Uncovering dark matter in cancer by identifying epigenetic drivers.

The complex relationship between chromatin accessibility, transcriptional regulation, and cancer transitions presents a daunting puzzle. Terekhanova et al. created a pan-cancer epigenetic and transcriptomic atlas at single-cell resolution, yielding important insights into the underlying chromatin architecture of cancer transitions and novel discoveries with the potential to advance precision medicine.

High-throughput characterization of functional variants highlights heterogeneity and polygenicity underlying lung cancer susceptibility.

Genome-wide association studies (GWASs) have identified numerous lung cancer risk-associated loci. However, decoding molecular mechanisms of these associations is challenging since most of these genetic variants are non-protein-coding with unknown function. Here, we implemented massively parallel reporter assays (MPRAs) to simultaneously measure the allelic transcriptional activity of risk-associated variants. We tested 2,245 variants at 42 loci from 3 recent GWASs in East Asian and European populations in the context of two major lung cancer histological types and exposure to benzo(a)pyrene. This MPRA approach identified one or more variants (median 11 variants) with significant effects on transcriptional activity at 88% of GWAS loci. Multimodal integration of lung-specific epigenomic data demonstrated that 63% of the loci harbored multiple potentially functional variants in linkage disequilibrium. While 22% of the significant variants showed allelic effects in both A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cell lines, a subset of the functional variants displayed a significant cell-type interaction. Transcription factor analyses nominated potential regulators of the functional variants, including those with cell-type-specific expression and those predicted to bind multiple potentially functional variants across the GWAS loci. Linking functional variants to target genes based on four complementary approaches identified candidate susceptibility genes, including those affecting lung cancer cell growth. CRISPR interference of the top functional variant at 20q13.33 validated variant-to-gene connections, including RTEL1, SOX18, and ARFRP1. Our data provide a comprehensive functional analysis of lung cancer GWAS loci and help elucidate the molecular basis of heterogeneity and polygenicity underlying lung cancer susceptibility.

A 584 bp deletion in CTRB2 inhibits chymotrypsin B2 activity and secretion and confers risk of pancreatic cancer.

Genome-wide association studies (GWASs) have discovered 20 risk loci in the human genome where germline variants associate with risk of pancreatic ductal adenocarcinoma (PDAC) in populations of European ancestry. Here, we fine-mapped one such locus on chr16q23.1 (rs72802365, p = 2.51 × 10-17, OR = 1.36, 95% CI = 1.31-1.40) and identified colocalization (PP = 0.87) with aberrant exon 5-7 CTRB2 splicing in pancreatic tissues (pGTEx = 1.40 × 10-69, ßGTEx = 1.99; pLTG = 1.02 × 10-30, ßLTG = 1.99). Imputation of a 584 bp structural variant overlapping exon 6 of CTRB2 into the GWAS datasets resulted in a highly significant association with pancreatic cancer risk (p = 2.83 × 10-16, OR = 1.36, 95% CI = 1.31-1.42), indicating that it may underlie this signal. Exon skipping attributable to the deletion (risk) allele introduces a premature stop codon in exon 7 of CTRB2, yielding a truncated chymotrypsinogen B2 protein that lacks chymotrypsin activity, is poorly secreted, and accumulates intracellularly in the endoplasmic reticulum (ER). We propose that intracellular accumulation of a nonfunctional chymotrypsinogen B2 protein leads to ER stress and pancreatic inflammation, which may explain the increased pancreatic cancer risk in carriers of CTRB2 exon 6 deletion alleles.

Transcriptional regulation by NR5A2 links differentiation and inflammation in the pancreas.

Chronic inflammation increases the risk of developing one of several types of cancer. Inflammatory responses are currently thought to be controlled by mechanisms that rely on transcriptional networks that are distinct from those involved in cell differentiation. The orphan nuclear receptor NR5A2 participates in a wide variety of processes, including cholesterol and glucose metabolism in the liver, resolution of endoplasmic reticulum stress, intestinal glucocorticoid production, pancreatic development and acinar differentiation. In genome-wide association studies, single nucleotide polymorphisms in the vicinity of NR5A2 have previously been associated with the risk of pancreatic adenocarcinoma. In mice, Nr5a2 heterozygosity sensitizes the pancreas to damage, impairs regeneration and cooperates with mutant Kras in tumour progression. Here, using a global transcriptomic analysis, we describe an epithelial-cell-autonomous basal pre-inflammatory state in the pancreas of Nr5a2+/- mice that is reminiscent of the early stages of pancreatitis-induced inflammation and is conserved in histologically normal human pancreases with reduced expression of NR5A2 mRNA. In Nr5a2+/-mice, NR5A2 undergoes a marked transcriptional switch, relocating from differentiation-specific to inflammatory genes and thereby promoting gene transcription that is dependent on the AP-1 transcription factor. Pancreatic deletion of Jun rescues the pre-inflammatory phenotype, as well as binding of NR5A2 to inflammatory gene promoters and the defective regenerative response to damage. These findings support the notion that, in the pancreas, the transcriptional networks involved in differentiation-specific functions also suppress inflammatory programmes. Under conditions of genetic or environmental constraint, these networks can be subverted to foster inflammation.

Inferred expression regulator activities suggest genes mediating cardiometabolic genetic signals.

Expression QTL (eQTL) analyses have suggested many genes mediating genome-wide association study (GWAS) signals but most GWAS signals still lack compelling explanatory genes. We have leveraged an adipose-specific gene regulatory network to infer expression regulator activities and phenotypic master regulators (MRs), which were used to detect activity QTLs (aQTLs) at cardiometabolic trait GWAS loci. Regulator activities were inferred with the VIPER algorithm that integrates enrichment of expected expression changes among a regulator's target genes with confidence in their regulator-target network interactions and target overlap between different regulators (i.e., pleiotropy). Phenotypic MRs were identified as those regulators whose activities were most important in predicting their respective phenotypes using random forest modeling. While eQTLs were typically more significant than aQTLs in cis, the opposite was true among candidate MRs in trans. Several GWAS loci colocalized with MR trans-eQTLs/aQTLs in the absence of colocalized cis-QTLs. Intriguingly, at the 1p36.1 BMI GWAS locus the EPHB2 cis-aQTL was stronger than its cis-eQTL and colocalized with the GWAS signal and 35 BMI MR trans-aQTLs, suggesting the GWAS signal may be mediated by effects on EPHB2 activity and its downstream effects on a network of BMI MRs. These MR and aQTL analyses represent systems genetic methods that may be broadly applied to supplement standard eQTL analyses for suggesting molecular effects mediating GWAS signals.

A pooled genome-wide association study identifies pancreatic cancer susceptibility loci on chromosome 19p12 and 19p13.3 in the full-Jewish population.

Jews are estimated to be at increased risk of pancreatic cancer compared to non-Jews, but their observed 50-80% excess risk is not explained by known non-genetic or genetic risk factors. We conducted a GWAS in a case-control sample of American Jews, largely Ashkenazi, including 406 pancreatic cancer patients and 2332 controls, identified in the dbGaP, PanScan I/II, PanC4 and GERA data sets. We then examined resulting SNPs with P < 10-7 in an expanded sample set, of 539 full- or part-Jewish pancreatic cancer patients and 4117 full- or part-Jewish controls from the same data sets. Jewish ancestries were genetically determined using seeded FastPCA. Among the full Jews, a novel genome-wide significant association was detected on chromosome 19p12 (rs66562280, per-allele OR = 1.55, 95% CI = 1.33-1.81, P = 10-7.6). A suggestive relatively independent association was detected on chromosome 19p13.3 (rs2656937, OR = 1.53, 95% CI = 1.31-1.78, P = 10-7.0). Similar associations were seen for these SNPs among the full and part Jews combined. This is the first GWAS conducted for pancreatic cancer in the increased-risk Jewish population. The SNPs rs66562280 and rs2656937 are located in introns of ZNF100-like and ARRDC5, respectively, and are known to alter regulatory motifs of genes that play integral roles in pancreatic carcinogenesis.

Cell-type-specific eQTL of primary melanocytes facilitates identification of melanoma susceptibility genes.

Most expression quantitative trait locus (eQTL) studies to date have been performed in heterogeneous tissues as opposed to specific cell types. To better understand the cell-type-specific regulatory landscape of human melanocytes, which give rise to melanoma but account for <5% of typical human skin biopsies, we performed an eQTL analysis in primary melanocyte cultures from 106 newborn males. We identified 597,335 cis-eQTL SNPs prior to linkage disequilibrium (LD) pruning and 4997 eGenes (FDR < 0.05). Melanocyte eQTLs differed considerably from those identified in the 44 GTEx tissue types, including skin. Over a third of melanocyte eGenes, including key genes in melanin synthesis pathways, were unique to melanocytes compared to those of GTEx skin tissues or TCGA melanomas. The melanocyte data set also identified trans-eQTLs, including those connecting a pigmentation-associated functional SNP with four genes, likely through cis-regulation of IRF4 Melanocyte eQTLs are enriched in cis-regulatory signatures found in melanocytes as well as in melanoma-associated variants identified through genome-wide association studies. Melanocyte eQTLs also colocalized with melanoma GWAS variants in five known loci. Finally, a transcriptome-wide association study using melanocyte eQTLs uncovered four novel susceptibility loci, where imputed expression levels of five genes (ZFP90, HEBP1, MSC, CBWD1, and RP11-383H13.1) were associated with melanoma at genome-wide significant P-values. Our data highlight the utility of lineage-specific eQTL resources for annotating GWAS findings, and present a robust database for genomic research of melanoma risk and melanocyte biology.

CLPTM1L/CRR9 ectodomain interaction with GRP78 at the cell surface signals for survival and chemoresistance upon ER stress in pancreatic adenocarcinoma cells.

Altered regulation of endoplasmic reticulum (ER) homeostasis has been implicated in many cancers and has recently become a therapeutic and chemosensitization target of interest. We have identified Cleft Lip and Palate Transmembrane 1-Like (CLPTM1L)/Cisplatin Resistance Related Protein 9 (CRR9) as an ER stress related mediator of cytoprotection in pancreatic cancer. We recently demonstrated that CLPTM1L is highly expressed in pancreatic ductal adenocarcinoma and associated with poor outcome. Furthermore, we have discovered that CLPTM1L interacts with phosphoinositol-3-kinase-alpha at the tumor cell surface and causes up-regulation of Bcl-xL and pAkt mediated survival signaling. Here, we demonstrate surface relocalization and survival signaling by CLPTM1L triggered by endoplasmic reticular (ER) stress. We demonstrate the interaction of CLPTM1L with the central ER stress survival mediator, Glucose Regulated Protein 78 (GRP78)/Binding Immunoglobulin Protein (BiP) and PI3K-alpha /p110α. This interaction and surface relocalization of CLPTM1L and GRP78 is induced by ER stress, including that caused by treatment with gemcitabine. We demonstrate that the extracellular loop of CLPTM1L is required for gemcitabine resistance and interaction with GRP78. This interaction and the chemoresistance effect conferred by this pathway is targetable with our recently developed inhibitory CLPTM1L antibodies, which may represent novel modalities of chemosensitization and treatment of pancreatic adenocarcinoma. Anchorage independent growth, GRP78-mediated chemoresistance, and Akt phosphorylation were abrogated by inhibition of CLPTM1L. These findings demonstrate a novel and potentially targetable mechanism of cytoprotection and chemoresistance in pancreatic tumors.

Winner's Curse Correction and Variable Thresholding Improve Performance of Polygenic Risk Modeling Based on Genome-Wide Association Study Summary-Level Data.

Recent heritability analyses have indicated that genome-wide association studies (GWAS) have the potential to improve genetic risk prediction for complex diseases based on polygenic risk score (PRS), a simple modelling technique that can be implemented using summary-level data from the discovery samples. We herein propose modifications to improve the performance of PRS. We introduce threshold-dependent winner's-curse adjustments for marginal association coefficients that are used to weight the single-nucleotide polymorphisms (SNPs) in PRS. Further, as a way to incorporate external functional/annotation knowledge that could identify subsets of SNPs highly enriched for associations, we propose variable thresholds for SNPs selection. We applied our methods to GWAS summary-level data of 14 complex diseases. Across all diseases, a simple winner's curse correction uniformly led to enhancement of performance of the models, whereas incorporation of functional SNPs was beneficial only for selected diseases. Compared to the standard PRS algorithm, the proposed methods in combination led to notable gain in efficiency (25-50% increase in the prediction R2) for 5 of 14 diseases. As an example, for GWAS of type 2 diabetes, winner's curse correction improved prediction R2 from 2.29% based on the standard PRS to 3.10% (P = 0.0017) and incorporating functional annotation data further improved R2 to 3.53% (P = 2×10-5). Our simulation studies illustrate why differential treatment of certain categories of functional SNPs, even when shown to be highly enriched for GWAS-heritability, does not lead to proportionate improvement in genetic risk-prediction because of non-uniform linkage disequilibrium structure.

Characterising cis-regulatory variation in the transcriptome of histologically normal and tumour-derived pancreatic tissues.

OBJECTIVE: To elucidate the genetic architecture of gene expression in pancreatic tissues. DESIGN: We performed expression quantitative trait locus (eQTL) analysis in histologically normal pancreatic tissue samples (n=95) using RNA sequencing and the corresponding 1000 genomes imputed germline genotypes. Data from pancreatic tumour-derived tissue samples (n=115) from The Cancer Genome Atlas were included for comparison. RESULTS: We identified 38 615 cis-eQTLs (in 484 genes) in histologically normal tissues and 39 713 cis-eQTL (in 237 genes) in tumour-derived tissues (false discovery rate <0.1), with the strongest effects seen near transcriptional start sites. Approximately 23% and 42% of genes with significant cis-eQTLs appeared to be specific for tumour-derived and normal-derived tissues, respectively. Significant enrichment of cis-eQTL variants was noted in non-coding regulatory regions, in particular for pancreatic tissues (1.53-fold to 3.12-fold, p≤0.0001), indicating tissue-specific functional relevance. A common pancreatic cancer risk locus on 9q34.2 (rs687289) was associated with ABO expression in histologically normal (p=5.8×10-8) and tumour-derived (p=8.3×10-5) tissues. The high linkage disequilibrium between this variant and the O blood group generating deletion variant in ABO (exon 6) suggested that nonsense-mediated decay (NMD) of the 'O' mRNA might explain this finding. However, knockdown of crucial NMD regulators did not influence decay of the ABO 'O' mRNA, indicating that a gene regulatory element influenced by pancreatic cancer risk alleles may underlie the eQTL. CONCLUSIONS: We have identified cis-eQTLs representing potential functional regulatory variants in the pancreas and generated a rich data set for further studies on gene expression and its regulation in pancreatic tissues.

Pancreatic cancer risk is modulated by inflammatory potential of diet and ABO genotype: a consortia-based evaluation and replication study.

Diets with high inflammatory potential are suspected to increase risk for pancreatic cancer (PC). Using pooled analyses, we examined whether this association applies to populations from different geographic regions and population subgroups with varying risks for PC, including variation in ABO blood type. Data from six case-control studies (cases, n = 2414; controls, n = 4528) in the Pancreatic Cancer Case-Control Consortium (PanC4) were analyzed, followed by replication in five nested case-control studies (cases, n = 1268; controls, n = 4215) from the Pancreatic Cancer Cohort Consortium (PanScan). Two polymorphisms in the ABO locus (rs505922 and rs8176746) were used to infer participants' blood types. Dietary questionnaire-derived nutrient/food intake was used to compute energy-adjusted dietary inflammatory index (E-DII®) scores to assess inflammatory potential of diet. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using multivariable-adjusted logistic regression. Higher E-DII scores, reflecting greater inflammatory potential of diet, were associated with increased PC risk in PanC4 [ORQ5 versus Q1=2.20, 95% confidence interval (CI) = 1.85-2.61, Ptrend < 0.0001; ORcontinuous = 1.20, 95% CI = 1.17-1.24], and PanScan (ORQ5 versus Q1 = 1.23, 95% CI = 0.92-1.66, Ptrend = 0.008; ORcontinuous = 1.09, 95% CI = 1.02-1.15). As expected, genotype-derived non-O blood type was associated with increased PC risk in both the PanC4 and PanScan studies. Stratified analyses of associations between E-DII quintiles and PC by genotype-derived ABO blood type did not show interaction by blood type (Pinteraction = 0.10 in PanC4 and Pinteraction=0.13 in PanScan). The results show that consuming a pro-inflammatory diet and carrying non-O blood type are each individually, but not interactively, associated with increased PC risk.

Functional characterization of a chr13q22.1 pancreatic cancer risk locus reveals long-range interaction and allele-specific effects on DIS3 expression.

Genome-wide association studies (GWAS) have identified multiple common susceptibility loci for pancreatic cancer. Here we report fine-mapping and functional analysis of one such locus residing in a 610 kb gene desert on chr13q22.1 (marked by rs9543325). The closest candidate genes, KLF5, KLF12, PIBF1, DIS3 and BORA, range in distance from 265-586 kb. Sequencing three sub-regions containing the top ranked SNPs by imputation P-value revealed a 30 bp insertion/deletion (indel) variant that was significantly associated with pancreatic cancer risk (rs386772267, P = 2.30 × 10-11, OR = 1.22, 95% CI 1.15-1.28) and highly correlated to rs9543325 (r2 = 0.97 in the 1000 Genomes EUR population). This indel was the most significant cis-eQTL variant in a set of 222 histologically normal pancreatic tissue samples (ß = 0.26, P = 0.004), with the insertion (risk-increasing) allele associated with reduced DIS3 expression. DIS3 encodes a catalytic subunit of the nuclear RNA exosome complex that mediates RNA processing and decay, and is mutated in several cancers. Chromosome conformation capture revealed a long range (570 kb) physical interaction between a sub-region of the risk locus, containing rs386772267, and a region ∼6 kb upstream of DIS3 Finally, repressor regulatory activity and allele-specific protein binding by transcription factors of the TCF/LEF family were observed for the risk-increasing allele of rs386772267, indicating that expression regulation at this risk locus may be influenced by the Wnt signaling pathway. In conclusion, we have identified a putative functional indel variant at chr13q22.1 that associates with decreased DIS3 expression in carriers of pancreatic cancer risk-increasing alleles, and could therefore affect nuclear RNA processing and/or decay.

Leucocyte telomere length, genetic variants at the TERT gene region and risk of pancreatic cancer.

OBJECTIVE: Telomere shortening occurs as an early event in pancreatic tumorigenesis, and genetic variants at the telomerase reverse transcriptase (TERT) gene region have been associated with pancreatic cancer risk. However, it is unknown whether prediagnostic leucocyte telomere length is associated with subsequent risk of pancreatic cancer. DESIGN: We measured prediagnostic leucocyte telomere length in 386 pancreatic cancer cases and 896 matched controls from five prospective US cohorts. ORs and 95% CIs were calculated using conditional logistic regression. Matching factors included year of birth, cohort (which also matches on sex), smoking status, fasting status and month/year of blood collection. We additionally examined single-nucleotide polymorphisms (SNPs) at the TERT region in relation to pancreatic cancer risk and leucocyte telomere length using logistic and linear regression, respectively. RESULTS: Shorter prediagnostic leucocyte telomere length was associated with higher risk of pancreatic cancer (comparing extreme quintiles of telomere length, OR 1.72; 95% CI 1.07 to 2.78; ptrend=0.048). Results remained unchanged after adjustment for diabetes, body mass index and physical activity. Three SNPs at TERT (linkage disequilibrium r2<0.25) were associated with pancreatic cancer risk, including rs401681 (per minor allele OR 1.33; 95% CI 1.12 to 1.59; p=0.002), rs2736100 (per minor allele OR 1.36; 95% CI 1.13 to 1.63; p=0.001) and rs2736098 (per minor allele OR 0.75; 95% CI 0.63 to 0.90; p=0.002). The minor allele for rs401681 was associated with shorter telomere length (p=0.023). CONCLUSIONS: Prediagnostic leucocyte telomere length and genetic variants at the TERT gene region were associated with risk of pancreatic cancer.

Prospective study of DNA methylation at chromosome 8q24 in peripheral blood and prostate cancer risk.

BACKGROUND: Chromosome 8q24 has emerged as an important genetic susceptibility region for several cancers, including prostate cancer; however, little is known about the contribution of DNA methylation in this region to risk. METHODS: We prospectively evaluated DNA methylation at 8q24 in relation to prostate cancer using pre-diagnostic blood samples from 694 prostate cancer cases (including 172 aggressive cases) and 703 controls in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. We used logistic regression to estimate odds ratios and 95% confidence intervals. RESULTS: Although none remained significant after adjustment for multiple testing (q>0.05), of the 50 CpG sites meeting quality control, we identified 8 sites that were nominally associated with prostate cancer (Ptrend<0.05), including 6 correlated (Spearman ρ: 0.20-0.52) sites in POU5F1B and 2 intergenic sites (most significant site: Chr8:128428897 in POU5F1B, Ptrend=0.01). We also identified two correlated (ρ=0.39) sites in MYC (Chr8:128753187 and Chr8:128753154) that were associated with aggressive (Ptrend=0.02 and 0.03), but not non-aggressive disease (Ptrend=0.70 and 0.20; Pheterogeneity=0.01 and 4.6 × 10-3). These findings persisted after adjustment for the top 8q24 prostate cancer variants in our study. CONCLUSIONS: Although requiring replication, our findings provide some evidence that 8q24 DNA methylation levels may be associated with prostate cancer risk.

Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24.

Prostate cancer is the most prevalent noncutaneous cancer in males in developed regions, with African American men having among the highest worldwide incidence and mortality rates. Here we report a second genetic variant in the 8q24 region that, in conjunction with another variant we recently discovered, accounts for about 11%-13% of prostate cancer cases in individuals of European descent and 31% of cases in African Americans. We made the current discovery through a genome-wide association scan of 1,453 affected Icelandic individuals and 3,064 controls using the Illumina HumanHap300 BeadChip followed by four replication studies. A key step in the discovery was the construction of a 14-SNP haplotype that efficiently tags a relatively uncommon (2%-4%) susceptibility variant in individuals of European descent that happens to be very common (approximately 42%) in African Americans. The newly identified variant shows a stronger association with affected individuals who have an earlier age at diagnosis.

A common variant associated with prostate cancer in European and African populations.

With the increasing incidence of prostate cancer, identifying common genetic variants that confer risk of the disease is important. Here we report such a variant on chromosome 8q24, a region initially identified through a study of Icelandic families. Allele -8 of the microsatellite DG8S737 was associated with prostate cancer in three case-control series of European ancestry from Iceland, Sweden and the US. The estimated odds ratio (OR) of the allele is 1.62 (P = 2.7 x 10(-11)). About 19% of affected men and 13% of the general population carry at least one copy, yielding a population attributable risk (PAR) of approximately 8%. The association was also replicated in an African American case-control group with a similar OR, in which 41% of affected individuals and 30% of the population are carriers. This leads to a greater estimated PAR (16%) that may contribute to higher incidence of prostate cancer in African American men than in men of European ancestry.

Genome-wide association study of survival in patients with pancreatic adenocarcinoma.

BACKGROUND AND OBJECTIVE: Survival of patients with pancreatic adenocarcinoma is limited and few prognostic factors are known. We conducted a two-stage genome-wide association study (GWAS) to identify germline variants associated with survival in patients with pancreatic adenocarcinoma. METHODS: We analysed overall survival in relation to single nucleotide polymorphisms (SNPs) among 1005 patients from two large GWAS datasets, PanScan I and ChinaPC. Cox proportional hazards regression was used in an additive genetic model with adjustment for age, sex, clinical stage and the top four principal components of population stratification. The first stage included 642 cases of European ancestry (PanScan), from which the top SNPs (p≤10(-5)) were advanced to a joint analysis with 363 additional patients from China (ChinaPC). RESULTS: In the first stage of cases of European descent, the top-ranked loci were at chromosomes 11p15.4, 18p11.21 and 1p36.13, tagged by rs12362504 (p=1.63×10(-7)), rs981621 (p=1.65×10(-7)) and rs16861827 (p=3.75×10(-7)), respectively. 131 SNPs with p≤10(-5) were advanced to a joint analysis with cases from the ChinaPC study. In the joint analysis, the top-ranked SNP was rs10500715 (minor allele frequency, 0.37; p=1.72×10(-7)) on chromosome 11p15.4, which is intronic to the SET binding factor 2 (SBF2) gene. The HR (95% CI) for death was 0.74 (0.66 to 0.84) in PanScan I, 0.79 (0.65 to 0.97) in ChinaPC and 0.76 (0.68 to 0.84) in the joint analysis. CONCLUSIONS: Germline genetic variation in the SBF2 locus was associated with overall survival in patients with pancreatic adenocarcinoma of European and Asian ancestry. This association should be investigated in additional large patient cohorts.

Transcriptome analysis of pancreatic cancer reveals a tumor suppressor function for HNF1A.

Pancreatic ductal adenocarcinoma (PDAC) is driven by the accumulation of somatic mutations, epigenetic modifications and changes in the micro-environment. New approaches to investigating disruptions of gene expression networks promise to uncover key regulators and pathways in carcinogenesis. We performed messenger RNA-sequencing in pancreatic normal (n = 10) and tumor (n = 8) derived tissue samples, as well as in pancreatic cancer cell lines (n = 9), to determine differential gene expression (DE) patterns. Sub-network enrichment analyses identified HNF1A as the regulator of the most significantly and consistently dysregulated expression sub-network in pancreatic tumor tissues and cells (median P = 7.56×10(-7), median rank = 1, range = 1-25). To explore the effects of HNF1A expression in pancreatic tumor-derived cells, we generated stable HNF1A-inducible clones in two pancreatic cancer cell lines (PANC-1 and MIA PaCa-2) and observed growth inhibition (5.3-fold, P = 4.5×10(-5) for MIA PaCa-2 clones; 7.2-fold, P = 2.2×10(-5) for PANC-1 clones), and a G0/G1 cell cycle arrest and apoptosis upon induction. These effects correlated with HNF1A-induced down-regulation of 51 of 84 cell cycle genes (e.g. E2F1, CDK2, CDK4, MCM2/3/4/5, SKP2 and CCND1), decreased expression of anti-apoptotic genes (e.g. BIRC2/5/6 and AKT) and increased expression of pro-apoptotic genes (e.g. CASP4/9/10 and APAF1). In light of the established role of HNF1A in the regulation of pancreatic development and homeostasis, our data suggest that it also functions as an important tumor suppressor in the pancreas.

Integrating gene expression and epidemiological data for the discovery of genetic interactions associated with cancer risk.

Dozens of common genetic variants associated with cancer risk have been identified through genome-wide association studies (GWASs). However, these variants only explain a modest fraction of the heritability of disease. The missing heritability has been attributed to several factors, among them the existence of genetic interactions (G × G). Systematic screens for G × G in model organisms have revealed their fundamental influence in complex phenotypes. In this scenario, G × G overlap significantly with other types of gene and/or protein relationships. Here, by integrating predicted G × G from GWAS data and complex- and context-defined gene coexpression profiles, we provide evidence for G × G associated with cancer risk. G × G predicted from a breast cancer GWAS dataset identified significant overlaps [relative enrichments (REs) of 8-36%, empirical P values < 0.05 to 10(-4)] with complex (non-linear) gene coexpression in breast tumors. The use of gene or protein data not specific for breast cancer did not reveal overlaps. According to the predicted G × G, experimental assays demonstrated functional interplay between lipoma-preferred partner and transforming growth factor-ß signaling in the MCF10A non-tumorigenic mammary epithelial cell model. Next, integration of pancreatic tumor gene expression profiles with pancreatic cancer G × G predicted from a GWAS corroborated the observations made for breast cancer risk (REs of 25-59%). The method presented here can potentially support the identification of genetic interactions associated with cancer risk, providing novel mechanistic hypotheses for carcinogenesis.

Genetic polymorphisms in the 9p21 region associated with risk of multiple cancers.

The chromosome 9p21 region has been implicated in the pathogenesis of multiple cancers. We analyzed 9p21 single nucleotide polymorphisms (SNPs) from eight genome-wide association studies (GWAS) with data deposited in dbGaP, including studies of esophageal squamous cell carcinoma (ESCC), gastric cancer (GC), pancreatic cancer, renal cell carcinoma (RCC), lung cancer (LC), breast cancer (BrC), bladder cancer (BC) and prostate cancer (PrC). The number of subjects ranged from 2252 (PrC) to 7619 (LC). SNP-level analyses for each cancer were conducted by logistic regression or random-effects meta-analysis. A subset-based statistical approach (ASSET) was performed to combine SNP-level P values across multiple cancers. We calculated gene-level P values using the adaptive rank truncated product method. We identified that rs1063192 and rs2157719 in the CDKN2A/2B region were significantly associated with ESCC and rs2764736 (3' of TUSC1) was associated with BC (P ≤ 2.59 × 10(-6)). ASSET analyses identified four SNPs significantly associated with multiple cancers: rs3731239 (CDKN2A intronic) with ESCC, GC and BC (P = 3.96 × 10(-) (4)); rs10811474 (3' of IFNW1) with RCC and BrC (P = 0.001); rs12683422 (LINGO2 intronic) with RCC and BC (P = 5.93 × 10(-) (4)) and rs10511729 (3' of ELAVL2) with LC and BrC (P = 8.63 × 10(-) (4)). At gene level, CDKN2B, CDKN2A and CDKN2B-AS1 were significantly associated with ESCC (P ≤ 4.70 × 10(-) (5)). Rs10511729 and rs10811474 were associated with cis-expression of 9p21 genes in corresponding cancer tissues in the expression quantitative trait loci analysis. In conclusion, we identified several genetic variants in the 9p21 region associated with the risk of multiple cancers, suggesting that this region may contribute to a shared susceptibility across different cancer types.