Monteiro, Álvaro N.A.

Abstract Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and, through physical DNA interactions, BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-seq regions, and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer. Significance: Mapping the 9p22.2 ovarian cancer risk locus identifies BNC2 as an ovarian cancer risk gene. See related commentary by Choi and Brown, p. 439

Abstract Genome Wide Association Studies (GWAS) are designed to identify single nucleotide polymorphisms (SNPs) mapping to genomic regions associated with a particular trait or disease. Currently, 19 genomic loci associated with risk for estrogen receptor negative breast cancer have been identified. However, for most of these loci the mechanisms driving susceptibility have remained elusive. The 2p23.2 locus, defined by SNP rs67073037 (p = 4.76 x 10-9), contains 79 SNPs exhibiting genome-wide significant associations as well as four candidate genes: SPDYA, TRMT61B, WDR43, and FAM179A. Post-GWAS functional analysis revealed two candidate causal regulatory elements: an enhancer-like element defined by SNP rs44017214 located in the first intron of the WDR43 gene; and a segment in the 3’-UTR region of WDR43 defined by SNPs rs11680458 and rs11331880 which are predicted to have allele specific binding of non-coding microRNAs miR-548 and miR-376, respectively. We validated allele specific transcription factor binding to rs44017214 using competitive binding of USF1/USF2 to the risk allele in both MCF10A and CAL51 cell lines in Electrophoretic Mobility Shift Assays (EMSA). Additionally, rs44017214 was removed using CRISPR-cas9, and expression of neighboring genes was evaluated by qRT-PCR. No change in expression was found for any gene in a 2 Mb region surrounding the SNP, with the exception of PLB1. This suggests that the enhancer element in the intron of WDR43 has PLB1 as its target. To assess the miRNA binding site impact on WDR43 expression the pMIR-REPORT Luciferase allele-specific vector was co-transfected with miR-141/200, miR548, or miR-376 in MCF10A cells. The rs1131880 allele C in the WDR43 3’-UTR lead to the suppression of luciferase expression by miR-376 compared to the G allele. This study reveals two independent regulatory mechanisms within the 2p23.2 locus; (i) modulation of enhancer activity by allele specific binding of transcription factors USF1/USF2 to rs4401714, and (ii) decreasing the expression of WDR43 through the formation of a miR-376 binding site at the 3’-UTR of the WDR43 gene. Elucidating the functional significance of these common variants in the novel susceptibility loci 2p23.2 furthers our knowledge of the etiology of estrogen receptor negative breast cancer. Citation Format: Carly M. Harro, Gustavo Mendoza-Fandino, Nicholas T. Woods, Xueli Li, Fergus J. Couch, Alvaro N. Monteiro. Two distinct regulatory mechanisms underlie estrogen receptor negative breast cancer susceptibility at the 2p23.2 locus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1306. doi:10.1158/1538-7445.AM2017-1306

Abstract Breast cancer risks conferred by many germline missense variants in the BRCA1 and BRCA2 genes, often referred to as variants of uncertain significance (VUS), have not been established. In this study, associations between 19 BRCA1 and 33 BRCA2 missense substitution variants and breast cancer risk were investigated through a breast cancer case–control study using genotyping data from 38 studies of predominantly European ancestry (41,890 cases and 41,607 controls) and nine studies of Asian ancestry (6,269 cases and 6,624 controls). The BRCA2 c.9104A>C, p.Tyr3035Ser (OR = 2.52; P = 0.04), and BRCA1 c.5096G>A, p.Arg1699Gln (OR = 4.29; P = 0.009) variant were associated with moderately increased risks of breast cancer among Europeans, whereas BRCA2 c.7522G>A, p.Gly2508Ser (OR = 2.68; P = 0.004), and c.8187G>T, p.Lys2729Asn (OR = 1.4; P = 0.004) were associated with moderate and low risks of breast cancer among Asians. Functional characterization of the BRCA2 variants using four quantitative assays showed reduced BRCA2 activity for p.Tyr3035Ser compared with wild-type. Overall, our results show how BRCA2 missense variants that influence protein function can confer clinically relevant, moderately increased risks of breast cancer, with potential implications for risk management guidelines in women with these specific variants. Cancer Res; 77(11); 2789–99. ©2017 AACR.

Abstract Background: Genome-wide association studies (GWAS) have identified multiple loci associated with epithelial ovarian cancer (EOC) susceptibility, but further progress requires integration of epidemiology and biology to illuminate true risk loci below genome-wide significance levels (P < 5 × 10−8). Most risk SNPs lie within non–protein-encoding regions, and we hypothesize that long noncoding RNA (lncRNA) genes are enriched at EOC risk regions and represent biologically relevant functional targets. Methods: Using imputed GWAS data from about 18,000 invasive EOC cases and 34,000 controls of European ancestry, the GENCODE (v19) lncRNA database was used to annotate SNPs from 13,442 lncRNAs for permutation-based enrichment analysis. Tumor expression quantitative trait locus (eQTL) analysis was performed for sub-genome-wide regions (1 × 10−5 > P > 5 × 10−8) overlapping lncRNAs. Results: Of 5,294 EOC-associated SNPs (P < 1.0 × 10−5), 1,464 (28%) mapped within 53 unique lncRNAs and an additional 3,484 (66%) SNPs were correlated (r2 > 0.2) with SNPs within 115 lncRNAs. EOC-associated SNPs comprised 130 independent regions, of which 72 (55%) overlapped with lncRNAs, representing a significant enrichment (P = 5.0 × 10−4) that was more pronounced among a subset of 5,401 lncRNAs with active epigenetic regulation in normal ovarian tissue. EOC-associated lncRNAs and their putative promoters and transcription factors were enriched for biologically relevant pathways and eQTL analysis identified five novel putative risk regions with allele-specific effects on lncRNA gene expression. Conclusions: lncRNAs are significantly enriched at EOC risk regions, suggesting a mechanistic role for lncRNAs in driving predisposition to EOC. Impact: lncRNAs represent key candidates for integrative epidemiologic and functional studies. Further research on their biologic role in ovarian cancer is indicated. Cancer Epidemiol Biomarkers Prev; 26(1); 116–25. ©2016 AACR.

Background The rarity of mutations in PALB2, CHEK2 and ATM make it difficult to estimate precisely associated cancer risks. Population-based family studies have provided evidence that at least some of these mutations are associated with breast cancer risk as high as those associated with rare BRCA2 mutations. We aimed to estimate the relative risks associated with specific rare variants in PALB2, CHEK2 and ATM via a multicentre case-control study. Methods We genotyped 10 rare mutations using the custom iCOGS array: PALB2 c.1592delT, c.2816T>G and c.3113G>A, CHEK2 c.349A>G, c.538C>T, c.715G>A, c.1036C>T, c.1312G>T, and c.1343T>G and ATM c.7271T>G. We assessed associations with breast cancer risk (42 671 cases and 42 164 controls), as well as prostate (22 301 cases and 22 320 controls) and ovarian (14 542 cases and 23 491 controls) cancer risk, for each variant. Results For European women, strong evidence of association with breast cancer risk was observed for PALB2 c.1592delT OR 3.44 (95% CI 1.39 to 8.52, p=7.1×10−5), PALB2 c.3113G>A OR 4.21 (95% CI 1.84 to 9.60, p=6.9×10−8) and ATM c.7271T>G OR 11.0 (95% CI 1.42 to 85.7, p=0.0012). We also found evidence of association with breast cancer risk for three variants in CHEK2, c.349A>G OR 2.26 (95% CI 1.29 to 3.95), c.1036C>T OR 5.06 (95% CI 1.09 to 23.5) and c.538C>T OR 1.33 (95% CI 1.05 to 1.67) (p≤0.017). Evidence for prostate cancer risk was observed for CHEK2 c.1343T>G OR 3.03 (95% CI 1.53 to 6.03, p=0.0006) for African men and CHEK2 c.1312G>T OR 2.21 (95% CI 1.06 to 4.63, p=0.030) for European men. No evidence of association with ovarian cancer was found for any of these variants. Conclusions This report adds to accumulating evidence that at least some variants in these genes are associated with an increased risk of breast cancer that is clinically important.

Abstract Breast, ovarian, and prostate cancers are hormone-related and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association (GWA) studies. Meta-analyses combining the largest GWA meta-analysis data sets for these cancers totaling 112,349 cases and 116,421 controls of European ancestry, all together and in pairs, identified at P < 10−8 seven new cross-cancer loci: three associated with susceptibility to all three cancers (rs17041869/2q13/BCL2L11; rs7937840/11q12/INCENP; rs1469713/19p13/GATAD2A), two breast and ovarian cancer risk loci (rs200182588/9q31/SMC2; rs8037137/15q26/RCCD1), and two breast and prostate cancer risk loci (rs5013329/1p34/NSUN4; rs9375701/6q23/L3MBTL3). Index variants in five additional regions previously associated with only one cancer also showed clear association with a second cancer type. Cell-type–specific expression quantitative trait locus and enhancer–gene interaction annotations suggested target genes with potential cross-cancer roles at the new loci. Pathway analysis revealed significant enrichment of death receptor signaling genes near loci with P < 10−5 in the three-cancer meta-analysis. Significance: We demonstrate that combining large-scale GWA meta-analysis findings across cancer types can identify completely new risk loci common to breast, ovarian, and prostate cancers. We show that the identification of such cross-cancer risk loci has the potential to shed new light on the shared biology underlying these hormone-related cancers. Cancer Discov; 6(9); 1052–67. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 932

Abstract Background: Epithelial Ovarian Cancer (EOC) is a lethal gynecologic malignancy. Mitochondria are cellular organelles that regulate energy metabolism and are the primary source of reactive oxygen species (ROS), which damage both the nuclear and mitochondria DNA (mtDNA), contributing to the pathogenesis of EOC. Previously, we identified statistically significant EOC risk associations with 54 single nucleotide polymorphisms (SNPs) from 11 mitochondria-related genes in a study of 1815 EOC cases and 1900 controls. In this replication study we expand our investigation of mitochondrial metabolism genetic variants and associations with EOC risk and overall survival (OS). Methods: In a discovery set of 3761 EOC cases and 2722 controls, we investigated risk associations in 4200 SNPs from 130 mitochondrial metabolism genes. We genotyped 431 of the most significant SNPs from 86 genes in 14,525 EOC cases and 23,447 controls (of European ancestry) from 43 studies in OCAC using an Illumina Infinium iSelect BeadChip. Risk analyses were conducted using unconditional logistic regression under a log-additive model and adjusted for study site and the first five principal components of population substructure. Both invasive cancers combined and the four main histological subtypes were analyzed. Survival analyses were conducted on the same SNPs using Cox hazards models to estimate hazard ratios (HRs) and 95% CIs considering overall survival in 10,084 cases from 28 OCAC studies. We adjusted for multiple tests using False Discovery Rate q<20%. Results: The strongest evidence of an association for risk of invasive cancers combined and the serous subtype was SNP rs3096 in the pyridoxamine 5'-phosphate oxidase (PNPO) gene (OR=1.08, p=2.8x10-6). In clear cell carcinoma, the strongest association was a missense coding SNP rs28379632 (Ile421Met) in the MT-CYB gene (OR=4.0, p=0.003). For endometrioid cancer rs9330440 in the UNQ6494 gene was most significant (OR=0.89, p=0.001) while rs10267797 in the dihydrolipoamide dehydrogenase (DLD) gene was most significant in the mucinous subtype (OR=1.15, p=0.004). The strongest association with overall survival was mitochondrial transcription termination factor 1 (MTERF) rs2540571 (HR=0.92, p= 0.00011). Conclusion: These results suggest that mitochondrial metabolism gene variants appear to be associated with EOC risk and survival. Citation Format: Ganna Chornokur, Hui-Yi Lin, Alvaro Monteiro, Joellen Schildkraut, Ellen L. Goode, Paul DP Pharoah, Simon A. Gayther, Susan Ramus, Thomas A. Sellers, Catherine M. Phelan. Mitochondrial metabolism gene variants are associated with risk and survival in epithelial ovarian cancer (EOC). [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A50.

Abstract Background: Epithelial Ovarian Cancer (EOC) is a lethal gynecologic malignancy and the fifth cause of cancer mortality in women in the US. Normal ovarian physiology is intricately connected to tightly regulated small GTP binding proteins of the Ras superfamily (Ras, Rac, Rho, Rab, Arf, and Ran) which regulate key cellular processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. These proteins function in a highly coordinated manner through signaling cascades and feedback loops within and among the small GTPase subfamilies. We hypothesized that single nucleotide polymorphisms (SNPs) in small GTPase genes are associated with epithelial ovarian cancer (EOC) risk and aberrant expression of these genes correlates with tumor characteristics including overall survival (OS). Methods: In a discovery set of 7931 EOC cases and 9206 controls we investigated 9,356 SNPs from 112 genes, 657 of which showed associations up to the significance level of p<0.05. We genotyped 407 of the most significant SNPs from 112 genes in a combined dataset (discovery and replication) which consisted of 18,736 EOC cases and 23,448 controls (of European ancestry) from 43 studies in the Ovarian Cancer Association Consortium (OCAC), using an Illumina Infinium iSelect BeadChip as part of the Collaborative Oncological Gene-environment Study (COGS). Odds ratios and 95% confidence intervals were calculated using unconditional logistic regression under log-additive models. A False Discovery Rate (FDR) q<0.2 was applied. Pearson's correlation tests were performed on the expression of genes with tumor characteristics and OS in 561 ovarian tumors: serous (n=464); endometrioid (n=52); clear cell carcinoma (n=28) and mucinous (n=17) subtypes. Results: The most significantly associated SNP associations with EOC were: AKAP6 rs1955513, (OR=0.9, p=3.3x10-4) in all invasive; ARGHEF10L rs10788679, (OR=1.05, p=2.6x10-4) in serous; RAB31 rs1166373, (OR=1.25, p=4.0x10-3) in endometrioid; KRAS rs4963872, (OR=1.35, P=4.5x10-4) in mucinous EOC and TNIK rs6780532, (OR=0.88, p=7.7x10-3) in clear cell carcinoma. Increased expression of AKAP6 and TNIK was marginally correlated with advanced EOC stage (p<0.05) while high KRAS expression was correlated with OS (HR=1.18, p=0.038). Conclusions: Genetic variation in the small GTPase genes appears to be associated with ovarian cancer risk and aberrant expression of these genes correlates with tumor characteristics and OS. Citation Format: Hui-Yi Lin, Yin Xiong, Jonathan Tyrer, Douglas C. Marchion, Alvaro NA Monteiro, Andrew Berchuck, Joellen M. Schildkraut, Ellen L. Goode, Susan J. Ramus, Simon A. Gayther, Paul DP Pharoah, Steven A. Narod, Thomas A. Sellers, Catherine M. Phelan. Investigation of small GTPase genes in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B27.