Research Article Frequent Silencing of the Candidate Tumor Suppressor PCDH20 by Epigenetic Mechanism in Non–Small-Cell Lung Cancers Issei Imoto,1,4 Hiroyuki Izumi,1,2 Sana Yokoi,1,4 Hiroshi Hosoda,5 Tatsuhiro Shibata,6,7 Fumie Hosoda,4,7 Misao Ohki,7 Setsuo Hirohashi,6 and Johji Inazawa1,3,4 1Department of Molecular Cytogenetics, Medical Research Institute and Graduate School of Biomedical Science, 2Thoracic and Cardiovascular Surgery, and 3Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone, Tokyo Medical and Dental University; 4Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation; 5Tokyo Kyosai Hospital; and 6Pathology Division and 7Cancer Genomics Project, National Cancer Center Research Institute, Tokyo, Japan Abstract varying considerably among members (1), whereas the amino acid Protocadherins are a major subfamily of the cadherin sequences of the other domains, in particular the cytoplasmic superfamily, but little is known about their functions and domain, vary significantly among members (2). More than 60 intracellular signal transduction. We identified a homozy- protocadherins have been identified and are currently a major gous loss of protocadherin 20 (PCDH20, 13q21.2) in the subfamily of the cadherin superfamily (3, 5). Protocadherins have course of a program to screen a panel of non–small-cell up to seven extracellular domains, a single transmembrane region, lung cancer (NSCLC) cell lines (1 of 20 lines) for genomic and divergent and distinct cytoplasmic portions, which also exhibit cell-to-cell adhesion activity, but the adhesion mechanism is copy number aberrations using an in-house array-based comparative genomic hybridization. PCDH20 mRNA was thought to be distinct from that of classic cadherins (6–8). expressed in normal lung tissue but was not expressed in Protocadherins are thought to have other important activities as the majority of NSCLC cell lines without a homozygous well, although the major functions of each have not been well deletion of this gene (10 of 19 lines, 52.6%). Expression of elucidated. A few members of the protocadherin family have been PCDH20 mRNA was restored in gene-silenced NSCLC cells suspected of involvement in the carcinogenesis of several tumors, after treatment with 5-aza 2V-deoxycytidine. The DNA such as colon cancer (9), hepatocellular carcinoma (9), renal cancer methylation status of the PCDH20 CpG-rich region correlat- (9, 10), and prostate cancer (11), through their overexpression or ed inversely with the expression of the gene and a putative inactivation. However, the association between protocadherin target region for methylation showed clear promoter proteins and the pathogenesis of many other cancers remains unclear. activity in vitro. Methylation of this PCDH20 promoter was frequently observed in primary NSCLC tissues (32 of 59 Genomic amplifications and homozygous deletions are believed tumors, 54.2%). Among our primary NSCLC cases, the to be useful for identifying oncogenes and tumor-suppressor genes, methylated PCDH20 seemed to be associated with a shorter respectively, critical to tumorigenesis. For example, several typical overall survival (P = 0.0140 and 0.0211 in all and stage I tumor-suppressor genes were originally pinpointed by mapping tumors, respectively; log-rank test), and a multivariate regions of biallelic loss in cancer cells (12–15) although the analysis showed that the PCDH20 methylation status was homozygous deletion of these genes is a rare event and other an independent prognosticator. Moreover, restoration of genetic and epigenetic mechanisms, such as mutation and PCDH20 expression in NSCLC cells reduced cell numbers in promoter methylation, mainly contribute to their functional colony formation and anchorage-independent assays. These inactivation. Therefore, the search for remarkable changes in copy results suggest that epigenetic silencing by hypermethylation number through the entire genome with high resolution will allow of the CpG-rich promoter region of PCDH20 leads to loss of precise and rapid identification of tumor-suppressor genes as well PCDH20 function, which may be a factor in the carcino- as oncogenes in cancer genomes. For this approach, we have applied several types of in-house bacterial artificial chromosome genesis of NSCLC. (Cancer Res 2006; 66(9): 4617-26) (BAC)–based array for array-based comparative genomic hybrid- ization (array-CGH) analysis (16–19). Introduction In the course of a program to screen a panel of non–small-cell Cadherins play an important role in the communication between lung cancer (NSCLC) cell lines for copy number aberrations in a adjacent cells. There are at least 80 members of the cadherin genome-wide manner using an in-house BAC array (16), we have superfamily in mammals including classic cadherins (1–3). They identified a homozygous loss of protocadherin 20 (PCDH20, are characterized by a unique domain called the cadherin motif or 13q21.2), of which the expression was absent in the majority of 2+ extracellular domain, containing Ca -dependent homophilic- NSCLC cell lines without homozygous loss, although it was present binding domains (4). The extracellular domain is tandemly in normal lung tissue. Further analysis using cell lines and primary repeated in the extracellular segment of all members of the tumors of NSCLC showed that the silencing of the PCDH20 gene cadherin superfamily, with the number of extracellular domains predominantly by epigenetic mechanisms may be involved in the pathogenesis of NSCLC. Requests for reprints: Johji Inazawa, Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Materials and Methods Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 81-3-5803-5820; Fax: 81-3-5803-0244; E-mail: Cell lines and primary tumors. Of the 20 NSCLC cell lines employed, [email protected]. I2006 American Association for Cancer Research. seven were derived from squamous-cell carcinomas (EBC-1, LK-2, PC10, doi:10.1158/0008-5472.CAN-05-4437 VMRC-LCP, LC-1 sq, ACC-LC-73, and KNS-62), seven from adenocarcinomas www.aacrjournals.org 4617 Cancer Res 2006; 66: (9). May 1, 2006 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Cancer Research Table 1. Loci of high-level amplification (log 2 ratio > 2.5) detected in NSCLC cell lines by array-CGHanalysis using MCG Whole Genome Array-4500 c No. BAC Locus* Cell line (total 20) Possible candidate gene No. known genes Chromosome band Position n Name 1 RP11-81F24 2p24.3chr2:15,177,504-15,178,217 1 VMRC-LCD MYCN 3 RP11-451A14 2p24.3chr2:15,927,694-15,928,209 1 VMRC-LCD MYCN 2 RP11-542D132q11.2 chr2:97,564,600-97,795,025 1 VMRC-LCD 16 3 RP11-62G13 5q34 chr5:160,654,723-160,806,043 1 VMRC-LCD 4 4 RP11-237F24 8q24.21 chr8:128,822,387-128,822,827 2 ABC-1 MYC 1 RP11-89L16 8q24.21 chr8:129,633,607-129,784,954 2 ABC-1, NCI-H460 MYC *Based on UCSC Genome Browser, May 2004 Assembly. cRepresentative candidate oncogene located around BAC. (11-18, A549, ABC-1, RERF-LC-OK, VMRC-LCD, SK-LC-3, and RERF-LC-KJ), products were digested with Taq 1orHha1 and electrophoresed. For and six from large-cell carcinomas (86-2, LU65, PC-13, ACC-LC-33, NCI-H460, bisulfite sequencing, PCR products were subcloned and then sequenced. and LU99A). All cell lines were maintained as previously described (13). Promoter reporter assay. We obtained by PCR several DNA fragments Primary tumor samples were obtained during surgery from 112 patients around the CpG-rich region of PCDH20 predicted by the CpGPLOT being treated at the National Cancer Center Hospital in Tokyo or the program.8 Each fragment was ligated into the vector pGL3-Basic Hokushin General Hospital in Nagano, Japan, with written consent from (Promega, Madison, WI) and an equal amount of each construct was each patient in the formal style and after approval by the local ethics introduced into cells along with an internal control vector (pRL-hTK, committees. Samples from 53patients with adenocarcinoma were Promega) using FuGENE 6 (Roche Diagnostics, Tokyo, Japan). A pGL3- embedded in paraffin for laser-captured microdissection after fixation in Basic vector without an insert served as a negative control. Firefly methanol for 24 hours, as described elsewhere (20). Tumors from the other luciferase and Renilla luciferase activities were each measured 36 hours 59 patients (Table 1), along with adjacent non-cancerous lung tissues from after transfection using the Dual-Luciferase Reporter Assay System 12 of them, were frozen immediately in liquid nitrogen and stored at À80jC (Promega); relative luciferase activities were calculated and normalized until required. None of the patients had received preoperative radiation, versus Renilla luciferase activity. chemotherapy, or immunotherapy. Methylation-specific PCR. Genomic DNA treated with sodium bisulfite Array-CGH analysis. MCG Whole Genome Array-4500 (16), which was amplified using primers specific to the methylated and unmethylated contains 4523BAC/PAC clones covering the entire genome at intervals of forms of DNA sequences of interest. DNAs from cell lines and peripheral f0.7 Mb, was used for analysis. Hybridizations were carried out as blood lymphocytes of a healthy male, recognized as unmethylated by described elsewhere (19). Hybridized slides were scanned with a GenePix bisulfite sequencing, were used as negative controls for the methylation- 4000B (Axon Instruments, Foster City, CA) and acquired images were specific PCR (MSP) assay, whereas those from cell lines recognized as analyzed with GenePix Pro 4.1 imaging software (Axon Instruments). methylated were used as positive controls for methylated alleles. PCR Fluorescence ratios were normalized so that the mean of the middle third of products were visualized on 3% agarose gels stained with ethidium log 2 ratios across the array was zero. Average ratios that deviated bromide. significantly (>2 SD) from zero were considered abnormal.