DcR3

DcR3 Avi Ashkenazi* Department of Molecular Oncology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080-4918, USA * corresponding author tel: 650-225-1853, fax: 650-225-6443, e-mail: [email protected] DOI: 10.1006/rwcy.2000.16013.

SUMMARY DcR3 is a novel secreted member of the TNFR family. Its closest relative is OPG, to which it shows 31% protein sequence homology. In vitro studies show that DcR3-Fc binds to FasL with an affinity that is comparable to that of Fas-Fc; binding of DcR3-Fc and Fas-Fc to FasL is mutually exclusive, which suggests that DcR3 is a decoy receptor that competes with Fas for FasL binding. DcR3-Fc blocks apoptosis induction by FasL in several cell-based assays. DcR3 binds also to the TNF family member LIGHT, and DcR3-Fc inhibits LIGHT-induced apoptosis in colon cancer cells. The DcR3 gene appears to be amplified frequently in cancer, and DcR3 mRNA is often detectable in malignant tissue. Thus, DcR3 may play a pathologic role in cancer, perhaps through its interaction with FasL and/or LIGHT.

BACKGROUND

Discovery Apoptosis is a physiological cell-suicide mechanism that enables metazoans to eliminate individual cells that threaten the organism's survival. Fas ligand (FasL, also called CD95 or Apo1 ligand) regulates mainly three types of apoptosis: (a) activationinduced cell death (AICD) of mature T lymphocytes; (b) elimination of inflammatory cells from immuneprivileged sites; and (c) immune-cytotoxic killing of damaged cells (Nagata, 1997). T cell AICD helps shut down the host's immune response once an infection has been cleared. Repeated stimulation of the T cell receptor (TCR) by antigen induces expression of FasL and Fas on the surface of T helper cells; subsequently,

FasL engages Fas and triggers apoptosis in the activated lymphocytes, leading to their elimination. Immune-privileged sites are tissues such as the eye, brain, or testis, in which any inflammatory immune response perturbs function; cells in immune-privileged sites constitutively express FasL, and eliminate infiltrating leukocytes that express Fas through Fasdependent apoptosis. Immune-cytotoxic cells such as natural killer (NK) cells and cytotoxic T lymphocytes eliminate cells that have been damaged by viral or bacterial infection or by oncogenic transformation. This elimination occurs through two major mechanisms: one pathway involves release of perforin and granzymes; an alternative pathway involves expression of FasL and apoptosis-induction through Fas on target cells (Nagata, 1997; Moretta, 1997). FasL belongs to a family of type 2 transmembrane proteins that are structurally related to tumor necrosis factor (TNF); its receptor, Fas, belongs to a family of type 1 transmembrane proteins that are structurally similar to TNF receptors (TNFRs) (Nagata, 1997). TNFR family members contain homologous extracellular cysteine-rich domains (CRDs) (Smith et al., 1994). A subset of the TNFR family, including TNFRI, Fas, death receptor 3 (DR3), DR4, and DR5, contain a related cytoplasmic region dubbed `death domain' (Itoh and Nagata, 1993; Tartaglia et al., 1993). The death domain of Fas mediates apoptosis signaling in response to FasL binding, by activating the apoptotic protease caspase 8 through the adapter molecule FADD/Mort1 (Ashkenazi and Dixit, 1998). Until the recent identification of decoy receptor 3 (DcR3) (Pitti et al., 1998), Fas was the only receptor which was known to bind to FasL. DcR3 was discovered by a search of expressed sequence tag (EST) databases for ESTs that showed sequence homology to members of the TNF receptor (TNFR) gene superfamily (Pitti et al., 1998). A set of

1744 Avi Ashkenazi overlapping ESTs with homology to TNFR was identified, and was used to isolate a full-length cDNA from human fetal lung. The cDNA encoded a previously unknown polypeptide that resembles the TNFR family, which was named DcR3 based upon functional studies.

Alternative names TNFRSF6B; TR6; OPG2.

Structure The N-terminal sequence of DcR3 contains a typical secretion signal, followed by four tandem cysteinerich domains (CRD), which are characteristic of the TNFR family. Unlike most TNFR family members, DcR3 lacks an apparent transmembrane sequence, suggesting that it is a soluble, rather than a membrane-associated molecule. This was confirmed by expressing a recombinant, histidine-tagged form of DcR3 in mammalian cells: the protein was secreted into the cell culture medium (Pitti et al., 1998). DcR3 has one potential N-linked glycosylation site (Asn173).

Main activities and pathophysiological roles DcR3 binds to FasL (Pitti et al., 1998). FasL plays a key role in regulating the immune response;

however, how the function of FasL is controlled is not fully understood. One mechanism involves the molecule cFLIP, which modulates the apoptosis signal transduction pathway downstream of Fas (Tschopp et al., 1998). A second mechanism involves proteolytic shedding of FasL from the cell surface (Tanaka et al., 1998). DcR3 acts as a decoy for FasL; hence, it may be involved in extracellular regulation of FasL activity. The closest known relative of DcR3, osteoprotegerin (OPG), appears to act as a decoy for the TNF family members OPGL (Simonet et al., 1997), as well as for Apo2L/TRAIL (Emery et al., 1998). Thus, DcR3 and OPG define a novel subset of TNFR homologs that may function as secreted decoys to modulate specific TNF-related ligands. A similar mechanism is used by certain Pox viruses, which produce soluble TNFR homologs, presumably to inhibit the host's antiviral immune response (Smith et al., 1994). The frequent amplification of the DcR3 gene in lung and colon tumors (Pitti et al., 1998) raises the possibility that DcR3 may have a pathological role in cancer.

GENE

Accession numbers AF104419.

Sequence See Figure 1.

Figure 1 Nucleotide sequence of the DcR3 gene. cDNA

Sequence

TCCGCAGGCGGACCGGGGGCAAAGGAGGTGGCATGTCGGTCAGGCACAGCAGGGTCCTGTGTCCGCGCTGAGCCGCGCTCTCCCTGCTC CAGCAAGGACCATGAGGGCGCTGGAGGGGCCAGGCCTGTCGCTGCTGTGCCTGGTGTTGGCGCTGCCTGCCCTGCTGCCGGTGCCGGCT GTACGCGGAGTGGCAGAAACACCCACCTACCCCTGGCGGGACGCAGAGACAGGGGAGCGGCTGGTGTGCGCCCAGTGCCCCCCAGGCAC CTTTGTGCAGCGGCCGTGCCGCCGAGACAGCCCCACGACGTGTGGCCCGTGTCCACCGCGCCACTACACGCAGTTCTGGAACTACCTGG AGCGCTGCCGCTACTGCAACGTCCTCTGCGGGGAGCGTGAGGAGGAGGCACGGGCTTGCCACGCCACCCACAACCGTGCCTGCCGCTGC CGCACCGGCTTCTTCGCGCACGCTGGTTTCTGCTTGGAGCACGCATCGTGTCCACCTGGTGCCGGCGTGATTGCCCCGGGCACCCCCAG CCAGAACACGCAGTGCCAGCCGTGCCCCCCAGGCACCTTCTCAGCCAGCAGCTCCAGCTCAGAGCAGTGCCAGCCCCACCGCAACTGCA CGGCCCTGGGCCTGGCCCTCAATGTGCCAGGCTCTTCCTCCCATGACACCCTGTGCACCAGCTGCACTGGCTTCCCCCTCAGCACCAGG GTACCAGGAGCTGAGGAGTGTGAGCGTGCCGTCATCGACTTTGTGGCTTTCCAGGACATCCCATCAAGAGGCTGCAGCGGCTGCTGCAG GCCCTCGAGGCCCCGGAGGGCTGGGGTCCGACACCAAGGGCGGGCCGCGCGGCCTTGCAGCTGAAGCTGCGTCGGCGGCTCACGGAGCT CCTGGGGGCGCAGGACGGGGCGCTGCTGGTGCGGCTGCTGCAGGCGCTGCGCGTGGCCAGGATGCCCGGGCTGGAGCGGAGCGTCCGTG AGCGCTTCCTCCCTGTGCACTGATCCTGGCCCCCTCTTATTTATTCTACATCCTTGGCACCCCACTTGCACTGAAAGAGGCTTTTTTTT AAATAGAAGAAATGAGGTTTCTTAAAAAAAAAAAAAAAAAAAAAA

DcR3 1745

Chromosome location and linkages Human chromosome 20q13. Nearest linked marker: AFM218xe7 (Pitti et al., 1998).

PROTEIN

Accession numbers AF104419_1

Sequence See Figure 2.

Description of protein The DcR3 polypeptide is 300 amino acids long; the mature secreted protein is predicted to be 277 amino acids long. Immunoprecipitation of native DcR3 from conditioned media of the HCT116 colon carcinoma cell line, which shows about 4-fold amplification of the DcR3 gene, reveals a relative molecular mass of aproximately 37 kDa (Figure 3).

Relevant homologies and species differences There is one other soluble, secreted TNFR homolog known, namely, OPG (Simonet et al., 1997). DcR3 shows sequence similarity in particular to OPG (31%) (Figure 4), and TNFRII (29%), and relatively less homology to Fas (17%). All the cysteines in the four CRDs of DcR3 and OPG are conserved; however, the C-terminal portion of DcR3 is 101 residues shorter. So far, only the human DcR3 gene has been reported.

Affinity for ligand(s) A recombinant DcR3-Fc fusion protein (immunoadhesin) was used to study ligand interactions (Pitti

et al., 1998). DcR3-Fc bound specifically to human 293 cells transfected with full-length FasL (Suda et al., 1993), but not to cells transfected with TNF (Pennica et al., 1984); Apo2L/TRAIL (Wiley et al., 1995; Pitti et al., 1996); Apo3L/TWEAK (Chicheportiche et al., 1997; Marsters et al., 1998); or TRANCE/ RANKL/OPGL (Anderson et al., 1997; Wong et al., 1997; Lacey et al., 1998). DcR3-Fc coimmunoprecipitated purified soluble FasL, or shed soluble FasL from cells transfected with full-length FasL (Pitti et al., 1998). Size-exclusion chromatography confirmed that DcR3-Fc and soluble FasL formed a stable complex. Equilibrium binding analysis indicated that DcR3-Fc bound to soluble FasL with a Kd of 0.8 nM, compared to a Kd of 1.1 nM for Fas-Fc. DcR3-Fc blocked binding of soluble FasL to Fas-Fc, suggesting that the binding sites for the two receptors on FasL overlap. Recent work (Yu et al., 1999) confirms the binding of DcR3 to FasL, and demonstrates that DcR3 also binds to LIGHT, a TNF family member that binds two other receptors: HVEM and LT R (Mauri et al., 1998).

Cell types and tissues expressing the receptor Northern blot analysis indicated expression of a predominant 1.2 kb DcR3 mRNA transcript in human fetal lung, brain, and liver, and in human adult spleen, colon, and lung (Pitti et al., 1998), as well as in human umbilical vein endothelial cells and in phorbol esterstimulated Jurkat T cells (Yu et al., 1999). In addition, a relatively high DcR3 mRNA level was observed in the human colon carcinoma cell line SW480 (Pitti et al., 1998). DcR3 protein was detected in conditioned medium of HCT116 human colon carcinoma cells, which overexpress the DcR3 mRNA, but not in human colon carcinoma Colo205 cells, which do not overexpress DcR3 (Figure 3). DcR3 mRNA was also detected in a number of primary tumor specimens by in situ hybridization; expression of DcR3 message was detected specifically over malignant areas of the tumors (Pitti et al., 1998).

Figure 2 Amino acid sequence of the DcR3 protein. Protein Sequence MRALEGPGLSLLCLVLALPALLPVPAVRGVAETPTYPWRDAETGERLVCAQCPPGTFVQRPCRRDSPTTCGPCPPRHYTQFWNYLERCR YCNVLCGEREEEARACHATHNRACRCRTGFFAHAGFCLEHASCPPGAGVIAPGTPSQNTQCQPCPPGTFSASSSSSEQCQPHRNCTALG LALNVPGSSSHDTLCTSCTGFPLSTRVPGAEECERAVIDFVAFQDISIKRLQRLLQALEAPEGWGPTPRAGRAALQLKLRRRLTELLGA QDGALLVRLLQALRVARMPGLERSVRERFLPVH

1746 Avi Ashkenazi

HCT116

kDa

Colo205

Figure 3 Migration of native DcR3 protein on SDS-PAGE. Human HCT116 or COLO205 cell lines were cultured for 4 days. Conditioned media were collected, and subjected to immunoprecipitation and western blot analysis with mouse anti-human DcR3 monoclonal antibody.

60

42 DcR3 30

Figure 4 Schematic representation of the DcR3 and OPG proteins. The cysteine-rich domains (CRDs) are indicated by ovals; cysteines are indicated by vertical lines.

primary lung tumors, and 9 of 17 primary colon tumors showed genomic amplification of DcR3, ranging from 2- to 18-fold. Epicenter analysis in the nine colon tumors that had DcR3 amplification showed that DcR3 was amplified significantly, in contrast to neighboring chromosomal regions; this result is consistent with the possibility that amplification of DcR3 is functionally relevant to tumorigenesis. Analysis of DcR3 mRNA expression in primary tumor tissue sections by in situ hybridization revealed clear DcR3 expression in 2/2 colon tumors, 1/4 lung tumors, 2/5 breast tumors, and 1/1 gastric tumor. DcR3 message was typically localized to malignant areas of the tumors, but was absent from surrounding stromal tissue, indicating tumor-specific expression. There are several modes through which DcR3 overproduction could potentially provide growth/ survival advantages to tumors. (a) Because immunecytotoxic cells use FasL (among other mechanisms) to trigger apoptosis in cancer cells, tumors that amplify DcR3 may use the decoy to evade FasL-dependent immune-surveillence (Pitti et al., 1998). (b) Recent evidence suggests that FasL is inducibly expressed in several tissues; if tumor cells induce FasL expression in nearby stromal cells, then DcR3 might serve to protect tumor cells against apoptosis-induction by stromal FasL (Green, 1988). (c) DNA-damaging agents can induce FasL expression in tumor cells; DcR3 amplification might protect tumor cells against chemotherapy-induced, FasL-dependent apoptosis (Green, 1988). (d) DcR3 might contribute directly to tumorigenesis, perhaps by cooperation with oncogenes such as Myc (Green, 1988).

Regulation of receptor expression There is frequent overexpression of DcR3 in certain types of cancer.

Release of soluble receptors DcR3 is a secreted soluble protein. A membraneassociated form of the protein has not been found.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Human abnormalities The DcR3 gene is amplified frequently in certain types of human cancer (Pitti et al., 1998). Eight of 18

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain Three cell-based assay systems were used to investigate whether binding of DcR3 inhibits FasL activity (Pitti et al., 1998). First, apoptosis-induction by transient transfection of full-length FasL in HeLa cells, which express Fas, was studied. FasL induced apoptosis in about 25% of the cells; Fc-tagged DcR3 or Fas completely blocked this effect. Second, AICD of peripheral blood T cells, a process that involves endogenous FasL (Nagata, 1997), was examined. Consistent with previous results (Dhein et al., 1995), T cell receptor engagement with anti-CD3 antibody increased the level of apoptosis in IL-2-stimulated CD4+ T cells by 2-fold. Fc-tagged DcR3 or Fas blocked this effect substantially. Third, the killing of

DcR3 1747 Fas-expressing target tumor cells by peripheral blood NK cells, which involves FasL (Arase et al., 1995; Medvedev et al., 1997), was investigated. NK cells triggered significant cell death in Jurkat T leukemia cells; Fc-tagged DcR3 or Fas inhibited target cell killing substantially, whereas control IgG did not. These results indicated that binding of DcR3 inhibits FasL activity. DcR3-Fc also was shown to inhibit apoptosis-induction in human colon carcinoma H29 cells by LIGHT or LIGHT plus IFN (Yu et al., 1999). Hence, DcR3-immunoadhesins may be useful for treating pathologic conditions that involve excessive activity of FasL and/or LIGHT.

Effects of inhibitors (antibodies) to receptors Because DcR3 overexpression appears to be functionally relevant to tumorigenesis, antibodies that block the interaction of DcR3 with FasL and LIGHT and/or augment clearance of DcR3 from the blood circulation might be useful for treating cancers that overproduce DcR3.

References Anderson, D. M., Maraskovsk, E., Billingsley, W. L., Dougall, W. C., Tometsko, M. E., Roux, E. R., Teepe, M. C., DuBose, R. F., Cosman, D., and Galibert, L. (1997). A homolog of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390, 175±179. Arase, H., Arase, N., and Saito, T. (1995). Fas-mediated cytotoxicity by freshly isolated natural killer cells. J. Exp. Med. 181, 1235±1238. Ashkenazi, A., and Dixit, V. M. (1998). Death receptors: signaling and modulation. Science 281, 1305±1308. Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu, Y., Scott, H., Hession, C., and Browning, J. L. (1997). TWEAK, a new secreted ligand in the TNF family that weakly induces apoptosis. J. Biol. Chem. 272, 32401±32410. Dhein, J., Walczak, H., Baumler, C., Debatin, K. M., and Krammer, P. H. (1995). Autocrine T-cell suicide mediated by Apo1/(Fas/CD95). Nature 373, 438±441. Emery, J. G., McDonell, P., Burke, M. C., Deen, K. C., Lyn, S., Silverman, C., Dul, E., Appelbaum, E. R., Eichman, C., DiPrinzio, R., Dodds, R. A., James, I. E., Rosenberg, M., Lee, J. C., and Young, P. R. (1998). Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J. Biol. Chem. 273, 14363± 14367. Green, D. (1988). Death deciever. Nature 396, 629±630. Itoh, N., and Nagata, S. (1993). A novel protein domain required for apoptosis: mutational analysis of human Fas antigen. J. Biol. Chem. 268, 10932±10937. Lacey, D. L., Timms, E., Tan, H. L., Kelley, M. J., Dunstan, C. R., Burgess, T., Elliot, R., and Boyle, W. J. (1998). Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165±176. Marsters, S. A., Sheridan, J. P., Pitti, R. M., Brush, J., Goddard, A., and Ashkenazi, A. (1998). Identification of a

ligand for the death-domain-containing receptor Apo3. Curr. Biol. 8, 525±528. Mauri, D., Ebner, R., Montgomery, R. I., Kochel, K. D., Cheung, T. C., Guo-Liang, Y., Ruben, S., Murphy, M., Eisenberg, R. J., Cohen, G. H., Spear, P. G., and Ware, C. F. (1998). LIGHT, a new member of the TNF superfamily, and lymphotoxin alpha are ligands for Herpesvirus entry mediator. Immunity 8, 21±30. Medvedev, A. E., Johnsen, A. C., Haux, J., Steinkjer, B., Egeberg, K., Lynch, D. H., Sundan, A., and Espevik, T. (1997). Regulation of Fas and Fas ligand expression in NK cells by cytokines and the involvement of Fas ligand in NK/ LAK cell-mediated cytotoxicity. Cytokine 9, 394±404. Moretta, A. (1997). Molecular mechanisms in cell-mediated cytotoxicity. Cell 90, 13±18. Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355±365. Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B., and Goeddel, D. V. (1984). Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 312, 724±729. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996). Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor receptor family. J. Biol. Chem. 271, 12697±12690. Pitti, R., Marsters, S. A., Lawrence, D. A., Roy, M., Kischkel, F. C., Dowd, P., Huang, A., Donahue, C. J., Sherwood, S. W., Baldwin, D. T., Godowski, P. J., Wood, W. I., Gurney, A. L., Hillan, K. J., Cohen, R. L., Goddard, A. D., and Botstein, D., A, A. (1998). Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396, 699±703. Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang, M. S., Luthy, R., Nguyen, H. Q., Wooden, S., Bennet, L., Boone, T., Shimamoto, G., DeRose, M., Elliot, R., Colombero, A., Tan, H. L., Trail, G., Sullivan, J., Davy, E., Bucay, N., and Boyle, W. J. (1997). Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89, 309±319. Smith, C. A., Farrah, T., and Goodwin, R. G. (1994). The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76, 959±962. Suda, T., Takahashi, T., Golstein, P., and Nagata, S. (1993). Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75, 1169±1178. Tanaka, M., Itai, T., Adachi, M., and Nagata, S. (1998). Downregulation of Fas ligand by shedding. Nature Med. 4, 31±36. Tartaglia, L. A., Ayers, T. M., Wong, G. H. W., and Goeddel, D. V. (1993). A novel domain within the 55 kd TNF receptor signals cell death. Cell 74, 845±853. Tschopp, J., Irmler, M. a., and Thome, M. (1998). Inhibition of Fas death signals by FLIPs. Curr. Opin. Immunol. 10, 552±558. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl, J. K., Sutherland, G. R., Davis-Smith, T., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995). Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673±682. Wong, B. R., Rho, J., Arron, J., Robinson, E., Orlinick, J., Chao, M., Kalachikov, S., Cayani, E., Bartlet, F. S., Frankel, W. N., Lee, S. W., and Choi, Y. (1997). TRANCE is a novel ligand of the TNFR family that activates c-Jun-N-terminal kinase in T cells. J. Biol. Chem. 272, 25190±25194. Yu, K., Kwon, B., Ni, J., Zhai, Y., Ebner, R., and Kwon, B. (1999). A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis. J. Biol. Chem. 274, 13733±13736.