Shen, Howard C.

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 An ovarian cancer Genome Wide Association Study (GWAS) identified 9p22.2 as a novel susceptibility locus with the most statistically significant single nucleotide polymorphisms (SNPs) located in an intergenic region near the Basonuclin 2 (BNC2) gene, which codes for a putative transcription regulator containing three pairs of zinc finger (ZF) domains. The minor alleles are protective in terms of ovarian cancer susceptibility. However, the molecular mechanisms by which these SNPs modify susceptibility remain largely unknown. The significant SNPs in the 9p22.2 locus lie in non-coding regions and therefore are hypothesized to affect the activity of regulatory elements and modify the expression of a target gene(s). In order to test this hypothesis we conducted fine mapping for the locus which delimited a ∼74kb region containing SNPs with a p-value less than 10-8. FAIRE-Seq and ChIP-Seq experiments for histone markers conducted in immortalized ovarian surface epithelial cells (IOSE) and fallopian tube epithelial cells (IFTE) were used to prioritize functional SNP candidates which overlap with regulatory elements. Luciferase assays tested the ability of these regulatory elements to activate transcription. Chromosome conformation capture (3C) experiments demonstrate a physical interaction between the BNC2 promoter and candidate regulatory elements containing risk-associated SNPs. Methylation Quantification at Trait Loci (mQTL) revealed an association between decreased methylation at the BNC2 promoter and the protective minor alleles. Expression analysis shows decreased expression of BNC2 in cancer versus normal tissue implicating tumor suppressor function of BNC2. Therefore the protective minor allele likely increases expression of BNC2 which in turn contains tumor suppressing properties that decrease ovarian cancer risk. BNC2 protein is in complex with transcriptional regulatory proteins, in particular the NURD complex components. Moreover, BNC2 acts as a transcriptional repressor in in vitro transfection assays indicating that it functions in transcriptional repression. We then used protein binding microarrays and CHIP-Seq experiments to identify its putative DNA binding motifs and its downstream target genes. Analysis of this data suggests that BNC2 functions in a regulatory transcription network that impacts on genes implicated in ovarian cancer with enrichment for genes in the TGF-beta response pathway. Citation Format: Melissa A. Buckley, Howard C. Shen, Gustavo A. Mendoza-Fandino, Nicholas T. Woods, Anxhela Gjyshi, Juliet French, Kate Lawrenson, Honglin Song, Jonathan Tyrer, Renato S. Carvalho, Alexandra Valle, Ann Chen, Sean Yoder, Gregory Bloom, Ya-Yu Tsai, Ally Yang, Timothy R. Hughes, Xiaotao Qu, Mine Cicek, Melissa Larson, Ellen Goode, Brooke Fridley, Susan Ramus, Georgia Chenevix-Trench, Paul Pharoah, Thomas A. Sellers, Simon Gayther, Alvaro N.A. Monteiro, Ovarian Cancer Association Consortium. Functional analysis of the 9p22 locus implicates the transcriptional regulation of BNC2 as a mechanism in ovarian cancer predisposition. [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 3285. doi:10.1158/1538-7445.AM2014-3285

Abstract The presence of regulatory T cells (Treg) in solid tumors is known to play a role in patient survival in ovarian cancer and other malignancies. We assessed inherited genetic variations via 749 tag single-nucleotide polymorphisms (SNP) in 25 Treg-associated genes (CD28, CTLA4, FOXP3, IDO1, IL10, IL10RA, IL15, 1L17RA, IL23A, IL23R, IL2RA, IL6, IL6R, IL8, LGALS1, LGALS9, MAP3K8, STAT5A, STAT5B, TGFB1, TGFB2, TGFB3, TGFBR1, TGRBR2, and TGFBR3) in relation to ovarian cancer survival. We analyzed genotype and overall survival in 10,084 women with invasive epithelial ovarian cancer, including 5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous carcinoma cases of European descent across 28 studies from the Ovarian Cancer Association Consortium (OCAC). The strongest associations were found for endometrioid carcinoma and IL2RA SNPs rs11256497 [HR, 1.42; 95% confidence interval (CI), 1.22–1.64; P = 5.7 × 10−6], rs791587 (HR, 1.36; 95% CI, 1.17–1.57; P = 6.2 × 10−5), rs2476491 (HR, = 1.40; 95% CI, 1.19–1.64; P = 5.6 × 10−5), and rs10795763 (HR, 1.35; 95% CI, 1.17–1.57; P = 7.9 × 10−5), and for clear cell carcinoma and CTLA4 SNP rs231775 (HR, 0.67; 95% CI, 0.54–0.82; P = 9.3 × 10−5) after adjustment for age, study site, population stratification, stage, grade, and oral contraceptive use. The rs231775 allele associated with improved survival in our study also results in an amino acid change in CTLA4 and previously has been reported to be associated with autoimmune conditions. Thus, we found evidence that SNPs in genes related to Tregs seem to play a role in ovarian cancer survival, particularly in patients with clear cell and endometrioid epithelial ovarian cancer. Cancer Immunol Res; 2(4); 332–40. ©2014 AACR.