Promoter Methylation of Genes in and around the Candidate Lung Cancer Susceptibility Locus 6q23-25 Mathewos Tessema, 1 Randy Willink, 1 Kieu Do, 1 Yang Y. Yu, 1 Wayne Yu, 3 Emi O. Machida, 3 Malcolm Brock, 3 Leander Van Neste, 4 Christine A. Stidley, 2 Stephen B. Baylin, 3 and Steven A. Belinsky 1 1 Lung Cancer Program, Lovelace Respiratory Research Institute; 2 Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico; 3 Cancer Biology Division, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland; and 4 Department of Molecular Biotechnology, Faculty of Biosciences Engineering, Ghent University, Ghent, Belgium Abstract Chromosomal aberrations associated with lung cancer are frequently observed in the long arm of chromosome 6. A candidate susceptibility locus at 6q23-25 for lung cancer was recently identified; however, no tumor suppressor genes inactivated by mutation have been identified in this locus. Genetic, epigenetic, gene expression, and in silico screening approaches were used to select 43 genes located in 6q12-27 for characterization of methylation status. Twelve (28%) genes were methylated in at least one lung cancer cell line, and methylation of 8 genes was specific to lung cancer cell lines. Five of the 8 genes with the highest prevalence for methyla- tion in cell lines (TCF21, SYNE1, AKAP12, IL20RA , and ACAT2 ) were examined in primary lung adenocarcinoma samples from smokers (n = 100) and never smokers (n = 75). The prevalence for methylation of these genes was 81%, 50%, 39%, 26%, and 14%, respectively, and did not differ by smoking status or age at diagnosis. Transcription of SYNE1, AKAP12 , and IL20RA was completely silenced by hypermethylation and could be restored after treatment with 5-aza-2-deoxycytidine. Significant associations were found between methylation of SYNE1 and TCF21, SYNE1 and AKAP12 , and AKAP12 and IL20RA , indicating a coordinated inactivation of these genes in tumors. A higher prevalence for methylation of these genes was not associated with early-onset lung cancer cases, most likely precluding their involvement in familial susceptibility to this disease. Together, our results indicate that frequent inactivation of multiple candidate tumor suppressor genes within chromosome 6q likely contributes to development of sporadic lung cancer. [Cancer Res 2008;68(6):1707–14] Introduction Lung cancer remains the leading cause of cancer death among both men and women, with an estimated 160,440 deaths and 173,000 new cases annually in the United States alone (1). It kills more people than breast, colon, and prostate cancer combined. Lung tumors develop through mutation or epigenetic silencing via promoter hypermethylation of numerous genes involved in cell cycle control, growth regulation, signaling, apoptosis, and adhe- sion (2). Some of the earliest changes detected in the bronchial epithelium of smokers include loss of heterozygosity (LOH) at chromosomes 3p21, 9p21, and 17p13 (3). Within these areas of allelic loss, inactivation of the remaining allele by promoter hypermethylation of RASSF1A and p16 genes and by mutation of the p53 gene is commonly seen in non–small cell lung cancers (NSCLC) (4). Methylation of the p16 gene is one of the earliest changes in lung cancer development, occurring in the field of epithelial damage induced by carcinogens within tobacco and increasing in prevalence during histologic progression of adeno- carcinoma and squamous cell carcinoma (5–7). Another chromosome region where LOH is frequently observed in adenocarcinomas from both smokers and never smokers is 6q . LOH within 6q22-27 occurs in 30% to 55% of tumors (8), whereas deletions between 6q14 and 6q24 have been detected in 60% of primary lung tumors (9). Moreover, this locus has been associated with lung cancer susceptibility. A recently conducted genome-wide linkage analysis of 52 extended pedigrees with a minimum of three family members with aerodigestive cancer identified a lung cancer susceptibility locus at chromosome 6q23-25 (10). Smoking at any level also was found to increase risk in carriers of the inherited susceptibility locus on chromosome 6q . The high frequency for LOH and the potential existence of a susceptibility locus within 6q23-25 supports the existence of tumor suppressor genes inactivated through the classic Knudson’s two-hit model in which complete loss of gene function arises through loss of one allele and mutation of the second allele (11). However, only one candidate tumor suppressor gene, p34 , localized to 6q25 has been identified, but this gene was not found to be associated with familial lung cancer susceptibility (12). This scenario is reminiscent of the chromosome 3p14-25 in which LOH is commonly seen in lung tumors, although no major tumor suppressor gene inactivated by mutation has been identified in this locus. Rather, genes inactivated by promoter hypermethylation at prevalences ranging from 30% to 58% have been identified within this locus. These include RASSF1A, BLU, SEMA38 , and retinoic acid receptor b (4). Chromosome 6 is one of the gene-rich and CpG island–rich chromosomes that contains f1,557 genes and 1,070 CpG islands (13, 14). The fact that chromosome 6q accumulates genetic aber- ration in the form of LOH could also make this region a hotspot for silencing of genes by promoter hypermethylation. Support for this supposition is growing as genes silenced by methylation in lung tumors are now being identified within 6q . Estrogen receptor a, which maps to 6q25 , has been shown by our laboratory to be silenced by promoter hypermethylation in 20% and 36% of lung tumors from smokers and never smokers, respectively (15). Re- cently, Smith et al. (16) identified a tumor suppressor gene, TCF21 , within the 6q23-24 locus that is normally expressed in lung airway epithelial cells but silenced in aerodigestive tumors. The purpose Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Steven A. Belinsky, Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive Southeast, Albuquerque, NM 87108. Phone: 505-348-9465; Fax: 505-348-4990; E-mail: sbelinsk@LRRI.org. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-6325 www.aacrjournals.org 1707 Cancer Res 2008; 68: (6). March 15, 2008 Research Article Research. on June 24, 2015. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from