Michael A. James

Principles for the post-GWAS functional characterization of cancer risk loci

Genome wide association studies (GWAS) have identified more than 200 mostly new common low-penetrance susceptibility loci for cancers. The predicted risk associated with each locus is generally modest (with a per-allele odds ratio typically less than 2) and so, presumably, are the functional effects of individual genetic variants conferring disease susceptibility. Perhaps the greatest challenge in the ‘post-GWAS’ era is to understand the functional consequences of these loci. Biological insights can then be translated to clinical benefits, including reliable biomarkers and effective strategies for screening and disease prevention. The purpose of this article is to propose principles for the initial functional characterization of cancer risk loci, with a focus on non-coding variants, and to define ‘post-GWAS’ functional characterization. By December 2010, there were 1,212 published GWAS studies1 reporting significant (P < 5 × 10−8) associations for 210 traits (Table 1), and the Catalog of Published GWAS states that by March 2011, 812 publications reported 3,977 SNP associations1. This is likely a small fraction of the common susceptibility loci of low penetrance that will eventually be identified. Despite these successes in identifying risk loci, the causal variant and/or the molecular basis of risk etiology has been determined for only a small fraction of these associations2–4. Plausible candidate genes can be based on proximity to risk loci, but few have so far been defined in a more systematic manner (Supplementary Table 1). Table 1 The genomic context in which a variant is found can be used as preliminary functional analysis Increased investment in post-GWAS functional characterization of risk loci5 has now been advocated across diseases and for cardiovascular disease and diabetes6. For cancer biology, the complex interplay between genetics and the environment in many cancers poses a particularly exciting challenge for post-GWAS research. Here we suggest a systematic strategy for understanding how cancer-associated variants exert their effects. We mostly refer to SNPs throughout the paper, but we recognize that other types of common genetic (for example, copy number variants) or epigenetic variation may influence risk. Our understanding of the way in which a risk variant initiates disease pathogenesis progresses from statistical association between genetic variation and trait or disease variation to functionality and causality. The functional consequences of variants in protein-coding regions causing most monogenic disorders are more readily interpreted because we know the genetic code. For non-Mendelian or multifactorial traits, most of the common DNA variants have so far mapped to non-protein–coding regions2, where our understanding of functional consequences and causality is more rudimentary. Our hypothesis is that the trait-associated alleles exert their effects by influencing transcriptional output (such as transcript levels and splicing) through multiple mechanisms. We emphasize appropriate assays and models to test the functional effects of both SNPs and genes mapping to cancer predisposition loci. Although much of what is written is applicable to alleles discovered for any trait, the section on modeling gene effects will emphasize measuring cancer-related phenotypes. At some loci, multiple, independently associated risk alleles rather than single risk alleles may be functionally responsible for the occurrence of disease. Genotyping susceptibility loci (and their correlated variants) in multiple populations with different linkage disequilibrium (LD) structures may prove effective in substantially reducing the number of potentially causative variants (that is, the same causal variant may segregate in multiple populations), as shown for the FGFR2 locus in breast cancer7, but for most loci there will remain a set of potentially causative variants that cannot be separated at the statistical level from case-control genotype data. A susceptibility locus should be re-sequenced to ascertain all genetic variation, identifying candidate functional or causal variants and identifying candidate causal genes. Ideally, the identification of a causal SNP would be the next step to reveal the molecular mechanisms of risk modification. Practically, however, it is unclear what the criteria for causality should be, particularly in non-protein–coding regions. Thus, although we propose a framework set of analyses (Box 1), we acknowledge that the techniques and methods will continue to evolve with the field. Box 1 Strategies to progress from tag SNP to mechanism Target resequencing efforts using linkage disequilibrium (LD) structure. Use other populations to refine LD regions (for example African ancestry with shorter LD and more heterogeneity). Determine expression levels of nearby genes as a function of genotype at each locus (eQTL). Characterize gene regulatory regions by multiple empirical techniques bearing in mind that these are tissue and context specific. Combine regulatory regions with risk loci using coordinates from multiple reference genomes to capture all variation within the shorter regulatory regions that correlates with the tag SNP at each locus. Multiple experimental manipulations in model systems are needed to progressively implicate transcription units (genes) in mechanisms relevant to the associated loci: Knockouts of regulatory regions in animal (difficult and may be limited by functional redundancy, but new targeting methods in rat are promising) models followed by genome-wide expression analysis. Use chromatin association methods (3C, CHIA-PET) of regulatory regions to determine the identity of target genes (compare with eQTL data). Targeted gene perturbations in somatic cell models. Explore fully genome-wide eQTL and miRNA quantitative variation correlation in relevant tissues and cells. Explore epigenetic mechanisms in the context of genome-wide genetic polymorphism. Employ cell models and tissue reconstructions to evaluate mechanisms using gene perturbations and polymorphic variants. The human cancer cell xenograft has re-emerged as a minimal in vivo validation of these models. Above all, resist the temptation to equate any partial functional evidence as sufficient. Published claims of functional relevance should be fully evaluated using the steps detailed above.

Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.

Human Papillomavirus Type 16 E6 Activates NF-κB, Induces cIAP-2 Expression, and Protects against Apoptosis in a PDZ Binding Motif-Dependent Manner

ABSTRACT Infection with human papillomavirus (HPV) is a critical factor in the pathogenesis of most cervical cancers and some aerodigestive cancers. The HPV E6 oncoprotein from high-risk HPV types contributes to the immortalization and transformation of cells by multiple mechanisms, including degradation of p53, transcriptional activation of human telomerase reverse transcriptase (hTERT), and degradation of several proteins containing PDZ domains. The ability of E6 to bind PDZ domain-containing proteins is independent of p53 degradation or hTERT activation but does correlate with oncogenic potential (R. A. Watson, M. Thomas, L. Banks, and S. Roberts, J. Cell Sci. 116:4925-4934, 2003) and is essential for induction of epithelial hyperplasia in vivo (M. L. Nguyen, M. M. Nguyen, D. Lee, A. E. Griep, and P. F. Lambert, J. Virol. 77:6957-6964, 2003). In this study, we found that HPV type 16 E6 was able to activate NF-κB in airway epithelial cells through the induction of nuclear binding activity of p52-containing NF-κB complexes in a PDZ binding motif-dependent manner. Transcript accumulation for the NF-κB-responsive antiapoptotic gene encoding cIAP-2 and binding of nuclear factors to the proximal NF-κB binding site of the cIAP-2 gene promoter are induced by E6 expression. Furthermore, E6 is able to protect cells from TNF-induced apoptosis. All of these E6-dependent phenotypes are dependent on the presence of the PDZ binding motif of E6. Our results imply a role for targeting of PDZ proteins by E6 in NF-κB activation and protection from apoptosis in airway epithelial cells.

EGFR-T790M Is a Rare Lung Cancer Susceptibility Allele with Enhanced Kinase Activity

The use of tyrosine kinase inhibitors (TKI) has yielded great success in treatment of lung adenocarcinomas. However, patients who develop resistance to TKI treatment often acquire a somatic resistance mutation (T790M) located in the catalytic cleft of the epidermal growth factor receptor (EGFR) enzyme. Recently, a report describing EGFR-T790M as a germ-line mutation suggested that this mutation may be associated with inherited susceptibility to lung cancer. Contrary to previous reports, our analysis indicates that the T790M mutation confers increased Y992 and Y1068 phosphorylation levels. In a human bronchial epithelial cell line, overexpression of EGFR-T790M displayed a growth advantage over wild-type (WT) EGFR. We also screened 237 lung cancer family probands, in addition to 45 bronchoalveolar tumors, and found that none of them contained the EGFR-T790M mutation. Our observations show that EGFR-T790M provides a proliferative advantage with respect to WT EGFR and suggest that the enhanced kinase activity of this mutant is the basis for rare cases of inherited susceptibility to lung cancer.

Fine mapping of chromosome 6q23-25 region in familial lung cancer families reveals RGS17 as a likely candidate gene

Purpose: We have previously mapped a major susceptibility locus influencing familial lung cancer risk to chromosome 6q23-25. However, the causal gene at this locus remains undetermined. In this study, we further refined this locus to identify a single candidate gene, by fine mapping using microsatellite markers and association studies using high-density single nucleotide polymorphisms (SNP). Experimental Design: Six multigenerational families with five or more affected members were chosen for fine-mapping the 6q linkage region using microsatellite markers. For association mapping, we genotyped 24 6q-linked cases and 72 unrelated noncancer controls from the Genetic Epidemiology of Lung Cancer Consortium resources using the Affymetrix 500K chipset. Significant associations were validated in two independent familial lung cancer populations: 226 familial lung cases and 313 controls from the Genetic Epidemiology of Lung Cancer Consortium, and 154 familial cases and 325 controls from Mayo Clinic. Each familial case was chosen from one high-risk lung cancer family that has three or more affected members. Results: A region-wide scan across 6q23-25 found significant association between lung cancer susceptibility and three single nucleotide polymorphisms in the first intron of the RGS17 gene. This association was further confirmed in two independent familial lung cancer populations. By quantitative real-time PCR analysis of matched tumor and normal human tissues, we found that RGS17 transcript accumulation is highly and consistently increased in sporadic lung cancers. Human lung tumor cell proliferation and tumorigenesis in nude mice are inhibited upon knockdown of RGS17 levels. Conclusion:RGS17 is a major candidate for the familial lung cancer susceptibility locus on chromosome 6q23-25.

Functional characterization of CLPTM1L as a lung cancer risk candidate gene in the 5p15.33 locus

Cleft Lip and Palate Transmembrane Protein 1-Like (CLPTM1L), resides in a region of chromosome 5 for which copy number gain has been found to be the most frequent genetic event in the early stages of non-small cell lung cancer (NSCLC). This locus has been found by multiple genome wide association studies to be associated with lung cancer in both smokers and non-smokers. CLPTM1L has been identified as an overexpressed protein in human ovarian tumor cell lines that are resistant to cisplatin, which is the only insight thus far into the function of CLPTM1L. Here we find CLPTM1L expression to be increased in lung adenocarcinomas compared to matched normal lung tissues and in lung tumor cell lines by mechanisms not exclusive to copy number gain. Upon loss of CLPTM1L accumulation in lung tumor cells, cisplatin and camptothecin induced apoptosis were increased in direct proportion to the level of CLPTM1L knockdown. Bcl-xL accumulation was significantly decreased upon loss of CLPTM1L. Expression of exogenous Bcl-xL abolished sensitization to apoptotic killing with CLPTM1L knockdown. These results demonstrate that CLPTM1L, an overexpressed protein in lung tumor cells, protects from genotoxic stress induced apoptosis through regulation of Bcl-xL. Thus, this study implicates anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis and resistance to chemotherapy.

HPV16-E6 associated hTERT promoter acetylation is E6AP dependent, increased in later passage cells and enhanced by loss of p300

The E6 oncoprotein from high‐risk HPV types activates human telomerase reverse transcriptase (hTERT) transcription in human keratinocytes. Studies on how E6 regulates hTERT have implicated E‐box or X‐box elements in the hTERT promoter (Veldman et al., Proc Natl Acad Sci USA 2003;100:8211–14; Oh et al., J Virol 2001;75:5559–66; Gewin et al., Genes Dev 2004;18:2269–82), but the mechanism of activation by E6 is still controversial and not well defined. Here, we demonstrate that induction of both hTERT expression and telomerase activity by HPV‐16 E6 in early passage keratinocytes is associated with acetylation of histone H3 at the hTERT promoter, is dependent on the E6 associated protein (E6AP) and is not exclusively reliant on E‐box or X‐box elements. Further increases in histone acetylation of the hTERT promoter and hTERT transcriptional activity in E6 expressing cells that had been passaged extensively in culture were found to occur only with the endogenous promoter and not with an exogenously introduced hTERT promoter construct. Telomerase activity at both early and late passages, however, was dependent on E6AP expression, implying a continued reliance on E6 function for telomerase activity. Our results demonstrate that E6 induces hTERT promoter acetylation, but that further increases in telomerase activity and histone acetylation in later passage E6 expressing cells are independent of E6 activation of the core hTERT promoter. We also provide evidence that the transcription factor p300 is a potential repressor of telomerase activation and histone acetylation in the context of E6 expression. These studies give insight into how immortalization by HPV results in upregulation of hTERT and furthers our understanding of how telomerase is activated during the process of malignant transformation.

RGS17, an Overexpressed Gene in Human Lung and Prostate Cancer, Induces Tumor Cell Proliferation Through the Cyclic AMP-PKA-CREB Pathway

We have identified RGS17 as a commonly induced gene in lung and prostate tumors. Through microarray and gene expression analysis, we show that expression of RGS17 is up-regulated in 80% of lung tumors, and also up-regulated in prostate tumors. Through knockdown and overexpression of RGS17 in tumor cells, we show that RGS17 confers a proliferative phenotype and is required for the maintenance of the proliferative potential of tumor cells. We show through exon microarray, transcript analysis, and functional assays that RGS17 promotes cyclic AMP (cAMP)-responsive element binding protein (CREB)-responsive gene expression, increases cAMP levels, and enhances forskolin-mediated cAMP production. Furthermore, inhibition of cAMP-dependent kinase prevents tumor cell proliferation, and proliferation is partially rescued by RGS17 overexpression. In the present study, we show a role for RGS17 in the maintenance of tumor cell proliferation through induction of cAMP signaling and CREB phosphorylation. The prevalence of the induction of RGS17 in tumor tissues of various types further implicates its importance in the maintenance of tumor growth.

Dietary administration of berberine or Phellodendron amurense extract inhibits cell cycle progression and lung tumorigenesis.

Phellodendron amurense extract is a Chinese herbal remedy that has recently been studied for its antitumor, antimicrobial and other biological activities. It is previously unknown if these agents are bioavailable and effective against tumors when delivered as a dietary component. It is also unknown if the anti‐tumorigenic properties of berberine, an isoquinoline alkaloid component of P. amurense, is equally effective when administered alone. There are contrasting reports on the cellular processes involved in anti‐tumorigenesis by P. amurense and berberine. Here we find that berberine, when administered orally through the diet, inhibits in vivo tumorigenesis of both p53 expressing and p53 null lung tumor xenografts equally whether administered in its pure form or as a part of P. amurense extract. We also show that berberine induces G1 cell cycle arrest, inhibits proliferative kinase signaling and arrests the growth of lung tumor cells in culture. Berberine administered in the diet was detectable by HPLC in the lungs of mice fed P. amurense or equivalent doses of berberine at concentrations of 455 and 518 ng/ml respectively and inhibited the growth of xenografted A549 cell tumors, which grew to 9.4 and 6.4 mm3 respectively, compared to 58.9 mm3 in control mice (P < 0.001). Phosphorylation of Akt, CREB and MAPK was inhibited in A549 cells by P. amurense. Demonstration of oral bioavailability and anti‐tumorigenic efficacy of dietary berberine, as well as further demonstration of signaling pathway modulation and cell‐cycle arrest, implicate this relatively safe, natural compound as a potentially important therapeutic and chemopreventive agent for lung cancer.

Haplotype and cell proliferation analyses of candidate lung cancer susceptibility genes on chromosome 15q24-25.1.

Recent genome-wide association studies have linked the chromosome 15q24-25.1 locus to nicotine addiction and lung cancer susceptibility. To refine the 15q24-25.1 locus, we performed a haplotype-based association analysis of 194 familial lung cases and 219 cancer-free controls from the Genetic Epidemiology of Lung Cancer Consortium (GELCC) collection, and used proliferation and apoptosis analyses to determine which gene(s) in the 15q24-25.1 locus mediates effects on lung cancer cell growth in vitro. We identified two distinct subregions, hapL (P = 3.20 x 10(-6)) and hapN (P = 1.51 x 10(-6)), which were significantly associated with familial lung cancer. hapL encompasses IREB2, LOC123688, and PSMA4, and hapN encompasses the three nicotinic acetylcholine receptor subunit genes CHRNA5, CHRNA3, and CHRNB4. Examination of the genes around hapL revealed that PSMA4 plays a role in promoting cancer cell proliferation. PSMA4 mRNA levels were increased in lung tumors compared with normal lung tissues. Down-regulation of PSMA4 expression decreased proteasome activity and induced apoptosis. Proteasome dysfunction leads to many diseases including cancer, and drugs that inhibit proteasome activity show promise as a form of cancer treatment. Genes around hapN were also investigated, but did not show any direct effect on lung cancer cell proliferation. We concluded that PSMA4 is a strong candidate mediator of lung cancer cell growth, and may directly affect lung cancer susceptibility through its modulation of cell proliferation and apoptosis.