Integrative Molecular Epidemiology of Human Cancer

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Project 2A: Evaluate the functional importance of gene and gene-environment interactions in the molecular epidemiology and cancer-related disparities between African- and European-Americans. Genome Wide Association Studies (GWAS) of African-American lung cancer will identify potential functional polymorphisms involved in health disparities. African-American men have higher age-adjusted cancer incidence rates (99.9 per 100,000) when compared with Caucasian and Asian men (76.4 and 52.2 per 100,000, respectively), as well as higher age-adjusted death rates (African American 82.6 per 100,000; Caucasian 65.3 per 100,000; Asian 35.9 per 100,000). To date, GWAS performed in populations of European and Asian descent have identified over 15 lung cancer risk loci. Although African Americans have higher lung cancer incidence and poorer lung cancer survival when compared with other racial and ethnic populations in the United States, no GWAS has been conducted in this population. In addition, African admixture in African Americans could provide an opportunity to identify new lung cancer risk alleles. In light of the current evidence suggesting a genetic contribution to lung cancer in African Americans, we initiated and conducted a first-in-kind two-stage GWAS, genotyping 1,024,001 single nucleotide polymorphisms (SNPs) in 1737 cases and 3602 controls (stage 1), followed by replication of the most significantly associated genotypes in an independent sample set of 866 cases and 796 controls (stage 2). Both stages of the study included samples from the NCI-MD case-control cohort, in addition to 10 other cohorts. Combining results from stages 1 and 2, two SNPs were significant at the genome-wide significance level, rs2036527, adjacent to CHRNA5. We confirmed previous results for two loci associated with lung cancer on 15q25.1 and 5p15.33, near candidate genes, CHRNA5 and TERT, respectively, in African Americans. Project 2B: Functional polymorphisms in microRNA, and their mRNA Targets are associated with lung cancer risk, diagnosis and prognosis. MicroRNAs (miRNA), a class of noncoding genes, modulate mRNA translation by primarily binding to the 3primeUTR of mRNA transcripts. MiRNAs temper the translation of up to 60% of mRNA transcripts through semiconservative binding. The seed region of a miRNA, which corresponds to nucleotides 2-8 from its 5 prime end, coordinates exact Watson-Crick binding to the target mRNA sequence. Genetic variation within a miRNA-binding site has two main consequences on target protein expression, i.e., quantitative and qualitative. Requirements for sequence complementarity and stable thermodynamics around the miRNA-3 prime UTR seed-binding site primes sequence variations within these regions as strong candidates for functional SNPs, thus we hypothesized that genetic variation in miRNA-binding sites is a contributing factor to lung cancer risk. We analyzed several key biologic pathways previously implicated in lung cancer etiology; Phase I/II metabolism, DNA repair, and inflammation. Initial genotyping was conducted on patients and population controls from the NCI-MD case-control study, followed by validation on a case-control series from our Japanese collaborators. rs1126579, in CXCR2, was found to be associated with a reduced risk of lung cancer in European Americans and in the Japanese population in adjusted models. Consistent with our bioinformatics prediction, we subsequently showed that rs1126579 disrupts a binding site for miR516a-3p and alters expression of CXCR2. We leveraged TCGA RNA-seq data to explore if CXCR2 isoforms are differentially modulated by rs1126579. We found expression of CXCR2 - UC002vha.1 to be higher in samples with T allele compared with C allele. Collectively, these data suggest that T allele increases CXCR2 expression in lung tissue by disrupting a binding site for miR-516a-3p. Consistent with the association of the T allele with reduced risk, loss of miRNA regulation and higherexpression of CXCR2, expression of the CXCR2 mRNA was also decreased in lung adenocarcinoma tissue compared with noninvolved lung tissue. As IL8 is the primary ligand for CXCR2, we analyzed serum levels of IL8 on controls and cases for which we had rs1126579 genotype and serum data. The TT genotype of rs1126579 was associated with a reduced risk of lung cancer in subjects with high IL8 levels, while no association was observed in subjects with low IL8 levels, suggesting that IL8 could be a potential effect modifier of the relationship between rs1126589 and lung cancer risk. SNP, rs1126579, in the 3 prime UTR of an inflammatory gene CXCR2 is associated with reduced risk of lung cancer among individuals with high levels of IL8. Project 2C: Germline variation in NCF4, an innate immunity gene, is associated with an increased risk of colorectal cancer. Chronic inflammation has been implicated in the etiology of colorectal adenoma and cancer however, few key inflammatory genes mediating this relationship have been identified. We examined 196 SNPs in 20 candidate innate immunity genes in a nested case-control study based within the prospective prostate, lung, colorectal and ovarian (PLCO) cancer screening trial (719 adenoma cases, 481 cancer cases and 719 controls), and investigated their association with risk of colorectal neoplasia. After Bonferroni correction, the AG/GG genotype of rs5995355, upstream of NCF4, was associated with an increased risk of colorectal cancer. NCF4 is part of the NAPDH complex, a key factor in the innate immune response. Notably, the genes involved in these pathways, including cholesterol and inositol biosynthesis, were downregulated, suggesting the NADPH function was somehow compromised in cells that carried the variant allele of the SNP. Additional studies on the functional consequences of rs5995355 may help to clarify the mechanistic link between inflammation and colorectal cancer. An analysis of publicly available gene expression datasets showed reduced NCF4 expression in colon cancer but our analyses suggest that the variant allele does not affect expression, but rather modulates activity of the NADPH complex. There is an association between NCF4, an innate immunity gene, with colorectal carcinoma risk.