Human Cancer Biology Identification and Characterization of a Novel Melanoma Tumor Suppressor Gene on Human Chromosome 6q21 Jackie Mei-Wah Fung,1Ross Smith,3 Melissa A. Brown,3 Sze Hang Lau,1Dan Xie,4 George K. Lau, 2 andXin-YuanGuan1,4 Abstract Purpose: By characterizing a complex chromosome rearrangement involving 6q and 17p in melanoma cell line UACC-930, we isolated a candidate tumor suppressor gene at 6q21, named prenyl diphosphate synthase subunit 2 (PDSS2), which was interrupted by an inversion breakpoint. The purpose of this study was to determine the tumor-suppressive potential of PDSS2 in the development of melanoma. Experimental Design: To isolate the rearranged 6q in UACC-930 cells, a bacterial artificial chromosome clone (RP1-67A8) covering the breakpoint at 6q21was digested with HindIII and each DNA fragment was used as the probe for the breakpoint in Southern blotting. The HindIII fragment probe covering the breakpoint was then used to screen an EcoRI-digested DNA library generated from UACC-930. To characterize the tumor-suppressive potential of PDSS2, PDSS2 was stably transfected into a highly tumorigenic melanoma cell line, UACC-903. The tumor-suppressive function of PDSS2 was shown by both in vitro and in vivo assays. The differential expression of PDSS2 in benign nevi and primary melanoma samples was also studied. Results: Down-regulation of PDSS2 was observed in 59 of 87 (67.8%) primary melanomas, which was significantly higher than that in benign nevi (7 of 66, 10.6%; P < 0.001). In addition, an overexpression of the PDSS2 in UACC-903 cells could inhibit tumor cell growth, decrease the colony-forming ability in soft agar, and totally abrogate the tumorigenicity of UACC-903 in nude mice. Conclusions: Our results support the proposal that PDSS2 is a novel tumor suppressor gene that plays an important role in the development of malignant melanoma. Melanoma is the most serious type of skin cancer (1). Genetic Several studies showed that 6q21 is the most frequent deleted alterations in melanoma have been widely studied and the region, suggesting the existence of a TSG at 6q21 (2, 3, 9). deletion of 6q is one of the most common chromosomal Although a couple of candidate TSGs at 6q, including AIM1 at changes (2–4). The reversion of the tumorigenic phenotype of 6q21 (10) and SOD2 at 6q25.3 (11), has been studied, the melanoma cells by introducing a normal human chromosome underlying mechanisms of these genes in melanoma develop- 6 provides the first biological evidence that chromosome 6 ment are unclear. In addition, the tumor-suppressive role of contains a tumor suppressor gene (TSG) associated with SOD2 is controversial. Miele et al. (12) found that SOD2 could melanoma pathogenesis (5). The abnormality of 6q has also not suppress tumorigenicity or metastasis of human melanoma been detected in other malignancies, including malignant C8161 cells. Therefore, more effort in looking for new candidate lymphoma (6), ovarian (7), and prostate carcinomas (8). TSGs at 6q related to melanoma pathogenesis is justified. In our previous study, we found a reciprocal-like transloca- tion between 6q21 and 17p13 in the melanoma cell line UACC- Authors’ Affiliations: Departments of 1Clinical Oncology and 2Medicine, The 930 (9). The translocation was further characterized by University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, chromosome microdissection and fluorescence in situ hybrid- 3 China; School of Molecular and Microbial Sciences,The University of Queensland, ization, and a complex chromosomal rearrangement was St. Lucia, Queensland, Australia; and 4State Key Laboratory of Oncology in Southern China, Cancer Center, SunYat-Sen University, Guangzhou, China detected that includes an inversion of 6q [inv(6)(q21q27)] Received 6/11/08; revised 10/23/08; accepted 10/25/08. and a translocation between the inverted 6q and 17p Grant support: Leung Kwok Tze Foundation, Sun Yat-Sen University ‘‘Hundred [t(6;17)(q27;p13)]. A bacterial artificial chromosome clone Talents Program’’ (85000-3171311), and The Major State Basic Research Program (RP1-67A8) spanning the inversion breakpoint at 6q21 was of China (2006CB910104). isolated by fluorescence in situ hybridization screening and 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 loss of one or more copies at 6q21 was detected in 10 of 12 with 18 U.S.C. Section 1734 solely to indicate this fact. melanoma cell lines (9). This result suggests that the bacterial Requests for reprints: Xin-Yuan Guan, Department of Clinical Oncology, The artificial chromosome clone may contain a TSG related to the University of Hong Kong, Room 56, 10/F Laboratory Block, 21 Sassoon melanoma development, which was interrupted by the inver- Road, Pokfulam, Hong Kong Special Administrative Region, China. Phone: 852-28199782; Fax: 852-28169126; E-mail: [email protected]. sion breakpoint. In the present study, we further characterized F 2009 American Association for Cancer Research. the breakpoints on the rearranged 6q in UACC-930 and found doi:10.1158/1078-0432.CCR-08-1472 a candidate TSG, named prenyl diphosphate synthase subunit 2 www.aacrjournals.org 797 Clin Cancer Res 2009;15(3) February 1, 2009 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2009 American Association for Cancer Research. Human Cancer Biology (Clontech). The open reading frame of PDSS2 flanked by the primers Translational Relevance 5¶-TACTGTAAGTTCCTCCTCTGC and 5¶-AATTTTGTTTGTAGCAGTTCC were amplified using PCR. Cycle sequencing reactions were set up using The incidence of melanoma has increased markedly over BigDye Terminator v3.1 Cycle Sequencing kits (Applied Biosystems) the past 40 years inWestern countries. Deletion of chromo- according to the manufacturer’s instructions. some 6q is one of the most frequently chromosomal Quantitative PCR. cDNA was subjected to quantitative PCR with a ¶ alterations in melanoma, suggesting the presence of tumor SYBR Green PCR kit (Applied Biosystems) using primers 5 -GAAT- CAGGTAGTGTCAGAGG and 5¶-GAGGCTATTCCAGCTGTCATG for suppressor gene(s) at 6q. In this article, we identified a PDSS2,whereas5¶-CTCTTAGCTGAGTGTCCCGC and 5¶-CTGAT- novel candidate tumor suppressor gene, prenyl diphos- CGTCTTCGAACCTCC for 18S rRNA. The amplification protocol con- phate synthase subunit 2 (PDSS2), which was interrupted sisted of incubations at 95jC for 15 s, 60jC for 1 min, and 72jC for by an inversion breakpoint in a melanoma cell line, UACC- 1 min for 40 cycles. Quantification was done using the ABI PRISM 930. Our study showed that PDSS2 was frequently 7900HT Sequence Detection System (Applied Biosystems). All quanti- down-regulated in primary melanomas and transfection of tative PCRs were done in triplicate. The CT of each gene of interest PDSS2 into a highly tumorigenic melanoma cell line [CT(gene of interest) test] in each sample was normalized with control C UACC-903 could completely reverse the tumorigenic 18S rRNA [ T(18S) test] for RNA amount variation and calibrator for C phenotypes. PDSS2 encodes an essential enzyme in- plate-to-plate variation using the following formula: D T(test) = C (gene of interest) test - C (18S) test. Relative expression level was volved in the coenzyme Q10 (CoQ10) biosynthetic pathway. T T Low CoQ level has been reported in different malignan- presented as the relative fold change and calculated using the following 10 -DDCT C C C cies, including melanoma. Supplement of CoQ has been formula: 2 =[ T(gene of interest) - T(18S)] test - [ T(gene of 10 interest) - C (18S)] calibrator. used in clinics for cancer treatment. We believe that this T Southern,Northern,and Western blot analysis. For Southern blot study provides a new insight in the melanoma develop- analysis, 10 Ag of genomic DNA from tumor cell lines were digested ment, which may lead to finding out new therapeutic with HindIII and EcoRV, fractionated on a 0.8% agarose gel, transferred targets in the future. to a nylon membrane (GE Healthcare), and hybridized with 32P-labeled probes overnight at 42jC. For Northern blot analysis, 10 Agof total RNA from each sample were fractionated on a 1% agarose gel, transferred to a nylon membrane, and hybridized with 32P-labeled (PDSS2), which was interrupted by the 6q21 breakpoint. The probes overnight at 65jC. sequence of PDSS2 (AF254956) was firstly submitted to For Western blot analysis, 10 Ag of protein extract were separated by National Cancer for Biotechnology Information by the authors 10% SDS-PAGE, transferred to a polyvinylidene difluoride Hybond-P in 2000 and was then named as C6orf210 by Human Genome membrane (GE Healthcare), and detected by a mouse monoclonal Organization. Recently, C6orf210 was found to be important in antibody specific against PDSS2 (1:1,000; Wolwo Biotech) and a goat h determining the length of the side chain of ubiquinone in polyclonal antibody against -actin (1:2,000; Santa Cruz Biotechnol- ogy). The antibody detects a single band at 37 kDa, which is the mammals and was named as PDSS2 (13). Mutation of PDSS2 estimated size of PDSS2 protein. had only been reported in Leigh syndrome with nephropathy Tissue microarray and immunohistochemistry. Archived paraffin and coenzyme Q10 (CoQ10) deficiency (14). In this article, the blocks from 99 melanomas and 82 benign nevi were collected from authors, for the first time, showed the tumor-suppressive ability the Cancer Center of Sun Yat-Sen University (Guangzhou, China). The of PDSS2 by both in vivo and in vitro assays. The results tissue microarray block was constructed according to the method indicated that PDSS2 may play an important role in the described previously (15). Immunohistochemistry was done using development of melanoma. standard antigen-antibody methods with the anti-PDSS2 antibody. As a negative control, the primary antibody was replaced with blocking serum in PBS. The antibody could specifically detect PDSS2 expression Materials and Methods (cytoplasmic staining) in most benign nevi and some of melanoma cells.