, 151 -1 52 p-catenin, 5 Caveolin, 94-95 CBP protein, 44-45 CD2,30 CD4,150 CD8,219,220 CD45,151 Cell cycle and chromosome changes, 196 and C-Src, 90, 91, 102 and DNA methylation patterns, 200-201 DNA repair and arrest, 190-191 and EGFR, 31-32,34-35 and M y 6 oncogenes, 31-32,34-35 Cell membranes, 7-8, 10-11, 69, 94 Cell migration, 10-1 1 Cells anchorage, 5-10 differentiation, 30-31, 81, 201 growth regulation, 10, 12 interactions extracellular matrix, 2-4, 9, 90, 100 other cells, 5,90, 102 invasive properties, 10-12, 70-71 motility, 10-11,66,68, 84, 91 tumorigenic to nontumorigenic conversion, 3-4, 13 Cervical cancer, 86 c-Fms, see Colony-stimulating factor-1 receptor

Index Chromosomes, 50-52,196-200 Cigarettes and alcohol, 171-172 and asbestos, 170, 176-177, 178-179, 180-181 carcinogenesis timing, 178 and colon cancer, 198 dose-response, 167-168, 175 education against, 161-162, 173-175, 182 and nucleotide excision repair, 198 and radiation, 177 and radon, 171,178-179,180-181 Circulation blood effect on tumor cells, 12-13 blood vessel basement membranes, 12 quantitation of tumor cells, 14 and vasculature “addresses,” 15 Cisplatinum, 202 c-kit growth factor receptor gene, 48-49 Colon cancer and bile acids, 195-196,206 and chromosome changes, 196-199 and c-Src gene, 82-84, 103 and c-Yes gene, 88 dietary factors, 165, 198,202, 204 and exogenous carcinogens, 207 and FAK, 83-84 and HERl, 73, 103 hereditary nonpolyposis, 192 location factors, 195-196, 198-200, 205-207 and methylation balance, 204-206, 207 Met tyrosine kinase expression, 65 and Myb oncogene, 30 and natural selection, 188-192 and nucleotide mismatch repair, 192-1 96 and p53 inactivation, 198 prognostic factors, 200 sFN effect, 1 3 and synergism, 165 Colony-stimulating factor-1 receptor, 66-68 Colorectal cancer, see Colon cancer Cortactin, 8, 68, 99-100 CSF-1, see Colony-stimulating factor-1 receptor c-Src, see also Basic fibroblast growth factor; c-Yes gene and CSF-I, 6 7 and EGFR, 73,75,89-90,91-96,102103

Index and estrogen receptors, 77 and FAK, 8,98-99 and integrins, 4-5 mechanism of action, 81-82, 84, 89-96 and receptor tyrosine kinases, 62 regulation, 79 structure, 78-79 targets, 96-102 therapeutic applications, 102-103 and types of cancer, 84-86 Cyclin D, 10,44,49-50 c-Yes gene, 82, 87-88 Cyp40 protein, 44 Cytochrome P-450,207 Cytoskeleton, 9

D Debrisoquine, 174, 180 Diet, 165,175, 198,202,204 Differentiation, 30-31, 81,201 DNA damage and repair, 178,190-191,192196,197 methylation balance, 200-203 Drosophilu, and Myb genes, 34-35

E EBV, see Epstein-Barr virus E-cadherin, 5 EGFR, see Epidermal growth factor receptors Endometrium cancer of, and CSF-1,67,68 and melanoma antigen gene, 230-232 Endothelial cells and angiogenesis, 15-16 and integrins, 8 and metastatic specificity, 15 vascular, 12,66 Environmental factors, 190-192, 198-199 Epidermal growth factor receptors and colon cancer, 73, 83, 84 and c-Src, 72-78, 89-90,91-96,102103 Epithelial cells c-Myb protooncogene expression, 3 1 and integrins, 2, 6, 11 Epstein-Barr virus, 88, 128 E-selectin, 15

245 Esophageal cancer and c-Src, 86 and EGFR, 73,75 smoking effect, 171-172, 177 Estrogen, 48-49, 179 receptors for, 76-78 Ethanol, 202,203 E26 virus, 23, 31,40 Extracellular matrix, 2-4, 5-6,9, 100

FAK, see Focal adhesion kinase Fas, 205,215 Fecapentanes, 207 FGF receptors, see Fibroblast growth factor receptors Fibrin, 11 Fibroblast growth factor receptors, 68-70, 100, see also Basic fibroblast growth factor Fibronectin and actin stress fiber formation, 9 and angiogenicity, 16 and circulating tumor cells, 13 in normal tissue, 3 Src and Rus effect on, 5 tumor suppression and enhancement, 3, 10 tumor-surrounding matrix formation, 11 5-Fluorouracil, 202 Focal adhesion kinase, 83-84, 98-99 Focal adhesions, 7-9,11,100 Free oxygen species, 196 Fyn, 87-88

G GAGE genes, 227 Gastric cancer and CSF-1, 67 and c-Src, 86 and EGFR, 73,75 location factors, 207 Met tyrosine kinase expression, 65 GBX2 gene, 49 Gender factor and colon cancer, 188 and KSHV, 134 Genetic susceptibility, see also Mutations to colorectal cancer, 188

246 Genetic susceptibility (continued) to lung cancer, 174-175, 178-180 Gene transcription, 96-98 Glioblastomas, 73 Glioma, 66, 74 Growth regulation, 10, 12

H Hairy cell leukemia, 67 Head and neck cancer, 100, 171-172,231 Hematopoiesis, 30-31 Hemophiliacs, and KSHV, 147 Hepatocyte growth factorkarter factor receptor, 64-66, 84 Hepatoma cells, 70; see also Liver cancer Heptad leuciiie repeat, 38-40 HERl, see Epidermal growth factor receptors Herpes simplex virus, 175 Herpes virus, see Kaposi’s sarcoma-associated herpesvirus HGF/SF. see Hepatocyte growth factorlkatter factor receptor HIV-1, and Kaposi’s sarcoma, 136-141 HLA molecules, 219-220,226, 227-229, 233-239 Hodgkin’s disease, 67, 88 Human herpesvirus 8, see Kaposi’s sarcomaassociated herpesvirus Human papilloma virus, 86, 214

I IGF-1, 31 1L-la, see Interleukin-la 11.-6, see Interleukin-6 Ileal cancer. 6.5 Immunosuppression and KSHV, 150 of melanoma, 214-239 Inflammatory bowel disease, 88 Insulin, 12 Insulin-like growth factor, 12 Inregrin-linked kinase, 5, 9 Integrins and actin cytoskeleton, 9-10 and anchorage dependence, 5-6 and angiogenesis, 16 and cell migration, 17 and fihronectin, 3 , 9

Index and insulin, 12 and invasion, 12 and melanoma, 11 and metastases redirection, 15 and pl30Cas, 99 specificity, 2, 6, 12 as targets for therapy, 16 Interleukin-la, 65 Interleukin-2, 216, 219,220, 225 Interleukin-6 and KSHV, 152,153 and melanoma, 220 and Met tyrosine kinase, 65 Interleukin-12,220 Invasive properties, 10-12, 70-71

K Kaposi’s sarcoma-associated herpesvirus in children, 143-145 disease associations, 145- 152 geographic distribution and endemic Kaposi’s sarcoma, 135137,154 PCR-based studies, 124-126 serological studies, 126-135 and HIV as cofactor, 133, 137-141, 147-151, 153-154 in uninfected persons, 125-126, 154 and immunosuppression, 150 and lymphoma, 151 molecular epidemiology, 122-124 in older persons, 133 serologic assays, 126-130 transmission routes, 137, 138-145, 150 in transplant recipients, 140-141, 145, 152 variants, 122-124, 154

L Laryngeal cancer, 73,171-172, 177 Latency-associated nuclear antigen, 126 135,138-145,149 Leucines, 38-40 Leukemias acute myeloid, 67, 232 hairy cell, 67 and leucines, 39

Index

and melanoma antigen gene, 23 1-232 murine myeloid, 40 M y 6 oncogene expression, 30 Leukocytes, and metastases, 15 Lewis lung carcinoma, 68, 71 Liposomes, 16 Liver cancer, 73, 175,204 Liver metastases, 14 Lung cancer and asbestos, 168-169 and CSF-1,67,68 and c-Src, 85-86, 103 and EGFR, 73,74,75,103 genetic susceptibility, 174-175, 179-180 and melanoma antigen gene, 230-232 and radon, 169 and smoking, 168 Lung metastases, 14 Lymph node proliferative disorders, 151152 Lymphomas B cell and avian leukosis virus, 26 body cavity-based, 151 Burkitt’s, 88 metastatic specificity, 15 M y b oncogene expression, 30 T cell and KSHV, 153 Marek’s, 26-30 and M y b oncogenes, 30

M MAGE genes, 214,220,226-227,232 Major histocompatibilitycomplex, see HLA molecules MAPK, see Mitogen-activated protein kinase Marek’s T-cell lymphoma, 26-30 Medulloblastomas, 74 Melanoma antigens, 214,219-227,230-232 genetics, 223-225,226,230 and basic FGF, 88 chemotherapy regimen, 216 and integrins, 11 metastases, 216,218,227-232 and PDGF receptors, 71 sFN effect, 13 and tyrosine kinases, 87 Mesotheliomas, 71

247 Met, see Hepatocyte growth factor/Scatter factor receptor Metastases of colon cancer, and c-Src, 83 CSF-1 role, 68, 102 and FGF receptor, 69-70 HER1 role, 74 HGF/SF receptor role, 66 and immunotherapy, 227-232 of melanoma, 216,218,227-232 and RGD cell attachment peptides, 13-14 site-specificity, 14-16 superfibronectin effect, 13 Methotrexate, 202 Methylation and colon cancer, 194-196, 199,204205,206 of DNA, 200-204,205 and melanoma, 227 melanoma antigen in testis, 232 Microsatellite instability, 192-1 96, 199200,205-206 Mim-1 gene, 48 Mitogen-activated protein kinase, 43,46, 70,98 Mitosis, see Cell cycle Moloney murine leukemia virus, 25 Mouse and CFS-1 expression, 67 and c-Src, 85, 86 DNA-MTase-deficient,205 EGFR studies, 73, 75, 89 and M y b oncogenes, 30-31 Mouth cancer, 171-172, 177 Multiple myeloma, 153 MUM-1 gene, 223-225 Mutations, see also Retroviral insertional mutagenesis and carcinogen type, 199-200 encoding melanoma antigens, 223 -225, 226,230 and methylation, 201 and natural selection, 189, 190-192 and nucleotide mismatch repair, 192196 and smoking, 177 and tumor antigen types, 214 My& oncogenes activation, 23-30 and cell cycle, 31-32 and differentiation, 30-31

Index

M y h oncogenes (continued) discovery, 22-23 evolution model, 35 expression. 30-33 related genes, 33-35, 40 Myb proteins carboxyl terminus, 40-41 and chromatin, 50-52 DNA-binding domain, 36-37 Myb repeat, 36 promoter effect, 32 transcription role, 38-40, 46-47, 49-SO v-Myb and c-Myb, 41-49 Myc oncogene, 179 Myelomonoc ytic rumors, 25

N N-acetyltransferase, 207 Nasopharyngeal cancer, 88 Natural selecrion and colon cancer, 188-192, 195-199, 203-204,205 and melanoma metastases, 227 Nephrobfastoma, 23. 86 NIH3T3 cells, 68, 73, 92 NK inhibitory receptors, 232-238 Nucleolar proteins, 46 Nucleotide repair excision, 196-197 mismatch, 192-196

Oncogenes, see also specific genes anchorage-affecting, 7 and integrin activity, 4-5 and lung cancer, 178-180 phorbol ester effect, 179 specificity, 22 Oncoproreins, 8 -9 Osteoblasts, and HGF, 65 Osteociasts, 65, 67 Osteogenic sarcoma, 13, 65 Osteopetrosis, 23 Ovarian cancer and c-Src, 86 and EGFR, 73,74, 75 Met tyrosine kinase expression, 65 sFN effect, 13

P Pancreatic cancer and CSF-1, 67 and M y b onogene, 30 and PDGF receptors, 71 and smoking, 177 Papillary carcinomas, 65 Paracrine signaling, 66, 71 Parotid gland cancer, 86 PDGF, see Platelet-derived growth factor PECAM- 1, 101-102 Peptidomimetics, 103 p58 receptors, 233, 235 P53 gene, 177 PS3 protein, 197-198 Phagocytes, 66-67 Pharyngeal cancer, 171-172 Phosphorylation and actin disassembly, 70 and anchorage, 8-9 and cell motility, 68, 84, 91 and corractin, 100 by C-Src, 8-9, 67, 73, 90, 103 of FAK, 84 of Myb proteins, 41-43,46 of Y845, and oncogenicity, 93, 103 of Y934, and mitogenesis, 91 Plasma membrane, 94 Plasniinogen activators, 66 Platelet-derived growth factor, 12 receptors for, 70-71, 90-91 Piatelets, 2, 13-14 pl30Cas, 8-9,99 pl00 protein, 46 plSORhoGAP, 101 p160 protein, 46 PRAME gene, 230-232,234-235 Prostate cancer, 14,69, 73, 75 Proteases, invasion role, 11 Proteins, see also Oncoproteins; specific proteins actin binding, 68 antiapoptotic, 6, 7 Public health education, 161-162, 173-175, 182

R Radiation, see also W radiation of melanoma tissue, 219 and smoking, 177,178

Index

Radon carcinogenesis timing, 178 dose-response, 169, 175 and smoking, 171,174,177,180 Rak protein, 89 Ras oncogene and aneuploidy, 199 and cell membrane, 7-8 cofactors, 179 and integrin activity, 4-5 and smoking/asbestos synergism, 177 Ras proteins, 10,101 Receptor tyrosine kinases and c-SRC, 62 and HGF/SF, 64-66 structure, 63-64 Renal cancer, see also Nephroblastoma and melanoma antigen gene, 230-232 Met tyrosine kinase expression, 65 and smoking, 177 Retroviral insertional mutagenesis, 25-30 RGD cell attachment sequence, 2, 13-14 Rhadinoviruses, 122,128 Rho proteins, 10, 101 Rous sarcoma virus, 22-23

S Sarcoma, 26, 179,230-232; see also Osteogenic sarcomas Selectins, 15 Serinelthreonine kinases, 9 sFN, see Superfibronectin Signaling molecules and cell membranes, 7-8 and integrins, 2-3,4-5,12 Shc, 12 Signaling pathways anchorage-related, 7 growth-related, 12 Ras, 12,65 Skin cancer, 86,152,153, 198; see also Melanoma Smoking, see Cigarettes Src, see c-Src Superfibronectin, 13 Syndecan-1,102 Synergism alcohol and smoking, 171-172

249 arsenic and smoking, 177 asbestos and smoking, 170, 176-177, 178-179 versus autosynergism, 163-164 definition, 162-164, 165 follow-up duration, 180-181 public health implications, 172-176 radon and smoking, 171,178-179 versus statistical interaction, 164-167 viruses with chemical carcinogens, 175

T Tamoxifen, 75 Targeting, of drugs, 16 T cells and melanoma, 218-232 NK inhibitory receptors, 232-238 Telomere binding, 50-52 Testis tissue, 230-232 TGF-a, 72,76,77 TGF-P, 6 Thymocytes, 31 Thyroid cancer, 65, 72 TNF-a, 83 TP53 gene, 197-198,199,200 Transplant recipients, 140-141,145, 152 Tyrosine kinases, see c-Src; Receptor tyrosine kinases

U Urokinase, 11 Urokinase plasminogen activator, 66, 98 W radiation, 196, 198

v Vaccine, for melanoma, 216-219,225,235238 Vascular endothelial cells, 66 Vascular endothelial growth factor, 98 VCAM-1,15 v-Crk protein, 9

Y Y845 phosphorylation site, 93,103

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