Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, Mirror image polydactyly 1, in nasopharyngeal carcinoma Arthur Kwok Leung Cheunga, Hong Lok Lungb, Josephine Mun Yee Kob, Yue Chenga,c, Eric J. Stanbridged, Eugene R. Zabarovskye, John M. Nichollsf, Daniel Chuab, Sai Wah Tsaog, Xin-Yuan Guanb, and Maria Li Lungb,1 aDepartment of Biology and Center for Cancer Research, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (SAR), People’s Republic of China; bDepartment of Clinical Oncology, University of Hong Kong, Pokfulam, Hong Kong (SAR), People’s Republic of China; cDepartment of Biology, City of Hope, Beckman Research Institute, Duarte,CA 91010; dDepartment of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697; eDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden; and Departments of fPathology and gAnatomy, University of Hong Kong, Pokfulam, Hong Kong (SAR), People’s Republic of China Edited by George Klein, Karolinska Institutet, Stockholm, Sweden, and approved July 7, 2009 (received for review January 7, 2009) Chromosome 14 allelic loss is common in nasopharyngeal carcinoma particular interest to us that chromosome 14 loss is associated with (NPC) and may reflect essential tumor suppressor gene loss in tumor- cancer metastasis in breast tumors (15) and with poor clinical prognosis igenesis. An intact chromosome 14 was transferred to an NPC cell line for other head and neck cancers (16). Thus, it is possible that a using a microcell-mediated chromosome transfer approach. Microcell chromosome 14 TSG may be a useful prognostic marker in NPC. hybrids (MCHs) containing intact exogenously transferred chromo- In this study, we obtained functional evidence showing definitively some 14 were tumor suppressive in athymic mice, demonstrating that that chromosome 14 is tumor suppressive in NPC. We used a microcell- intact chromosome 14 NPC MCHs are able to suppress tumor growth medicated chromosome transfer (MMCT) approach to investigate in mice. Comparative analysis of these MCHs and their derived tumor whether intact chromosome 14 can functionally complement existing segregants identified 4 commonly eliminated tumor-suppressive CRs. defects in an NPC cell line. CRs and candidate genes were identified. Here we provide functional evidence that a gene, Mirror-Image A presumptive TSG is up-regulated in microcell hybrids (MCHs) and down-regulated in tumor revertants arising after a long period of POLydactyly 1 (MIPOL1), which maps within a single 14q13.1–13.3 CR selection in vivo (17). A candidate TSG, Mirror-Image POLydactyly 1 and that hitherto has been reported to be associated only with a (MIPOL1), was identified in this study. MIPOL1 was first mapped in a developmental disorder, specifically suppresses in vivo tumor forma- patient with a genetic disease resulting in mirror-image polydactyly of tion. MIPOL1 gene expression is down-regulated in all NPC cell lines hands and feet (18) and mild craniofacial and acallosal central nervous and in Ϸ63% of NPC tumors via promoter hypermethylation and system midline defects (19). Of perhaps only incidental interest is a allelic loss. SLC25A21 and FOXA1, 2 neighboring genes mapping to recent transcriptome screening study identifying the insertion of the this region, did not show this frequent down-regulated gene expres- DGKB and neighboring ETV1 genes into the MIPOL1 gene, resulting sion or promoter hypermethylation, precluding possible global meth- in up-regulation of the ETV1 oncogene in prostate cancer (20). To date, ylation effects and providing further evidence that MIPOL1 plays a however, the function of MIPOL1 is still unknown. This study links this unique role in NPC. The protein localizes mainly to the nucleus. gene, associated with a developmental disorder, to cancer. We inves- Re-expression of MIPOL1 in the stable transfectants induces cell cycle tigated its clinical relevance, cytolocalization, possible mechanisms of arrest. MIPOL1 tumor suppression is related to up-regulation of the inactivation, and its ability to suppress tumor formation in vivo. p21(WAF1/CIP1) and p27(KIP1) protein pathways. This study provides compelling evidence that chromosome 14 harbors tumor suppressor Results genes associated with NPC and that a candidate gene, MIPOL1,is Transfer of Intact Chromosome 14 Suppresses HONE1 Tumor Forma- associated with tumor development. tion. The MMCT approach was used to transfer an intact human chromosome 14 into the tumorigenic NPC cell line, HONE1, using microcell-mediated chromosome transfer ͉ MIPOL1 ͉ cell cycle donor MCH-D14-C2. Microsatellite typing and whole-chromo- arrest ͉ promoter hypermethylation some FISH were used to confirm the successful transfer of chro- mosome 14 into all 5 MCH cell lines (Fig. 1A and supporting information (SI) Fig. S1). Fig. 1B shows representative results from asopharyngeal carcinoma (NPC) is a unique malignancy that is the microsatellite analysis. Nparticularly prevalent among the southern Chinese but is rare The recipient HONE1 cell line is highly tumorigenic in nude mice, elsewhere. Numerous molecular alterations have been detected in this with palpable tumors consistently formed within 21 days and reaching cancer (1). Allelic loss of chromosome 14 and alterations in chromo- a size greater than 900 mm3 by 6 weeks after injection (Fig. 1C and some copy numbers are found commonly in NPC (1–3) as well as in a Table S1). The 5 MCHs suppress tumor growth in vivo, and only small wide variety of other cancers (4–7). Alterations in chromosome 14 also tumors were observed 6 weeks after injection. have been reported in both early-onset colon cancers (8) and menin- gioma progression (9). Hypermethylation and down-regulation of chro- Tumor Segregant Analysis Delineates Commonly Eliminated Regions mosome 14 genes are observed in glioblastomas (10) and oligodendro- and Demonstrates MIPOL1 Association with Tumor Suppression. Small 3 glial tumors (11). These findings suggest that potential tumor suppressor tumors (less than 250 mm ) form 6 weeks after the injection of genes (TSGs) mapping to chromosome 14 have a role in tumorigenesis. Deletion mapping of gastrointestinal (12), bladder (13), and ovarian Author contributions: A.K.L.C., H.L.L., J.M.Y.K., and M.L.L. designed research; A.K.L.C., (14) tumors identified chromosome 14 loci associated with tumor H.L.L., J.M.Y.K., and J.M.N. performed research; Y.C., E.J.S., E.R.Z., D.C., S.W.T., and X.-Y.G. suppression at 14q11.1-q12, 14q12–13, 14q23–24.3, and 14q32.1-q32.2. contributed new reagents/analytic tools; A.K.L.C., H.L.L., J.M.Y.K., J.M.N., and M.L.L. ana- In our previous NPC study, 2 critical regions (CRs) at 14q11.2–13.1 and lyzed data; and A.K.L.C., H.L.L., J.M.Y.K., E.J.S., and M.L.L. wrote the paper. 14q32.1 also were identified as being associated with growth suppres- The authors declare no conflict of interest. sion. Although CRs presumably harboring candidate TSGs have been This article is a PNAS Direct Submission. identified, no candidate TSGs mapping to chromosome 14 have been 1To whom correspondence should be addressed. E-mail: [email protected]. identified as yet in NPC. Thus, further investigation of the role of This article contains supporting information online at www.pnas.org/cgi/content/full/ chromosome 14 in NPC tumorigenesis is warranted. It is also of 0900198106/DCSupplemental. 14478–14483 ͉ PNAS ͉ August 25, 2009 ͉ vol. 106 ͉ no. 34 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900198106 Downloaded by guest on September 29, 2021 A B C Fig. 1. Chromosome 14 microsatellite typing and tumorigenicity studies. (A) Microsatellite typing analysis of 5 MCH cell lines (14-E, -F, -M, -T, and -U) and their corresponding TSs. A total of 22 microsatellite markers were used. The donor MCH-D14-C2 (D), recipient cell line HONE1 (H), presence (open circles), absence (filled circles), and uninformative (U) status of markers and CRs are as indicated. The locations of the MIPOL1, SLC25A21, and FOXA1 genes and the RP11–460G19 and RP11–1116E12 BAC clones are shown on the left, and CRs 1–4 are shown on the right. (B) Representative microsatellite analysis of marker D14S75 for donor, HONE1, MCH-NPC-14T, and MCH-NPC-14T-TS2. An inverted open triangle designates the exogenous donor allele transfer, and an inverted filled triangle indicates the loss of an allele in MCH-NPC-14T-TS2, respectively. (C) In vivo tumorigenicity assay of the recipient NPC cell line, HONE1, 5 chromosome 14 MCHs (MCH-NPC-14-E, -F, -M, -T, and -U) and MCH-NPC-14E-TS10, -14T-TS2, and -14U-TS1. The curves represent an average tumor volume of all sites inoculated for each cell population. Statistically significant differences in tumor growth were observed for HONE1 compared with 5 MCHs and their TSs. chromosome 14 MCHs. All tumor revertants were excised and tent with the loss of MIPOL1 and the nearby CR in the TS being GENETICS established as tumor segregant (TS) cell lines for subsequent associated with a subsequent increased tumorigenicity but do not analysis. The long period required for the emergence of these exclude the possibility that other regional genes may be involved. tumors is consistent with occurrence of in vivo selection from a majority population of non-tumorigenic cells. Using a panel of 22 MIPOL1 Gene and Protein Expression Levels in MCHs, TSs, and NPC Cell microsatellite markers spanning the entire chromosome 14q arm, Lines and Patient Tumors. Up-regulation of MIPOL1 at both protein we performed PCR-based microsatellite typing to compare the and mRNA levels was observed in the MCHs when compared with genotyping of the MCHs and their matched TSs. HONE1 cells, and extensive down-regulation of MIPOL1 was We defined 4 commonly eliminated CRs associated with tumor observed in the corresponding TSs (Fig.