Monochromosome Transfer and Microarray Analysis Identify a Critical Tumor-Suppressive Region Mapping to Chromosome 13q14 and THSD1 in Esophageal Carcinoma Josephine M.Y. Ko,1 Pui Ling Chan,1 Wing Lung Yau,1 Ho Kin Chan,1 King Chi Chan,1 Zhuo You Yu,1 Fung Mei Kwong,1 Lance D. Miller,6 Edison T. Liu,6 Li Chun Yang,1 Paulisally H.Y. Lo,1 Eric J. Stanbridge,7 Johnny C.O. Tang,2,3 Gopesh Srivastava,3 Sai Wah Tsao,4 Simon Law,5 and Maria L. Lung1 1Department of Biology and Center for Cancer Research, Hong Kong University of Science and Technology; 2Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University; Departments of 3Pathology, 4Anatomy, and 5Surgery, University of Hong Kong, Hong Kong (Special Administrative Region), People’s Republic of China; 6Cancer Biology and Pharmacology, Genome Institute of Singapore, Biomedical Sciences Institutes, Singapore; and 7Department of Microbiology and Molecular Genetics, University of California, Irvine, California Abstract 100% of ESCC and other cancer cell lines. Mechanisms Loss of chromosome 13q regions in esophageal for THSD1 silencing in ESCC involved loss of squamous cell carcinoma (ESCC) is a frequent event. heterozygosity and promoter hypermethylation, as Monochromosome transfer approaches provide analyzed by methylation-specific PCR and clonal direct functional evidence for tumor suppression by bisulfite sequencing. Transfection of wild-type THSD1 chromosome 13 in SLMT-1, an ESCC cell line, and into SLMT-1 resulted in significant reduction of identify critical regions at 13q12.3, 13q14.11, and colony-forming ability, hence providing functional 13q14.3. Differential gene expression profiles of three evidence for its growth-suppressive activity. These tumor-suppressing microcell hybrids (MCH) and findings suggest that THSD1 is a good candidate TSG. their tumorigenic parental SLMT-1 cell line were (Mol Cancer Res 2008;6(4):592–603) revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed Introduction down-regulation in SLMT-1, compared with NE1, an Esophageal cancer is geographically diverse, with only a immortalized normal esophageal epithelial cell line; 10.7% 5-year survival rate (1). Esophageal squamous cell carci- their average gene expression was restored in MCHs noma (ESCC) is the major histologic form. ESCC molecular compared with SLMT-1. Reverse transcription-PCR pathogenesis still remains poorly understood. validated gene expression levels in MCHs and a Chromosome 13q deletions are frequent events in several panel of ESCC cell lines. Results suggest that the human cancers, including ESCC (2, 3), nasopharyngeal (4), and tumor-suppressing effect is not attributed to RB1, lung (5) cancers. This current study is the first functional study but instead likely involves thrombospondin type I of the tumor-suppressive role of chromosome 13 in ESCC and domain-containing 1 (THSD1), a novel candidate TSG is initiated by the high-frequency ESCC chromosome 13q mapping to 13q14. Quantitative reverse transcription- losses detected by comparative genomic hybridization (3) and PCR detected down-regulation of THSD1 expression in loss of heterozygosity (LOH) studies (2, 6-9). Comparative genomic hybridization analysis showed 100% losses on chromosome 13q in 17 ESCC cases (3). The extremely high 13q loss was independently verified in genome-wide LOH Received 4/4/07; revised 11/4/07; accepted 12/20/07. studies (2), with 95% of 77 13q markers showing LOH. Thus, Grant support: Research Grants Council of the Hong Kong Special Administration Region, China (M.L. Lung), for The Hong Kong University of functional inactivation of TSGs on chromosome 13q is likely Science and Technology 6415/06M grant. key to ESCC development. The costs of publication of this article were defrayed in part by the payment of Monochromosome transfer into tumorigenic cell lines allows page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. functional complementation of existing defects and study of Note: Supplementary data for this article are available at Molecular Cancer tumor-suppressive effects driven by native endogenous regula- Research Online (http://mcr.aacrjournals.org/). tory environments, with control of single gene copy number Requests for reprints: Maria Li Lung, Department of Biology and Center for Cancer Research, Hong Kong University of Science and Technology, Clear Water gains more closely mimicking normal physiologic levels. Our Bay, Kowloon, Hong Kong (Special Administrative Region), People’s Republic previous studies identified tumor-suppressive regions localized of China. Phone: 852-2358-7307; Fax: 852-2358-1559. E-mail: [email protected] Copyright D 2008 American Association for Cancer Research. to 3p14 (10), 9q33-34 (11), and 14q32 (12) in the ESCC cell doi:10.1158/1541-7786.MCR-07-0154 line SLMT-1. This present study examines the tumor-suppressive 592 Mol Cancer Res 2008;6(4). April 2008 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. 13q14 Critical Region and THSD1 in Esophageal Cancer 593 FIGURE1. Chromosome 13 allelotyping of SLMT-1 with 50 markers revealed two regions of contiguous homozygosity in SLMT-1, one with 10 markers at 13q12.11-13q12.3 and the other with 27 markers from the 13q13.3 region to the telomeric end of chromosome 13q. Microsatellite typing – deletion mapping analysis of chromosome 13 MCHs and TSs delineated four CRs within regions 13q12.3 (CR1 at D13S1299-D13S1229 and CR2 at D13S1226), 13q14.11 (CR3 at D13S263), and 13q14.3 (CR4 at D13S133, which is 0.292 Mb from THSD1) due to nonrandom loss at specific markers in the TSs. The presence (o), endogenous loss ( ), and exogenous loss (.) of markers are depicted. 1, homozygous allelic pattern; 2, heterozygous alleles are present in NE1, EC18, KYSE180, and SLMT-1;*, genes mapping to this region. Mol Cancer Res 2008;6(4). April 2008 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. 594 Ko et al. role of chromosome 13 in SLMT-1. Comparative differential 13 transfer into MCH13-111, MCH13-113, and MCH13-117 gene expression observed after competitive hybridization in (Supplementary Fig. S1). cDNA oligonucleotide microarrays of the tumorigenic parental PCR microsatellite typing confirmed the presence of donor cell line and three tumor-suppressing chromosome 13 micro- and recipient alleles, validating the successful transfer of cell hybrids (MCH) identified novel candidate TSGs. chromosome 13 into SLMT-1 in three MCH13 cell lines. Of Thrombospondin type I domain-containing 1 (THSD1), at 50 microsatellite markers, 37 were informative. An ideogram 13q14.3, showed 100% down-regulation in a panel of 18 ESCC summarizes the genotyping results (Fig. 1). and other cancer cell lines. Transfection of wild-type THSD1 into SLMT-1 significantly reduced colony formation, provid- Tumorigenicity Assay of MCH Cell Lines ing functional evidence for growth-suppressive activity. Statistically significant delays in tumor growth kinetics and The current data indicate that the mechanism for THSD1 reduced tumor sizes were observed with all three chromosome down-regulation in ESCC involved both LOH and epigenetic 13 hybrids compared with the parental cell line (Fig. 2A). The silencing. Demethylation treatment restored THSD1 expression tumorigenic potentials of MCH13-111 and MCH13-113 in THSD1 down-regulated ESCC cell lines; results from methylation-specific PCR (MSP) analysis of ESCC cell lines and bisulfite sequencing of the promoter region of THSD1 in both cell lines and primary tissues showed that loss of THSD1 expression could be partially attributed to hypermethylation in ESCC. Results Chromosome 13 Allelotyping of Cell Lines Fifty markers were used for allelotyping ESCC SLMT-1 and human chromosome 13 donor cell line, MCH204.3 (Fig. 1). SLMT-1 contained two regions of contiguous homozygosity, one with 10 markers at 13q12.11-13q12.3 and another with 27 markers at the 13q13.3 region to the telomeric end of chromosome 13q. The two consecutive homozygous regions only contain a single allele in each of the 37 loci studied. The random chance for this occurring in a diploid genome is small. These results suggest nearly a complete deletion of a single copy of chromosome 13 in SLMT-1 cells. The loss of 13q was not only observed molecularly by allelotyping but also by comparative genomic hybridization analysis (data not shown). Loss of one copy of nearly the entire chromosome 13 in SLMT-1 strongly suggested the presence of TSGs, based on the assumption that the relevant genes in the remaining allele would also be inactivated. Transfer of Human Chromosome 13 into an ESCC Cell Line Fluorescence in situ hybridization (FISH) of SLMT-1 using human chromosome 13 WCP probe showed one intact signal of chromosome 13 and six signals representing translocations to other chromosomes (Supplementary Fig. S1). None of the morphologies of chromosome 13 detected in the meta- phase spreads appeared normal. Using microcell-mediated chromosome transfer, eight chromosome 13 MCH cell lines were obtained. Screening by DNA slot blot hybridization confirmed that five MCHs were mouse DNA-free (data not shown). FIGURE2. A . Tumor growth kinetics of the tumorigenic recipient, SLMT-1, were compared with chromosome 13 MCHs: MCH13-111, FISH and Microsatellite Typing Analysis of Chromosome MCH13-113, and MCH13-117. Points, average volume of six inoculation sites. All three chromosome 13 MCH cell lines were tumor suppressive. 13 MCHs The tumor growth kinetics of the chromosome 13 MCHs were compared The distinctive acrocentric pattern of the transferred exo- with their corresponding tumor segregants, MCH13-111/TS2R and genous chromosome 13 in the metaphases of chromosome 13 MCH13-117/TS1L. B. Representative results of D13S133 microsatellite typing at CR4 for the recipient SLMT-1, donor MCH204.3, hybrid MCH13- MCHs enabled its precise identification. Whole chromosome 111, and tumor segregant MCH13-111/TS2R.