The 19q12 bladder cancer GWAS signal: association with cyclin E function and aggressive disease

Abstract

A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell-cycle protein. We performed genetic fine-mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r(2) ≥ 0.7) associated with increased bladder cancer risk. From this group, we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWASs, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele OR = 1.18 [95% confidence interval (CI), 1.09-1.27, P = 4.67 × 10(-5)] versus OR = 1.01 (95% CI, 0.93-1.10, P = 0.79) for nonaggressive disease, with P = 0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (P = 0.013) and, independently, with each rs7257330-A risk allele (P(trend) = 0.024). Overexpression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E overexpression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models.

Figure 1. Genetic and genomic landscape of the CCNE1 region

Genomic plot of the CCNE1 region is based on information from the UCSC browser (www.genome.ucsc.edu) and RNA-sequencing of 7 tumors and 5 adjacent normal bladder tissue samples. The plot shows the main CCNE1 transcripts detected by our RNA-sequencing – WT1, WT2, cyclin E_S and cyclin E_T. Alternative exons missing in forms cyclin E_S and cyclin E_T are marked by red boxes. Arrows indicate translation start sites and direction of protein synthesis. Genetic variants on the plot are coding non-synonymous CCNE1 variants (marked in red), GWAS marker rs8102137, promoter variant rs7257330, an ovarian cancer candidate variant rs3218036, and several variants previously associated with various cancers (Supplementary Table 7). Histone H3 Lysine 27 acetylation marks (H3K27Ac) in 7 cell lines are a part of ENCODE track presented by the UCSC browser.

Figure 2. Forest plot for association of rs8102137 and rs7257330 in bladder cancer subgroups in the combined set of NCI-GWAS1 and NCI-GWAS2 samples

The plot shows allelic odds ratios and 95% confidence intervals for SNPs. Analysis was performed only on subjects with genotype information available for both SNPs, where rs8102137 was from actual genotyping in NCI-GWAS1 and NCI-GWAS2, and rs7257330 was converted from imputed allelic dosage with ≥ 90% probability and validated by TaqMan genotyping in a subset of samples. Bladder cancer sub-groups were defined in three categories (Supplementary Table 4): low-grade NMIBC (Ta with G1/G2), high-grade NMIBC (Ta with G3/G4 or all T1) and MIBC (all T2-T4)(3). Incomplete clinical information category includes cases with missing or incomplete tumor stage or grade data.

Figure 3. Association results, linkage disequilibrium (r²) and recombination plots of the CCNE1 region in combined NCI-GWAS1 and NCI-GWAS2 samples

Left y-axis (–log₁₀ scale) presents p-values for GWAS (filled circles) and well-imputed markers (IMPUTE2-score ≥ 0.9, open circles), adjusted for age, gender, study sites, smoking status (ever/never) and top eigenvectors (EV1, EV5, EV6) from principal component analysis. The color scheme is based on r² values between rs8102137 and corresponding markers. Right y-axis presents likelihood ratio of putative recombination hotspots based on five sets of 100 randomly selected controls from NCI-GWAS1 and shown as connected gray lines. The GWAS marker rs8102137 and the promoter variant rs7257330 are shown as diamonds. Pair-wise r² values based on all control samples are displayed at the bottom of the plot for all 165 SNPs included in our analyses. Genomic coordinates are based on the NCBI Human Genome Build 37.1/UCSC hg19 assembly. (A) Association analysis in a subset of patients with non-aggressive bladder cancer (stage Ta with grade G1 or G2, n = 1,870) vs. all controls (n = 10,857). (B) Association analysis in a subset of patients with aggressive bladder cancer (stage Ta with grade G3+ or stages T1-T4 with any grade, n = 1,930) vs. all controls (n = 10,857).

Figure 4. Analysis of CCNE1 mRNA expression in bladder tumors and adjacent normal tissue and in relation to rs8102137 and rs7257330 genotypes

Expression of total CCNE1 (all transcripts), cyclin E_S and cyclin E_T transcripts was measured with TaqMan assays. CCNE1 expression was normalized to a geometric mean of endogenous controls (B2M, GAPDH and PPIA) and presented on the log 2 scale. Less negative values correspond to higher relative expression of CCNE1 transcripts. The association between normalized CCNE1 mRNA expression and risk allele counts (0, 1 and 2) of both SNPs was evaluated with multivariable linear regression models adjusting for age and gender. Most of bladder tumors used for expression analysis (30 of 41 tumors) are of aggressive type (Supplementary Table 2).

Figure 5. Immunohistochemistry (IHC) analysis of cyclin E expression in bladder tissues

(A) Images of cyclin E expression in one pair of tumor-adjacent normal bladder tissue samples, representative of 8 pairs tested. Immunostaining was done with an anti-CCNE1 rabbit polyclonal antibody (HPA018169, Sigma, dilution 1:85). Images are presented at 10× magnification with 0.1 mm scale bars. (B) Representative images of bladder tumors with nuclear cyclin E expression scores of 0, 2, 4 and 6, which is a sum of nuclear positivity (scored 0-3) and intensity (scored 0-3). The samples are a part of a TMA that includes 265 bladder tumors, immunostaining was done with an anti-CCNE1 rabbit monoclonal antibody (1655-1, Epitomics, dilution 1:250). (C) Association between IHC cyclin E nuclear scores in 265 bladder tumors, bladder cancer aggressiveness and genotypes of rs8102137 and rs7257330. The scores are shown as least squares mean values with 95% confidence intervals, based on multivariable linear regression models assuming additive effects of SNPs, with adjustment for age, gender, two study sub-sites, smoking, and categories of tumor aggressiveness, when appropriate: non-aggressive and aggressive.