[CANCER RESEARCH 63, 432– 440, January 15, 2003] Melanoma Metastasis Suppression by Chromosome 6: Evidence for a Pathway Regulated by CRSP3 and TXNIP 1 Steven F. Goldberg, 2 Mary E. Miele, Naohito Hatta, Minoru Takata, 3 Carrie Paquette-Straub, Leonard P. Freedman, 4 and Danny R. Welch 5 Jake Gittlen Cancer Research Institute, Penn State College of Medicine, Hershey, Pennsylvania 17033 [S. F. G., D. R. W.]; Department of Medical Technology, University of Delaware, Newark, Delaware 19716 [M. E. M., C. P-S.]; The Penn State-National Foundation for Cancer Research Center for Metastasis Research, Hershey, Pennsylvania 17033 [D. R. W.]; Department of Dermatology, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa, 920-8640 Japan [N. H., M. T.]; and Cell Biology Program, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division, Joan and Sanford I. Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10021 [L. P. F.] ABSTRACT Loss of genetic material on chromosome 6 has been associated with progression of human melanomas. We showed previously that introducing chromosome 6 into metastatic human melanoma cell lines suppresses metastasis without affecting the ability of the hybrids to form progres- sively growing tumors. By subtractive hybridization comparing nonmeta- static chromosome 6-containing (neo6/C8161) versus parental (C8161) metastatic cells, the KISS1 metastasis suppressor gene was isolated. How- ever, KISS1 mapped to chromosome 1q32. To identify upstream regula- tor(s) of (and downstream effectors of) KISS1, microarray hybridization comparing C8161 and neo6/C8161 variants was performed. TXNIP/ VDUP1, a thioredoxin-binding protein, was expressed more highly in neo6/C8161 and in nonmetastatic melanomas. Increased TXNIP expres- sion inhibited metastasis and up-regulated KISS1. Surprisingly, TXNIP also mapped to chromosome 1q. PCR karyotyping that refined the region on chromosome 6 identified CRSP3/DRIP130, a transcriptional coactiva- tor, as a metastasis suppressor. CRSP3 transfectant cells had up-regulated KISS1 and TXNIP expression and were suppressed for metastasis. Quan- titative real-time reverse-transcription PCR of clinical melanoma samples showed that loss of CRSP3 expression correlated with decreased KISS1 expression and increased metastasis. Thus, we implicated a specific gene on chromosome 6 in the etiology of melanoma metastasis and identified potential up-stream regulators of KISS1 and TXNIP. INTRODUCTION Metastasis is the culmination of neoplastic progression toward autonomy from host regulation. Acquiring metastatic potential is apparently “driven” by genomic instability with coincident or super- imposed selection pressures (1). To be metastatic, tumor cells must complete a sequential, multistep cascade involving complex interac- tions between tumor and host. Cells must navigate a series of obsta- cles, including immune surveillance, loss of original tissue context, invasion through the basement membrane, lymphatic or hematoge- nous circulation, extravasation, and growth at the secondary site. Any cell shed from the primary neoplasm that fails to complete all requisite steps cannot form metastases (2, 3). We have shown that the MMCT 6 of human chr6 into metastatic human melanoma cell lines C8161 or MelJuSo suppressed metastasis without blocking tumor formation (4, 5). Metastasis-suppressed hy- brids of C8161 (neo6/C8161) were blocked at steps in the metastatic cascade affecting the survival and proliferation at secondary sites (6). We used subtractive hybridization to identify a metastasis suppressor gene, KISS1, which mapped to 1q32 (7–10). Tumor cells transfected with KISS1 cDNA were nonmetastatic, but remained tumorigenic. MMCT of chr6 with a deletion (40 Mb) spanning 6q16.3-q23 (neo6qdel) did not suppresses metastasis (11). Because KISS1 was cloned from C8161 cells, the genetic defect must be at the level of KISS1 regulation, not in KISS1 itself. Also, because neo6qdel hybrids did not express KISS1, the upstream regulator(s) of KISS1 was mapped between 6q16.3-q23 (11). Shirasaki et al. verified this hy- pothesis in melanoma clinical samples by showing loss of heterozy- gosity of markers in that region correlated with decreased KISS1 expression (12). KISS1 is a precursor for secreted neuropeptide ligands [designated Metastin (13) or Kisspeptins (14)] for a G-protein coupled [named hOT7T175 (13), AXOR12 (15), or hGPR54 (14)]. Although hOT7T175-transfected B16/BL6 melanoma-injected mice treated with Metastin developed fewer lung metastases (13), the mechanism of KISS1 suppression is still unknown. Recent publications suggest possible mechanisms for KISS1 metastasis suppression. Metastin induces Ca 2+ in receptor-transfected CHO cells (13), as well as phosphorylation of ERK1/2 and weak phosphorylation of p38/MAPK but not of SAPK/JNK (14). Metastin inhibits motility, chemotaxis, and invasion in vitro (13, 16), possibly by repressing the transcription of MMP-9 [via induction of cytosolic IB (17)]. Metastin also induces excessive formation of focal adhesions and stress fibers in hOT7T175-transfected B16/BL6 and induces the phosphorylation of FAK and paxillin (13), possibly through Rho (14). The primary objective of the current study was to identify the upstream regulator(s) of KiSS1 on chr6. Additionally, microarray hybridizations using metastatic (C8161) and nonmetastatic (neo6/ C8161) variants of a human melanoma cell line could be useful in identifying some additional downstream regulators of KISS1. MATERIALS AND METHODS Cell Lines and Culture. C8161 is a human melanoma cell line that metasta- sizes after orthotopic or i.v. injection into athymic mice (18). C8161.9 is a highly metastatic subclone of C8161 (7). Nonmetastatic neo6/C8161.1, neo6/C8161.2, neo6/C8161.3, and neo6/C8161.6 were derived by MMCT using the MCH262A1.D6 donor cell line (4). Metastatic neo6(del)(q16.3-q23)/C8161 hy- brids (neo6qdel/C8161.9.1, neo6qdel/C8161.9.2, neo6qdel/C8161.9.7, neo6qdel/ Received 8/6/02; accepted 11/14/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA66128 (to D. R. W.), CA87728 (to D. R. W.), and CA88876 (to M. E. M.); the National Foundation for Cancer Research (to D. R. W.); a Grant-in-Aid for Scientific Research (C2-12670812) from the Japan Society for the Promotion of Science (to N. H. and M. T.); a predoctoral fellowship from the Foreman Foundation (to S. F. G. and D. R. W.); and the Jake Gittlen Memorial Golf Tournament (to D. R. W.). 2 Present address: Laboratory of Biosystems and Cancer, National Cancer Institute, Building 37/Room 5046/MSC 4264, 37 Convent Drive, Bethesda, Maryland 20892. 3 Present address: Toyama Prefectural Central Hospital, 2-2-78 Nishinagae, Toyama, 930-8550 Japan. 4 Present address: Department of Bone Biology, Merck Research Laboratories, WP26A-1000, West Point, Pennsylvania 19486. 5 To whom correspondence should be addressed at: Department of Pathology, Uni- versity of Alabama at Birmingham, 1670 University Boulevard, Volker Hall G-038, Birmingham, AL 35294-0019. E-mail: dwelch@path.uab.edu; Phone: (205) 934-2956; Fax: (205) 934-1775. 6 The abbreviations used are: MMCT, microcell-mediated chromosome transfer; chr, chromosome; DME-F12, 1:1 mixture of DMEM and Ham’s F-12 medium; cDME-F12, mixture of DME-F12 and 5% fetal bovine serum; CMF-DPBS, Ca 2+ /Mg 2+ -free Dulbec- co’s PBS; TRX, thioredoxin; VGP, vertical growth phase; MTD, mean tumor diameter; RT-PCR, reverse-transcription PCR; RTQ, quantitative real-time RT-PCR; FAM, 6- carboxyfluorescein; TAMRA, 6-carboxy-tetramethyl-rhodamine. 432 Research. on June 25, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from