Integrative Molecular Epidemiology of Human Cancer

项目2A：评估基因和基因-环境相互作用在非洲裔和欧裔美国人的分子流行病学和癌症相关差异中的功能重要性。非裔美国人肺癌的全基因组关联研究（GWAS）将确定与健康差异有关的潜在功能多态性。非裔美国男性的年龄调整癌症发病率（99.9 / 10万）高于白种人和亚洲男性（分别为76.4 / 10万和52.2 / 10万），年龄调整死亡率（非裔美国人82.6 / 10万；白种人65.3 / 10万；亚洲人35.9 / 10万）。迄今为止，在欧洲和亚洲血统人群中进行的GWAS已经确定了超过15个肺癌风险位点。尽管与美国其他种族和族裔人群相比，非洲裔美国人的肺癌发病率更高，肺癌生存率更低，但尚未在该人群中进行GWAS。此外，非洲裔美国人的非洲混血儿可能为识别新的肺癌风险等位基因提供机会。鉴于目前的证据表明遗传对非裔美国人肺癌的影响，我们启动并进行了一项同类首次两阶段GWAS，在1737例病例和3602例对照（第一阶段）中对1,024,001个单核苷酸多态性（snp）进行基因分型，然后在866例病例和796例对照（第二阶段）的独立样本集中复制最显著相关的基因型。研究的两个阶段都包括来自NCI-MD病例对照队列的样本，以及其他10个队列。结合第一阶段和第二阶段的结果，两个snp在全基因组显著水平上显著，rs2036527，邻近CHRNA5。我们证实了先前在非裔美国人中15q25.1和5p15.33两个与肺癌相关的基因座的结果，它们分别接近候选基因CHRNA5和TERT。2B项目：microRNA的功能多态性及其mRNA靶点与肺癌风险、诊断和预后相关。MicroRNAs （miRNA）是一类非编码基因，主要通过与mRNA转录物的3primeUTR结合来调节mRNA的翻译。mirna通过半保守结合调节高达60%的mRNA转录物的翻译。miRNA的种子区对应于其5 '端2-8个核苷酸，协调与目标mRNA序列的精确沃森-克里克结合。mirna结合位点内的遗传变异对靶蛋白表达有两个主要影响，即定量和定性。对miRNA-3引物UTR种子结合位点的序列互补和稳定热力学的要求使得这些区域内的序列变异成为功能性snp的强有力候选，因此我们假设mirna结合位点的遗传变异是肺癌风险的一个因素。我们分析了先前涉及肺癌病因的几个关键生物学途径；I/II期代谢，DNA修复和炎症。对来自NCI-MD病例对照研究的患者和人群对照进行了初始基因分型，随后对来自日本合作者的病例对照系列进行了验证。在调整后的模型中，发现CXCR2中的rs1126579与欧洲、美国和日本人群肺癌风险降低有关。与我们的生物信息学预测一致，我们随后发现rs1126579破坏miR516a-3p的结合位点并改变CXCR2的表达。我们利用TCGA RNA-seq数据来探索CXCR2亚型是否受到rs1126579的差异调节。我们发现CXCR2 - UC002vha表达。1在T等位基因样品中比C等位基因样品高。总的来说，这些数据表明T等位基因通过破坏miR-516a-3p的结合位点来增加肺组织中CXCR2的表达。与T等位基因与风险降低、miRNA调控缺失和CXCR2高表达的关联一致，肺腺癌组织中CXCR2 mRNA的表达也比未受损伤的肺组织低。由于IL8是CXCR2的主要配体，我们分析了对照组和rs1126579基因型和血清数据的病例的血清IL8水平。rs1126579的TT基因型与高il - 8水平受试者肺癌风险降低相关，而在低il - 8水平受试者中未观察到相关，提示il - 8可能是rs1126589与肺癌风险关系的潜在效应修饰因子。炎症基因CXCR2 3 ' UTR中的rs1126579 SNP与il - 8水平高的个体中肺癌风险降低相关。项目2C：先天免疫基因NCF4的种系变异与结直肠癌风险增加有关。慢性炎症与结直肠腺瘤和癌症的病因学有关，然而，很少有关键的炎症基因介导了这种关系。我们在前瞻性前列腺、肺、结直肠和卵巢（PLCO）癌症筛查试验（719例腺瘤病例，481例癌症病例和719例对照）的巢式病例对照研究中，检测了20个候选先天免疫基因的196个snp，并调查了它们与结直肠肿瘤风险的关系。经Bonferroni校正，NCF4上游的rs5995355的AG/GG基因型与结直肠癌风险增加相关。NCF4是NAPDH复合体的一部分，NAPDH复合体是先天免疫反应的关键因子。值得注意的是，参与这些途径的基因，包括胆固醇和肌醇生物合成，被下调，这表明在携带SNP变异等位基因的细胞中，NADPH功能以某种方式受到损害。对rs5995355功能影响的进一步研究可能有助于阐明炎症与结直肠癌之间的机制联系。对公开的基因表达数据集的分析显示，结肠癌中NCF4的表达减少，但我们的分析表明，变异等位基因不影响表达，而是调节NADPH复合物的活性。先天性免疫基因NCF4与结直肠癌风险之间存在关联。

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.