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Association between CCNE1 variants and bladder cancer

Supplementary Figure 1: Electrophoretic mobility shift assays (EMSA) for CCNE1 SNPs rs8102137 and rs7257330. Supplementary Figure 2: Alignment of cyclin E protein isoforms - WT1, WT2 and ES and ET. Supplementary Figure 3: Functional analysis of cyclin E isoforms.Supplementary Table 1: Description of sub-studies included in NCI-GWAS1 and GWAS2 of bladder cancer. Supplementary Table 2: Characteristics of bladder tissue samples used for mRNA expression analysis. Supplementary Table 3: PCR primers, genotyping and gene expression assays, EMSA probes and antibodies. Supplementary Table 4: Bladder cancer stage and grade information for patients in the combined GWAS1+2 set. Supplementary Table 6: Association with bladder cancer risk with mutual adjustment for CCNE1 variants. Supplementary Table 7: Association with bladder cancer risk for CCNE1 variants previously associated with other cancers and for two non-synonymous coding variants. Supplementary Table 8: Association between cyclin E protein expression (IHC scores), bladder cancer patient characteristics and CCNE1 variants.

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 r2 ≥ 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 (Ptrend = 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. Cancer Res; 74(20); 5808–18. ©2014 AACR.

Background: 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. Methods: Genetic fine mapping analysis of the CCNE1 region was performed using data from two bladder cancer GWAS, which included a total of 5,942 cases and 10,857 controls. Cyclin E protein expression was evaluated by immunohistochemistry analysis in 265 bladder tumors in relation to CCNE1 genetic variants and tumor characteristics. Functional effects of cyclin E over-expression were evaluated with cell cycle assays. Findings: The original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r2≥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 GWAS, rs7257330 was associated only with aggressive bladder cancer (muscle-invasive or high-grade non-muscle-invasive cancer), with a per-allele odds ratio (OR) =1.18 (95%CI=1.09-1.27, p=4.67×10-5). Cyclin E protein expression was increased in aggressive bladder tumors (p=0.013) and, independently, with each rs7257330-A risk allele (ptrend=0.024). Over-expression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. Conclusions: Molecular mechanisms linking the CCNE1 GWAS signal and aggressive bladder cancer risk are related to cyclin E over-expression and cell cycle regulation. In combination with other genetic and clinical markers, CCNE1 genetic variants may be translationally useful for early prediction of aggressive bladder cancer. Citation Format: Yi-Ping Fu, Indu Kohaar, Lee Moore, Petra Lenz, Jonine D. Figueroa, Wei Tang, Patricia Porter-Gill, Stephen Chanock, Stephen M. Hewitt, Debra T. Silverman, Nathaniel Rothman, NCI-GWAS Bladder Cancer Consortium, Ludmila Prokunina-Olsson. Translational implications of the 19q12 bladder cancer GWAS signal for aggressive bladder cancer. [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 944. doi:10.1158/1538-7445.AM2014-944

Background: A recent GWAS identified a SNP rs2294008 within the prostate stem cell antigen (PSCA) at 8q24.3 as a risk factor for bladder cancer, [OR=1.13 (1.09-1.17), p=4E-11, Rothman, 2010]. PSCA encodes a GPI-anchored cell membrane glycoprotein expressed in the prostate, bladder, stomach and pancreas. A transcript with the non-risk C allele of rs2294008 generates a protein of 114 amino acids, while the risk T allele creates a novel upstream translation start site (acg->aTg) that extends the N-terminal leader peptide by 9 amino acids, to generate a protein of 123 amino acids. A humanized monoclonal anti-PSCA antibody is already used in clinical trials for prostate and pancreatic cancer, but the functional roles of PSCA and its genetic variants remain unknown. Methods: We performed RNA-sequencing in 3 matched normal-tumor bladder tissue samples and mRNA expression analysis in 42 tumor and 41 normal bladder tissue samples (24 matched normal-tumor pairs). Genotyping and AEI (Allelic Expression Imbalance) was performed using an allelic discrimination genotyping assay. PSCA isoforms with T and C alleles were cloned into a pFC14A CMV Flexi expression vector. Protein PSCA expression in normal/tumor bladder tissues and transfected cells was analyzed with Western blot and immunohistochemistry (IHC). Results and Conclusion: RNA-sequencing detected PSCA expression in all bladder tissue samples. Analysis of three samples heterozygous for rs2294008 showed evidence of strong AEI - 90% of all PSCA transcripts carried the risk T allele, while only 10% showed the non-risk C allele. Similar pattern of expression was observed for 11 other transcribed SNPs within the PSCA, but not for SNPs located in neighboring genes, Ly6K and JRK, implying that the AEI is specific for PSCA. By using an allele-specific expression assay that measured allelic ratio of rs2294008 in PSCA transcripts, we also confirmed AEI detected by RNA-seq. PSCA mRNA expression was strongly increased in individuals with risk T allele in both normal (ptrend =0.0155 for rs2294008) and tumor samples rs2294008 (ptrend=0.0054), which is further indicative of effect of AEI. IHC on a panel of paired normal-tumor bladder tissue microarrays (TMA) confirmed mRNA expression results and showed PSCA protein expression only in carriers of risk genotype (TT) but not in non-risk genotype (CC), both in tumor and normal bladder tissue sections. Analysis of recombinant PSCA expression in transfected cells showed stronger surface expression of the risk T allele, compared to C allele. In conclusion, we show preferential mRNA and protein expression of PSCA in individuals with risk allele T of rs2294008. Thus, genotyping information for PSCA rs2294008 may emerge as an important decisive factor for response to PSCA antibody mediated treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5126. doi:1538-7445.AM2012-5126

A single nucleotide polymorphism (SNP), rs8102137, located 6 Kb upstream from the cyclin E1 gene (CCNE1) on chromosome 19q12, has recently been identified as a risk factor for bladder cancer (OR, p=1.7×10-11, Rothman et al, Nat Gen, 2010). We found a higher expression of CCNE1 mRNA in cDNA samples from bladder tumors (n=42) compared to adjacent normal bladder tissue (n=41, 3.7 fold, p=2.7×10-12). However, the expression did not correlate with genotypes of rs8102137. We also performed allelic expression imbalance (AEI) using a coding synonymous variation in the last exon (rs7257694, Ser390Ser) that is in high LD with the GWAS rs8102137 (normal bladder tissue samples, n = 41, D’=1.0, r2=0.815 and in HapMap CEU samples, n=60, D’=0.95, r2=0.68). In normal and tumor tissue samples heterozygous for both SNPs, the risk variant of rs8102137 was associated with lower expression of allele T of rs7257694 (p=2.2×10-4 in normal samples and 1.11×10-10 in tumor samples). Western blot protein analysis with bladder tissue and prostate cell lines lysates showed that this allele-specific difference in mRNA expression is likely to be related to two protein isoforms that showed a differential pattern of expression dependent on rs8102137 and rs7257694 genotypes. CCNE1 regulates cell cycle G1/S transition, and alternative protein forms may have differential functions in cell cycle regulation, thereby affecting the risk of cancer. Alternative splicing forms of CCNE1 have been cloned and their functional analysis is ongoing. In conclusion, our results suggest that bladder cancer associated genetic variants within the CCNE1 gene may affect mRNA splicing and protein structure of the gene, contributing to altered regulation of cell cycle and risk of bladder and other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4974. doi:10.1158/1538-7445.AM2011-4974

Genome-wide association studies (GWAS) identified allele T of SNP rs2294008 within the prostate stem cell antigen (PSCA) gene as a risk factor for diffuse gastric cancer (OR=1.67, p=2.2×10-15) (The Study Group of Millennium Genome Project for Cancer, Nat Genet, 2008) and bladder cancer (OR=1.13, p=4.4×10-11) (Rothman et al, Nat Genet, 2010). In the present study we used data from the Stage 1 bladder cancer GWAS, which was performed on 8,801 individuals of European ancestry (3,529 cases and 5,115 controls), and information from the 1000 Genomes and HapMap 3 projects to impute all other SNPs within +/- 50kb region surrounding PSCA. Association analysis on the combined set of all genotyped and imputed SNPs (n=375) revealed a novel bladder cancer association signal for a variant genotyped in all samples. The association was found only in males (n=7,359, crude OR =1.14, p=1.15×10-4; adjusted for all covariates and rs2294008, OR=1.13, p=1.32×10-3), while no association was observed in females (n=1,442, crude OR=0.98, p=0.81; adjusted for all covariates and rs2294008, OR=0.98, p=0.82). The low linkage disequilibrium between these two SNPs (D’=0.202 and r2=0.019 in 8801 GWAS samples) suggests these variants are independent. A joint analysis showed that these two SNPs have a compound association with bladder cancer dependent on the number of risk genotypes (0, 1 or 2) in males (adjOR=1.18, p=3.93×10-6) but not in females (adjOR=1.01, p=0.95). The risk allele T of rs2294008 is functional as it introduces a novel translation start site that creates a PSCA protein with a leader peptide extended by 11 amino acids. The novel SNP that was found 10 Kb upstream of rs2294008, lies within an alternative untranslated first exon of PSCA that is marked by a H3K4me3 signal and is in a vicinity of an androgen receptor binding site, suggesting a possible role for this variant in regulation of PSCA promoter activity. In conclusion, a novel independent functional variant associated with bladder cancer risk was found within the PSCA gene, which might be important for male-specific regulation of PSCA expression and carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2783. doi:10.1158/1538-7445.AM2011-2783

A recent genome-wide association study (GWAS) for urinary bladder cancer (UBC) has identified multiple novel genetic risk factors (Rothman et. al, Nat Gen, 2010). One of these factors was an intronic SNP rs11892031 (p=1.0×10-7) located within the UGT1A locus on chromosome 2q37. The human cellular detoxification uridine 5’ diphosphate (UDP)-glucuronosyltransferases (UGTs) belong to a superfamily of proteins that conjugate diverse endo and exotoxins, increase their solubility and facilitate their removal via bile and urine. The UGT1A locus includes nine protein-coding genes and four pseudogenes. Each first exon of these genes is regulated by its own promoter and is spliced to four constant exons producing nine UGT1A proteins. Due to the complexity of the region and ∼90-95% similarity between the substrate-binding exon 1 sequences of UGT1A genes, this region is poorly represented in public databases (HapMap, 1000 Genomes, dbSNP). To ensure specificity of detection, we generated long-range amplicons and sequenced all UGT1A exons in HapMap individuals (CEU) and in 44 bladder cancer patients and identified 25 non-synonymous coding variations. All these variations have been genotyped in 1000 bladder cancer cases and 1000 controls from the Spanish Bladder Cancer Study (SBCS). We found one exonic SNP that showed association stronger than the original GWAS variant, and multiple variants that showed some evidence of gene and cigarette smoking interactions. Having ∼20 coding and potentially functional variants within the UGT1A region, we are exploring methods of simultaneous analysis of individual variants and their haplotypes. In conclusion, association of genetic variants within the UGT1A region confirms that altered cellular detoxification of environmental substrates is an important factor for development of bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4680. doi:10.1158/1538-7445.AM2011-4680

Biomarkers that can differentiate between normal and tumor state, and can be measured in easily accessible body fluids such as blood and urine, can be very important for cancer diagnostics and disease monitoring. MicroRNAs (miRNAs), short non-coding regulatory RNA molecules, are attractive as biomarkers because they are stable in different conditions and are easy to measure with quantitative PCR (qPCR) methods. In this study we aimed to identify a universal panel of miRNAs for cancer screening that can be easily tested in the serum of healthy individuals and patients with different types of cancer. First, we measured expression of ∼800 miRNAs in 40 controls and 60 patients with bladder, breast or prostate cancer using the low density TaqMan expression arrays (Applied Biosystems) and starting from 250 ul of serum. Based on these results we selected a panel of 24 miRNAs that showed best discrimination between normal and cancer samples. These miRNAs were then re-tested as a custom-designed mini-panel in serum samples of 44 healthy controls and cancer patients (31 bladder, 25 breast and 28 prostate) and in relevant normal and tumor tissue samples (42 normal bladder and 43 bladder tumors, 44 normal breast and 42 breast tumors and in 50 normal prostate and 20 prostate tumors). Only miRNAs that expressed in the same direction in serum and tissue samples and showed significant association with cancer in both sample types were used for further analysis. The current panel consists of 16 miRNAs – 14 targets, one positive control and one negative control. Using this panel on serum samples from 44 controls, 31 bladder cancer patients, 25 breast cancer patients and 28 prostate cancer patients we performed ROC analysis and achieved complete discrimination (AUC ∼1.0) between all types of cancers and controls and good discrimination between different types of cancers (minimal AUC 0.89 for breast and bladder samples). Our results prove that miRNA detection from serum might be a promising method of cancer detection. Currently, we are performing validation studies in independent sets of samples. A combination of information on genetic susceptibility factors identified by genome-wide association studies (GWAS), other cancer-specific factors such as PSA for prostate cancer and miRNA expression profiling, might provide additional tools for early disease diagnostics and help to guide treatment options. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-350. doi:10.1158/1538-7445.AM2011-LB-350

Genome-wide association studies (GWAS) identified single nucleotide polymorphism (SNP) rs10486567 within the JAZF1 gene on chromosome 7p15 as being associated with increased risk of prostate cancer in individuals of European ancestry (p=2.14×10-6, Thomas, 2008; p=7.79×10-11, Prokunina-Olsson, 2010). We now examined two additional SNPs, rs7808935 and rs67152137 that are in strong linkage disequilibrium with rs10486567 (r2∼1.0) and located in a 2 Kb non-coding genomic region of intron 2 of JAZF1. By chromatin immunoprecipitation (ChIP) assays in LNCaP and DU-145 prostate cancer cell lines, we established that all three SNPs regions bind androgen receptor, but only rs7808935 increased allele-specific binding, with the risk T allele enhancing binding (1.4-fold, p=0.021). We cloned the 2Kb genomic region encoding various haplotypes of the three SNPs into a luceferase reporter assay, and discovered that the risk haplotype increased the reporter activity by 2-fold (p=6.98×10-5). mRNA expression of JAZF1 in normal prostate tissue samples was increased in carriers of the risk haplotype (1.51-fold, p=0.015, n=59). The three associated SNPs are located in large intron 2 and about 250 Kb away from JAZF1 promoter region. To further explore the molecular relationships between the three prostate-cancer associated SNPs, mRNA expression of JAZF1 and risk of prostate cancer, we are currently performing analysis of long-distance chromatin interactions that involves the associated JAZF1genomic region. In conclusion, we hypothesize that the prostate-cancer associated variants of JAZF1 influence androgen receptor transcriptional activity and its effect on JAZF1. Our current research should identify mechanisms of how this affects JAZF1 mRNA expression and carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3816. doi:10.1158/1538-7445.AM2011-3816

Background: A recent genome-wide association study (GWAS) has identified a single nucleotide polymorphism (SNP) rs11249433 in the 1p11.2 region as a novel genetic risk factor for breast cancer, and this association was stronger with estrogen receptor (ER)-positive than with ER-negative cancer. We aimed to identify a biologically relevant molecular phenotype of this association. Methods: NOTCH2 expression was measured with custom-designed TaqMan assays on 226 breast tumor samples and 319 blood samples of breast cancer patients. PANTHER analysis based on expression in NCI-60 cell lines (Affymetrix HG-U133 arrays) was used to examine enrichment of pathways, biological processes, or molecular functions for transcripts significantly correlated with NOTCH2 expression. Results: We found evidence of a functional relationship between SNP rs11249433 and expression of the NOTCH2 gene located in the 1p11.2 region. NOTCH2 expression differed in subgroups of 226 breast tumor samples, being lowest in tumors with mutations in the p53 gene and highest in p53 wild-type/ER-positive tumors (p=0.0059). In the latter group, the NOTCH2 expression was particularly increased in carriers of risk genotypes (AG/GG) of rs11249433 when compared to the non-risk AA genotype (p=0.0062). This effect is tissue-specific since rs11249433 was not associated with NOTCH2 expression in blood samples of 319 breast cancer patients. Pathway enrichment analysis of NOTCH2 in the NCI-60 cell lines indicated role of NOTCH2 in integrin signaling, cell-cell interactions, and cell structure and motility. Conclusion: This is the first study to explain genetic association between SNP rs11249433 and breast cancer risk by showing that NOTCH2 expression in breast tumors differs in subgroups of patients and between patients with the risk rs11249433 genotypes when compared to the non-risk genotype. The NOTCH pathway has key functions in stem cell differentiation of ER-positive luminal cells in the breast. Therefore, increased expression of NOTCH2 in carriers of rs11249433 may promote development of ER-positive luminal ductal tumors. The exact mechanisms of regulation of NOTCH2 expression by rs11249433 as well as the role of NOTCH2 in breast cancer development remain to be determined. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4742.