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Main unadjusted effects of smoking, childhood and adult exposure to tobacco smoke and lung cancer risk.

Lung cancer has a familial component which suggests a genetic contribution to its etiology. Given the strong evidence linking smoking with lung cancer, we studied miRNA-related loci in genes associated with smoking behavior. CHRNA, CHRNB gene families, CYP2A6, and DRD1 (dopamine receptor D1) were mined for SNPs that fell within the seed region of miRNA binding sites and then tested for associations with risk in a three-stage validation approach. A 3′UTR (untranslated region) SNP in DRD1 was associated with a lower risk of lung cancer among individuals exposed to secondhand smoke during childhood [OR, 0.69; 95% confidence interval (CI), 0.60–0.79; P < 0.0001]. This relationship was evident in both ever (OR, 0.74; 95% CI, 0.62–0.88; P = 0.001) and never smokers (OR, 0.61; 95% CI, 0.47–0.79; P < 0.0001), European American (OR, 0.65; 95% CI, 0.53–0.80; P < 0.0001), and African American (OR, 0.73; 95% CI, 0.62–0.88; P = 0.001) populations. Although much remains undefined about the long-term risks associated with exposure to secondhand smoke and heterogeneity between individuals in regard to their susceptibility to the effects of secondhand smoke, our data show an interaction between an SNP in the 3′UTR of DRD1 and exposure to secondhand smoke during childhood. Further work is needed to explore the mechanistic underpinnings of this SNP and the nature of the interaction between DRD1 and exposure to secondhand smoke during childhood. Cancer Prev Res; 7(12); 1210–8. ©2014 AACR.

Characteristics of cases and controls from three studies combined.

Association between rs4809294 and rs2292975 and lung cancer risk stratified by exposure to secondhand smoke during adulthood in the NCI-MD Cohort (European Americans).

Association between 3'UTR SNPs and lung cancer in the NCI-MD Cohort (European Americans).

NCI-MD Case-Control Study Genomic DNA was isolated from buffy coat samples containing white blood cells using Flexigene DNA Kit (Qiagen) according to the manufacturer's instructions.

Flowchart detailing the selection process of SNPs. Rs686 in DRD1 was selected from the literature as being a miRNA-binding modulating SNP (Huang and Li, 2009 Biol Pschiatry, 65 9 702-705).

Test for departure from additivity for rs686 and exposure to secondhand smoke during childhood.

Association between rs686 and lung cancer risk stratified by exposure to secondhand smoke during adulthood.

Relationship between rs686 and pack-years of smoking (A) in the NCI-MD (B) and Wayne State (C) studies. Relationship between rs686 and age at smoking initiation (D) in the NCI-MD study.

SNP Selection.

Relationship between rs1126579 and lung cancer survival.

Representative images of CXCR2 staining in lung cancer specimens from the NCI-MD case control study.

Effect modification between IL-8 and rs1126579.

List of SNPs successfully genotypes in the NCI/MD Case Control study.

Risk associations stratified by smoking status.

CXCR2 isoform expression in human lung adenocarcinoma and squamous cell carcinoma tissue.

CXCR2 isoform expression predicted by Emsembl.

List of non-polymorphic SNPs.

PDF file - 123K

Predicted modulation of miR-138-1* binding to CXCR2 mRNA by rs1126579.

List of SNPs that failed primer design.

List of genes included in the study.

Lung cancer has a familial component which suggests a genetic contribution to its etiology. Given the strong evidence linking smoking with lung cancer, we studied miRNA-related loci in genes associated with smoking behavior. CHRNA, CHRNB gene families, CYP2A6, and DRD1 (dopamine receptor D1) were mined for SNPs that fell within the seed region of miRNA binding sites and then tested for associations with risk in a three-stage validation approach. A 3′UTR (untranslated region) SNP in DRD1 was associated with a lower risk of lung cancer among individuals exposed to secondhand smoke during childhood [OR, 0.69; 95% confidence interval (CI), 0.60–0.79; P < 0.0001]. This relationship was evident in both ever (OR, 0.74; 95% CI, 0.62–0.88; P = 0.001) and never smokers (OR, 0.61; 95% CI, 0.47–0.79; P < 0.0001), European American (OR, 0.65; 95% CI, 0.53–0.80; P < 0.0001), and African American (OR, 0.73; 95% CI, 0.62–0.88; P = 0.001) populations. Although much remains undefined about the long-term risks associated with exposure to secondhand smoke and heterogeneity between individuals in regard to their susceptibility to the effects of secondhand smoke, our data show an interaction between an SNP in the 3′UTR of DRD1 and exposure to secondhand smoke during childhood. Further work is needed to explore the mechanistic underpinnings of this SNP and the nature of the interaction between DRD1 and exposure to secondhand smoke during childhood. Cancer Prev Res; 7(12); 1210–8. ©2014 AACR.

Because chronic intestinal inflammation is a risk factor for colorectal cancer, we hypothesized that genetic variants of inflammatory mediators, such as mannose-binding lectin 2 (MBL2), are associated with colon cancer susceptibility. Here, we report the association of 24 MBL2 single-nucleotide polymorphisms (SNP) and corresponding haplotypes with colon cancer risk in a case–control study. Four SNPs in the 3′-untranslated region (UTR) of the gene (rs10082466, rs2120132, rs2099902, and rs10450310) were associated with an increased risk of colon cancer in African Americans. ORs for homozygous variants versus wild-type ranged from 3.17 [95% confidence interval (CI), 1.57–6.40] to 4.51 (95% CI, 1.94–10.50), whereas the 3′-UTR region haplotype consisting of these four variants had an OR of 2.10 (95% CI, 1.42–3.12). The C allele of rs10082466 exhibited a binding affinity of miR-27a and this allele was associated with both lower MBL plasma levels and activity. We found that 5′ secretor haplotypes known to correlate with moderate and low MBL serum levels exhibited associations with increased risk of colon cancer in African Americans, specifically as driven by two haplotypes, LYPA and LYQC, relative to the referent HYPA haplotype (LYPA: OR, 2.60; 95% CI, 1.33–5.08 and LYQC: OR, 2.28; 95% CI, 1.20–4.30). Similar associations were not observed in Caucasians. Together, our results support the hypothesis that genetic variations in MBL2 increase colon cancer susceptibility in African Americans. Cancer Res; 72(6); 1467–77. ©2012 AACR.

Global changes in gene expression accompany the development of cancer. Thus, inherited variants in miRNA-binding sites are likely candidates for conferring inherited susceptibility. Using an in silico approach, we compiled a comprehensive list of SNPs predicted to modulate miRNA binding in genes from several key lung cancer pathways. We then investigated whether these SNPs were associated with lung cancer risk in two independent populations. In general, SNPs in miRNA-binding sites are rare. However, some allelic variation was observed. We found that rs1126579 in CXCR2 was associated with a reduced risk of lung cancer in both European American [ORTT vs. CC 0.56 (0.37–0.88); P = 0.008] and Japanese [ORTT vs. CC 0.62 (0.38–1.00); P = 0.049] populations. Furthermore, we found that the SNP disrupted a novel binding site for miR-516a-3p, led to a moderate increase in CXCR2 mRNA and protein expression, and increased MAPK signaling. Moreover, analysis of rs1126579 with serum levels of IL8, its endogenous ligand, supported an interaction whereby rs1126579-T and high serum IL8 conferred synergistic protection from lung cancer. Our findings demonstrate a function for a 3′UTR SNP in modulating CXCR2 expression, signaling, and susceptibility to lung cancer. Cancer Res; 75(3); 566–75. ©2014 AACR.

Rare cancer types are not only understudied, but are typically represented by formalin-fixed paraffin-embedded (FFPE) (rather than freshly-frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non-surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole-transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 22 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog ...

Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.

Nat. Genet. 46, 722–725 (2014); published online 25 May 2014; corrected after print 12 November 2014 In the version of this article initially published, the introductory paragraph mistakenly stated that ameloblasts were “cells in the tooth roots of the upper (maxilla) and lower (mandible) jaw responsible for depositing enamel during tooth development (odontogenesis).

Rare cancer types are not only understudied, but are typically represented by formalin-fixed paraffin-embedded (FFPE) (rather than freshly-frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non-surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole-transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 21 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog pathway mutation confirmed increased basal pathway activity, and defined the response profile to various pharmacologic Hedgehog inhibitors. Together, our results define new ameloblastoma drivers and nominate new molecularly-directed therapies for this rare but disfiguring disease. More generally, our findings validate a robust approach for discovering driver mutations in rare cancers. Citation Format: Andrew C. McClary, Robert T. Sweeney, Jewison Biscocho, Benjamin R. Myers, Lila Neahring, Kevin A. Kwei, Kunbin Qu, Xue Gong, Tony Ng, Carol D. Jones, Sushama Varma, Justin I. Odegaard, Brian Rubin, Megan L. Troxell, Robert J. Pelham, James L. Zehnder, Philip A. Beachy, Jonathan R. Pollack, Robert B. West. Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2014-3436

In searching for a role for genetic variation in susceptibility to lung cancer, we postulated that miRNA modulating SNPs within smoking behavior associated genes, such as the nicotinic acetylcholine and dopamine receptor families, could modulate susceptibility to tobacco carcinogens and lung cancer risk. Bioinformatic analyses of these gene families identified 5 SNPs predicted to modulate a miRNA binding site. We assessed these SNPs in the National Cancer Institute-MD case-control study (European American [EA] & African Americans [AA] n=1,438). rs686 in DRD1 (dopamine receptor D1) was associated with a reduced risk of lung cancer after adjustment for age, gender and race (OR 0.63, 95% C.I. 0.47-0.85; P=0.002). As DRD1 mediates dopamine signaling following nicotine exposure, we reasoned that the rs686 variant associates with lung cancer risk via modulation of the nicotine reward pathway. However, adjustment for smoking both in terms of smoking status and pack-years did not significantly modulate the association (OR 0.57, 95% C.I. 0.40-0.81, P=0002). In addition, age at smoking initiation, pack-years of smoking and levels of urinary nicotine metabolites (cotinine and hydroxycotinine) did not vary across rs686 genotypes. Moreover, in an analysis of a never smoking cohort from Mayo Clinic, rs686 was also associated with a reduced risk of lung cancer (OR 0.77, 95% C.I. 0.62-0.97; P=0.027; n=629) (pooled analysis OR 0.61, 95% C.I. 0.47-0.78; P Citation Format: Brid M. Ryan, Ana I. Robles, Andrew C. McClary, Elise Bowman, Kirsi Vahakangas, Susan Olivo-Marston, Ping Yang, Jin Jen, Curtis C. Harris. Interaction between DRD1 and childhood exposure to environmental tobacco smoke modulates lung cancer risk in smokers and never smokers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4581. doi:10.1158/1538-7445.AM2013-4581

Background Colorectal cancer is the third most incident cancer and cause of cancer-related death in the United States. MicroRNAs, a class of small non-coding RNAs, have been implicated in the pathogenesis and prognosis of colorectal cancer, although few studies have examined the relationship between germline mutation in the microRNAs with risk and prognosis. We therefore investigated the association between a SNP in hsa-mir-608, which lies within the 10q24 locus, and colorectal cancer. Methods and Results A cohort consisting of 245 cases and 446 controls was genotyped for rs4919510. The frequency of the GG genotype was significantly higher in African Americans (15%) compared to Caucasians (3%) controls. There was no significant association between rs4919510 and colorectal cancer risk (African American: ORGG vs. CC 0.89 [95% CI, 0.41–1.80]) (Caucasian: ORGG vs. CC 1.76, ([95% CI, 0.48–6.39]). However, we did observe an association with survival. The GG genotype was associated with an increased risk of death in Caucasians (HRGG vs. CC 3.54 ([95% CI, 1.38–9.12]) and with a reduced risk of death in African Americans (HRGG vs. CC 0.36 ([95% CI 0.12–1.07). Conclusions These results suggest that rs4910510 may be associated with colorectal cancer survival in a manner that is dependent on race.

Colon cancer (CC) is estimated to be the third most common cause of cancer deaths in the United States in 2010 and the incidence is higher among African Americans compared with Caucasians. Chronic intestinal inflammation is a risk factor for CC. Moreover, mediators of chronic inflammation have been implicated in CC development. MBL, a mannose-binding protein, is a critical component of the innate immune system, and it serves as a front-line protection against pathogens. MBL deficiency is common and present in approximately 5% of the population, predisposing individuals to bacterial and fungal infections. A previous resequencing analysis of the 10.0kbp region that includes the MBL2 gene showed a recombination hot-spot in the 3′ end of the gene, dividing MBL2 into two separate haplotype blocks: 5′UTR and 3′UTR haplotypes. In our previous work, we used a case-control study from the greater Baltimore, Maryland area (261 cases; 537 controls) and observed that haplotypes in both regions are associated with CC risk in African Americans, but not in Caucasians. Moreover, we showed that four single nucleotide polymorphisms (SNPs) comprising a 3′UTR region haplotype are individually associated with increased CC risk in African Americans: rs10082466, rs2120132, rs2099902, and rs10450310. In this study, we investigated whether the SNPs in this haplotype block fell within putative microRNA (miRNA) site due to the observed association of 3′UTR-region haplotypes and plasma MBL levels, using publicly available web-tools. We hypothesized that rs10082466 association with CC is driven by hsa-miR-27a binding to the C allele, resulting in decreased plasma MBL2 levels and activity. We tested the interaction of hsa-miR-27a with the 3′UTR region of MBL2 and its modulation by 3′UTR haplotypes in vitro using reporter constructs via luciferase activity. The increased binding affinity for the C allele of rs10082466 was reflected by a significant decrease in normalized luciferase activity, compared with the T allele. We also showed the C allele to be associated with lower plasma MBL2 levels and activity in cases and controls. Moreover, the fold increase of hsa-mir-27a precursor is significantly higher in African Americans compared to Caucasians in colon cancer. Therefore, 3′UTR haplotype association with CC may be driven by hsa-miR-27a binding to the C allele of rs10082466, which results in decreased plasma MBL2 levels and activity. In addition to the supporting evidence linking low levels of circulating MBL2 to increased susceptibility to infection, these results suggest that a genetic variant in MBL2 may increase colon cancer susceptibility in African Americans by creating a miRNA binding site. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B60.

Differential microRNA expression and function has been implicated in a variety of human malignancies. Genetic variation in this network was recently proposed to explain the contribution of this family of non-coding RNAs to cancer. To test this hypothesis in lung cancer in a systematic manner, we collated a list of single nucleotide polymorphisms (SNPs) that lie within pre and mature miRNA genes. We then generated a list of SNPs from 600 genes in loci and pathways with relevance to lung cancer. The 3’ UTR of each of these genes was bioinformatically interrogated for overlap between putative microRNA binding sites and SNPs. The latter could either create or destroy a microRNA binding site and therefore impact protein translation. The SNPs were subsequently analyzed in a study of 300 Caucasian cases and 300 controls. Odds ratios for lung cancer risk were generated using unconditional logistic analyses and adjusted for age, gender, race, smoking status and pack years of smoking. Our study highlighted a number of novel associations. From our inflammation panel of genes, a SNP in the 3’ UTR of CXCR2 (IL8 receptor) was protective against lung cancer risk (OR: 0.44; 95% C.I. 0.26 – 0.78). As CXCR2 is implicated in oncogene induced senescence, we predict that this SNP gives rise to lower levels of the receptor and are currently testing this possibility both in vitro and in human tissues. Furthermore, a SNP in DRD1 (dopamine receptor DI) is also protective against lung cancer (OR: 0.45; 95% C.I. 0.24 – 0.85), thus intimating that the variant allele gives rise to decreased levels of DRD1 in the brain and therefore a negative re-enforcement of the reward pathway. Several metabolic genes were implicated from our study, the most notable of which was a SNP in ALDH1A3 (OR: 1.79; 95% C.I. 1.01 – 3.17). Apart from the delineated role in metabolism, this gene is also interesting due to its putative role in lung cancer stem cell biology. Although SNPs in microRNA genes are generally rare, a SNP in miR-585 was significantly associated with an increased risk of lung cancer (OR: 1.69; 95% C.I. 1.10 – 2.59), and most interestingly, the association was strongest for heterozygous carriers. We are currently investigating the effect of the miR-585 SNP on expression of the mature miRNA as well as the role of miR-585 in lung cancer etiology, as nothing is known in this regard at present. This study is presently being extended to a cohort of African American patients and a nested analysis of never-smokers is also underway. Moreover, we are endeavoring to determine the functional consequences of these epidemiological results and link risk-associated genotypes to oncogenic phenotypes. Our data provides a comprehensive evaluation of the association between genetic variation in the microRNA network and lung cancer risk, and has highlighted several novel associations that may help in our understanding of the genetic basis of lung cancer and the role that microRNAs play. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1171. doi:10.1158/1538-7445.AM2011-1171

two cohorts by incorporating the resulting coefficients of multivariable Cox regression model into a score that utilizes linear gene expression values. This gene expression classifier was validated in 6 additional publicly available datasets of stage I/II lung SCC (N 1⁄4 358). The classifier identified high-risk patients in multiple large-scale and geographically diverse cohorts (N 1⁄4 570). The results appear to be independent of race and gene expression platform. Canonical pathways associated with this signature encompass proteins involved in signal transduction, tissue remodeling, and cell motility that would broadly lead to cancer cell migration, invasion and proliferation, suggesting that the gene signature identifies molecular subsets of patients with clinical relevance. This gene classifier could be used to guide clinical decisions after surgical resection. Thus, we would advocate that this classifier be incorporated into prospective trials for further evaluation of its clinical effectiveness.