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Implication of a Chromosome 15Q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung Shuguang Leng1, Guodong Wu1, Leonard B

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Implication of a Chromosome 15Q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung Shuguang Leng1, Guodong Wu1, Leonard B Published OnlineFirst July 16, 2015; DOI: 10.1158/0008-5472.CAN-15-0243 Cancer Prevention and Epidemiology Research Implication of a Chromosome 15q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung Shuguang Leng1, Guodong Wu1, Leonard B. Collins2, Cynthia L.Thomas1, Carmen S.Tellez1, Andrew R. Jauregui3, Maria A. Picchi1, Xiequn Zhang1, Daniel E. Juri1, Dhimant Desai4, Shantu G. Amin4, Richard E. Crowell5, Christine A. Stidley5,Yushi Liu1, James A. Swenberg2, Yong Lin1, Marc G. Wathelet3, Frank D. Gilliland6, and Steven A. Belinsky1 Abstract O6-Methylguanine-DNA methyltransferase (MGMT) is a risk prediction for MGMT methylation. We found that the DNA repair enzyme that protects cells from carcinogenic effects predisposition to MGMT methylation arising from the of alkylating agents; however, MGMT is silenced by promoter 15q15.2 locus involved regulation of the ubiquitin protein hypermethylation during carcinogenesis. A single-nucleotide ligase E3 component UBR1. UBR1 attenuation reduced turn- polymorphism (SNP) in an enhancer in the MGMT promoter over of MGMT protein and increased repair of O6-methylgua- was previously identified to be highly significantly associated nine in nitrosomethylurea-treated human bronchial epithelial with risk for MGMT methylation in lung cancer and sputum cells, while also reducing MGMT promoter activity and abol- from smokers. To further genetic investigations, a genome-wide ishing MGMT induction. Overall, our results substantiate association and replication study was conducted in two smoker reduced gene transcription as a major mechanism for predis- cohorts to identify novel loci for MGMT methylation in sputum position to MGMT methylation in the lungs of smokers, and that were independent of the MGMT enhancer polymorphism. support the importance of UBR1 in regulating MGMT homeo- Two novel trans-acting loci (15q15.2 and 17q24.3) that were stasis and DNA repair of alkylated DNA adducts in cells. Cancer identified acted together with the enhancer SNP to empower Res; 75(15); 1–10. Ó2015 AACR. Introduction some (1, 2). The recovery of MGMT activity after its inactivation with alkylating agents is slow and results entirely from de novo O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA protein synthesis (3–5). repair enzyme that protects cells from carcinogenic effects of The predominant mechanism for MGMT gene inactivation in alkylating agents by removing adducts from the O6 position of carcinogenesis is hypermethylation of a CpG island within its guanine (1). This repair mechanism is conserved in all organisms promoter–enhancer region that leads to transcriptional silencing and involves transfer of methyl and other alkyl groups from the in lymphoma and several solid tumors, including brain, colon, O6 position of guanine in DNA to a cysteine residue within lung, and head and neck cancers (6). Silencing of this gene is enzymatic active site of MGMT protein. The irreversible binding associated with an increased prevalence for G:C to A:T transition of the alkyl group to the MGMT protein functionally inactivates mutations in K-ras and p53 genes in colorectal and brain tumors the enzyme and leads to a structural alteration resulting in the respectively, consistent with the important role for this gene in recognition by ubiquitin ligases and degradation by the protea- removing the highly promutagenic adduct O6-methylguanine (O6MG; refs. 7 and 8). Although alkylating adducts such as 1Lung Cancer Program, Lovelace Respiratory Research Institute, Albu- O6MG can be derived from endogenous methylation by S-ade- querque, New Mexico. 2Department of Environmental Sciences and nosylmethionine, knockouts of MGMT failed to show enhanced Engineering, University of North Carolina at Chapel Hill, Chapel Hill, tumor formation unless treated with exogenous alkylating carci- 3 North Carolina. Lung Fibrosis Program, Lovelace Respiratory nogens (9). MGMT methylation is a common event in lung Research Institute, Albuquerque, New Mexico. 4Department of Phar- macology, Penn State College of Medicine, Hershey, Pennsylvania. adenocarcinomas and associated with tumor progression (10). 5Department of Internal Medicine, University of New Mexico, Albu- Concomitant methylation of a panel of cancer-relevant genes, 6 querque, New Mexico. Keck School of Medicine, University of South- including MGMT, detected in lung epithelial cells present in ern California, Los Angeles, California. sputum predicts risk for subsequent lung cancer incidence in Note: Supplementary data for this article are available at Cancer Research moderate and heavy smokers (11, 12). In addition, MGMT Online (http://cancerres.aacrjournals.org/). methylation is also a prognostic biomarker for response of glio- Corresponding Author: Steven A. Belinsky, Lung Cancer Program, Lovelace blastoma patients to the alkylating agent temozolomide (13, 14). Respiratory Research Institute, Albuquerque, NM 87108. Phone: 505-348-9465; Genome-wide profiling of allele-specific methylation (ASM) Fax: 505-348-4990; E-mail: [email protected] using a methylation-sensitive single nucleotide polymorphism doi: 10.1158/0008-5472.CAN-15-0243 (SNP) array identified >35,000 ASM sites across the genomes of Ó2015 American Association for Cancer Research. multiple tissue types that were likely mediated by parental-origin www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst July 16, 2015; DOI: 10.1158/0008-5472.CAN-15-0243 Leng et al. (imprinting) or short distance cis-acting SNPs (15–18). This effort Sputum collection and measurement of MGMT promoter was further expanded by running methylation quantitative trait methylation loci (meQTL) analyses that associated genome-wide SNP array Induced and spontaneous morning sputum samples were data with methylation array data to identify short and long collected in LSC and VSC, respectively. Sputum samples were distance cis- and trans-acting germline meQTL in normal tissues washed multiple times using Saccomanno's fixative prior to long- that included lung, adipose, peripheral blood lymphocyte, and term storage at À80C. Sputum adequacy defined as the presence brain (19–25). In normal lung, 34,304 cis- and 585 trans-acting of deep lung macrophages or Curschmann's spiral (12) was meQTL were identified and enriched in CCTC-binding factor assessed by cytotechnologists and subjects with cancer cells (CTCF)-binding sites, DNase I hypersensitivity regions, and his- detected in sputum slides were removed from this study. Sputum tone marks (19). Four of five established lung cancer risk loci DNA was isolated and bisulfite converted. Methylation status for among persons with European ancestry are cis-acting meQTL (19). CpGs around the transcriptional start site of MGMT was assessed The findings of SNP/haplotype dependent ASM and meQTL using two stage methylation-specific PCR (33). Methylation status contribute to understanding genetic-epigenetic interactions and was scored as 0 (unmethylated) or 1 (methylated). Our assay can also provide functional relevance for disease risk loci discovered reproducibly detect one methylated allele in a background of from genome-wide association studies (GWAS). 10,000 unmethylated alleles (12). In addition to germline meQTL, somatic meQTL were also fi identi ed that affected the predisposition of cancer-relevant genes GWAS genotyping and quality assurance for promoter hypermethylation acquired in carcinogenesis for The HumanOmni2.5-4v1-H BeadChip (Illumina) was used to – multiple tumor types (13, 26 32). This is illustrated by our genotype 2,450,000 SNPs in 1200 Caucasian smokers from the studies and others for MGMT methylation. An enhancer SNP LSC. We removed 37 subjects due to low call rate (<95%, n ¼ 7), fi – rs16906252 located at the rst exon intron boundary of MGMT low heterozygosity (n ¼ 1), low Caucasian ancestry (<85%, n ¼ 2), fi was highly signi cantly associated with risk for MGMT methyl- and high relatedness with other samples (n ¼ 27). Furthermore, ation in colorectal cancer, pleural mesothelioma, lung adenocar- SNPs were excluded if they had a call rate of <90%, a minor allele À cinoma, glioblastoma, and diffuse large B-cell lymphoma frequency (MAF) <0.008, or P < 10 8 for Hardy–Weinberg equi- – fi (13, 27, 29 32). Signi cant association was also seen for MGMT librium test, or were on Y or pseudo-autosomal region of X methylation in sputum from smokers (27). Functional charac- chromosomes. The MAF cutoff is a technical one to identify at fi terization con rmed the ASM pattern in heterozygotes for least 20 heterozygotes for accurate genotype clustering required rs16906252 with the "A" allele preferentially silenced by meth- by GenomeStudio. After quality assessment, 1,163 subjects with ylation in primary lung tumors and lung cancer cell lines (27). 1,599,980 SNPs remained in the genetic association analysis Although rs16906252 is a major determinant for acquisition of (Supplementary Table S1). The genomic coordinate is based on MGMT methylation in lung carcinogenesis, 20% of current and hg18 unless mentioned otherwise. former smokers carrying wild homozygote (G/G) of rs16906252 are methylated for MGMT in their sputum (27), suggesting that SNP selection for replication other genetic or environmental factors may contribute to MGMT Four SNPs (rs72887860, rs62287262, rs16957091, and methylation. To address this issue, a GWAS discovery and repli- rs997781) associated with risk for MGMT methylation in
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