Epigenetic analysis leads to identification of HNF1B as a subtype-specific susceptibility gene for ovarian cancer

Abstract

HNF1B is overexpressed in clear cell epithelial ovarian cancer, and we observed epigenetic silencing in serous epithelial ovarian cancer, leading us to hypothesize that variation in this gene differentially associates with epithelial ovarian cancer risk according to histological subtype. Here we comprehensively map variation in HNF1B with respect to epithelial ovarian cancer risk and analyse DNA methylation and expression profiles across histological subtypes. Different single-nucleotide polymorphisms associate with invasive serous (rs7405776 odds ratio (OR)=1.13, P=3.1 × 10(-10)) and clear cell (rs11651755 OR=0.77, P=1.6 × 10(-8)) epithelial ovarian cancer. Risk alleles for the serous subtype associate with higher HNF1B-promoter methylation in these tumours. Unmethylated, expressed HNF1B, primarily present in clear cell tumours, coincides with a CpG island methylator phenotype affecting numerous other promoters throughout the genome. Different variants in HNF1B associate with risk of serous and clear cell epithelial ovarian cancer; DNA methylation and expression patterns are also notably distinct between these subtypes. These findings underscore distinct mechanisms driving different epithelial ovarian cancer histological subtypes.

Figure 1. Identification of HNF1B as a subtype-specific candidate gene for ovarian cancer and its establishment as a susceptibility gene.

(a) The scatterplot compares the mRNA expression (y axis) versus DNA methylation (x axis) in serous ovarian tumours from TCGA (see Methods). Each blue dot is a serous tumour sample, whereas each pink dot is one of the ten normal fallopian tube samples. The HNF1B promoter is silenced in the majority of these tumours, either by an epigenetic (bottom right, high DNA methylation and low mRNA expression) or an unknown alternative mechanism. The mRNA expression data were integrated from three platforms (online Methods) and interpreted as log ratios, and we observe the same pattern with each individual expression platform (Supplementary Fig. S1). (b) HNF1B-promoter DNA methylation differs by histological subtype. Although unmethylated in the normal fallopian tissue, this locus is hypermethylated (beta value >0.2) in approximately 50% of the TCGA (n=576; see Methods) serous cases as well as another independent set of 32 serous tumour samples (OCRF panel; see Methods), but remains unmethylated in clear cell tumours (OCRF panel; see Methods) (n=4). These data are consistent with reported HNF1B expression in the clear cell tumours. (c) Genetic variants in the HNF1B locus are associated with risk of ovarian cancer histological subtypes. Plotted in each panel is the −log10 (P-value) from the SNP association with risk for each subtype (Manhattan plots) located in the 150-kb region described in the text. Imputed SNPs are indicated with a relatively lighter colour, whereas the genotyped SNPs are indicated with a darker colour. Dashed lines indicate the genome-wide significance threshold (5 × 10⁻⁸). The linkage disequilibrium plot on the bottom shows the r² between the SNPs. Genomic coordinates are based on hg19 (Build37).

Figure 2. HNF1B-promoter DNA methylation, protein expression and global DNA-methylation pattern by subtype.

Each row is a tissue sample collected at the Mayo Clinic that belongs to one of the three categories: normal ovarian tissue (n=7), clear cell ovarian tumours (n=17) or serous ovarian tumours (n=196). Endometrioid (n=49) and mucinous (n=7) tumours are not included in this figure. Each column represents a CpG locus, either from the region flanking the HNF1B transcription start site (panel A, ordered by genomic locations with an arrow indicating the transcription start site) or from a global panel of 1,003 CpG loci mapped to autosomal CpG island regions that distinguish clear cell and serous subtypes (panel B, ordered by average DNA methylation across the samples). For each horizontal panel group, the samples (rows) are ordered by HNF1B IHC status. The heatmap shows the DNA-methylation beta value, with blue indicating low DNA methylation and red indicating high methylation. Clear cell tumours showed less DNA methylation at the HNF1B-promoter region and correspondingly higher HNF1B protein expression. The clear cell tumours generally show a CIMP where there is extensive gain of aberrant promoter methylation in a correlated manner. CIMP status (left side bar, defined as methylated at >80% of the 1,003 loci) is highly correlated HNF1B expression. Also noteworthy is that the HNF1B-promoter DNA methylation (panel a) is the opposite from the global pattern (panel b, Supplementary Fig. S8). This suggests HNF1B DNA methylation is not a passenger event of global DNA-methylation changes.

Figure 3. Correlation of serous risk-associated SNPs with HNF1B-promoter DNA-methylation level.

Plotted is the linkage disequilibrium region defined as r²>0.2 with the top serous SNP rs7405776. (a) Annotation of the region in terms of (from top to bottom:) UCSC genes, FANTOM mark, PRC marks (PRC2 and PRC1), the chromatin status determined in stem cells, the conservation score across this region and the CpG island information, on top of the location of the HM450 probe used in b boxplots of promoter DNA-methylation level of HNF1B (cg14487292) by SNP genotype with position indicated in c. This DNA-methylation probe was selected based on inverse association with mRNA expression for HNF1B, and does not contain any SNP with MAF >1% in its probe sequence. Each boxplot shows the distribution of DNA-methylation level by genotype (homozygous major—white; heterozygous—grey; and homozygous minor—black, where the minor alleles are the risk alleles). Two-sided P-values testing for trend are presented, and are computed for 231 Mayo Clinic high-grade, high-stage serous tumours to avoid confounding by histological subtypes, and also to be consistent with the TCGA data (primarily high-grade, high-stage serous). Results were similar with all subtypes combined. The risk alleles are associated with significantly increased DNA methylation. The association of rs11658063 genotype with promoter methylation is consistent across the entire region flanking HNF1B transcription start site, and stronger for the upstream promoter region (Supplementary Fig. S8).

Figure 4. Phenotypic effects and downstream targets of HNF1B overexpression in immortalized EECs.

(a) Morphological changes in EECs expressing a HNF1B GFP fusion protein (EEC^GFP.HNF1B). GFP-positive cells were sorted using flow cytometry. The arrows indicate five nuclei contained within a single EEC^GFP.HNF1B cell, showing the aberrant polynucleation that we observed in these cells. Using flow cytometry, we quantified the increase in polynucleation in EEC^GFP.HNF1B to be around eightfold compared with controls (data not shown). (b) Gene-expression analysis of HNF1B-target genes and clear cell ovarian cancer associated genes. *P>0.01.