Ashley Paquin

Association of germline variants in the APOBEC3 region with cancer risk and enrichment with APOBEC-signature mutations in tumors

High rates of APOBEC-signature mutations are found in many tumors, but factors affecting this mutation pattern are not well understood. Here we explored the contribution of two common germline variants in the APOBEC3 region. SNP rs1014971 was associated with bladder cancer risk, increased APOBEC3B expression, and enrichment with APOBEC-signature mutations in bladder tumors. In contrast, a 30-kb deletion that eliminates APOBEC3B and creates an APOBEC3A–APOBEC3B chimera was not important in bladder cancer, whereas it was associated with breast cancer risk and enrichment with APOBEC-signature mutations in breast tumors. In vitro, APOBEC3B expression was predominantly induced by treatment with a DNA-damaging drug in bladder cancer cell lines, and APOBEC3A expression was induced as part of the antiviral interferon-stimulated response in breast cancer cell lines. These findings suggest a tissue-specific role of environmental oncogenic triggers, particularly in individuals with germline APOBEC3 risk variants.

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

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.

Expression of Interferon Lambda 4 Is Associated with Reduced Proliferation and Increased Cell Death in Human Hepatic Cells.

Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be generated only in individuals carrying a ΔG frame-shift allele of an exonic genetic variant (rs368234815-ΔG/TT). The rs368234815-ΔG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-λ4 expressed in human hepatic cells—a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-λ4 but also detected secreted IFN-λ4 in the culture media of expressing cells. Secreted IFN-λ4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-λ4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-λ4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-λ4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-λ4-induced phenotypes—activation of ISGs, decreased proliferation, and increased cell death—could be inhibited by an anti-IFN-λ4-specific antibody. Our study offers new insights into biology of IFN-λ4 and its possible role in HCV clearance.

Modification of Occupational Exposures on Bladder Cancer Risk by Common Genetic Polymorphisms

Few studies have demonstrated gene/environment interactions in cancer research. Using data on high-risk occupations for 2258 case patients and 2410 control patients from two bladder cancer studies, we observed that three of 16 known or candidate bladder cancer susceptibility variants displayed statistically significant and consistent evidence of additive interactions; specifically, the GSTM1 deletion polymorphism (P interaction ≤ .001), rs11892031 (UGT1A, P interaction = .01), and rs798766 (TMEM129-TACC3-FGFR3, P interaction = .03). There was limited evidence for multiplicative interactions. When we examined detailed data on a prevalent occupational exposure associated with increased bladder cancer risk, straight metalworking fluids, we also observed statistically significant additive interaction for rs798766 (TMEM129-TACC3-FGFR3, P interaction = .02), with the interaction more apparent in patients with tumors positive for FGFR3 expression. All statistical tests were two-sided. The interaction we observed for rs798766 (TMEM129-TACC3-FGFR3) with specific exposure to straight metalworking fluids illustrates the value of integrating germline genetic variation, environmental exposures, and tumor marker data to provide insight into the mechanisms of bladder carcinogenesis.

Comparative Functional Analysis of 12 Mammalian IFN-λ4 Orthologs.

IFN-λ4 is a novel type-III interferon with strong clinical significance in humans. Only a subset of individuals—up to 10% of Asians, 50% of Europeans, and 90% of Africans—carry the ΔG allele of a genetic variant rs368234815-TT/ΔG and are genetically able to produce IFN-λ4 protein. Carriers of the ΔG allele have impaired ability to clear infection with hepatitis C virus (HCV). IFN-λ4 is also predicted to exist and be functionally important in several nonhuman mammals. In this study, we present the first comparative analysis of 12 mammalian IFN-λ4 orthologs in a human hepatic cell line, HepG2, which supports signaling of the human IFN-λ4. We show that despite differences in protein sequences, functional properties of the recombinant human and nonhuman IFN-λ4 proteins are comparable—they are all expressed as predominantly cytoplasmic proteins that are biologically active for induction of interferon signaling. We show that several IFN-λ4 orthologs can be detected by Western blotting, flow cytometry, and confocal imaging using a monoclonal antibody developed for the human IFN-λ4. Studies of IFN-λ4 in animals should help improve our understanding of the biology of this novel clinically important interferon in normal and disease conditions.

Abstract 4617: An alternatively spliced isoform of TMEM129 shows association with bladder cancer GWAS marker rs798766

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Genome-wide association studies (GWAS) have identified a number of single nucleotide polymorphisms (SNPs) associated with bladder cancer risk. Here, we explored the bladder cancer GWAS signal SNP rs798766 (C/T), which is located in the intron of TACC3 upstream of FGFR3 at 4p16.3 locus. This region represents a high linkage disequilibrium (LD) block and includes two more genes, TMEM129 and SLBP. Several SNPs in r2 = 1 with rs798766 are located within coding and non-coding regions of these genes; however, the coding SNPs are synonymous. Previously, genetic association of this GWAS marker has been shown by eQTL analysis with FGFR3 and TACC3 mRNA expression in adipose tissue. However, genetic association for all four genes and their several alternatively spliced isoforms remains largely unexplored in primary bladder normal and tumor tissue. We used genotype and RNA-seq data from The Cancer Genome Atlas (TCGA), bladder cancer data set (n = 242). Within the 1000 Kb region surrounding rs798766 we imputed genotypes of 13,529 SNPs in the TCGA data set based on the 1000 Genomes reference panel. Next, we performed eQTL analysis of FGFR3, TACC3, SLBP and TMEM129 both at gene level and at individual isoform level with genotypes of GWAS and several other markers. For validation, we used an independent panel of 42 bladder tumor and 42 adjacent normal tissue samples, using TaqMan expression assays. Expression was analyzed by general linear regression based on an additive genetic model, adjusting for age, sex and race. At the gene level in the TCGA data, we found significant association with TACC3 expression (p = 1.9 × 10−2unadj and p = 3.5 × 10−2 adj). The association was stronger for expression of an alternatively spliced isoform of TMEM129, designated TMEM129β, (p = 4.57×10−4unadj and p = 3.5 × 10−3adj). Computational annotation revealed that SNP rs2236786, r2 = 1 with GWAS marker, is located within a cassette exon that is an intron for isoform TMEM129β. Further analysis revealed that rs2236786 alleles affect a binding motif for alternative splicing factors SF2ASF, hnRNPF and CUG-BP. The functional exploration of this finding is underway. In conclusion, we suggest that the molecular phenotype of the 4p16.3 bladder cancer GWAS signal could be associated with differential expression of more than one gene (FGFR3, TACC3, SLBP and TMEM129) and their alternatively spliced isoforms. We present the first evidence for possible association of alternative splicing of TMEM129 and bladder cancer GWAS signal. Note: This abstract was not presented at the meeting. Citation Format: A. Rouf Banday, Ashley Paquin, Candace Middlebrooks, Eniko Kiss, Ludmila Prokunina-Olsson. An alternatively spliced isoform of TMEM129 shows association with bladder cancer GWAS marker rs798766. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4617. doi:10.1158/1538-7445.AM2015-4617

The 19q12 Bladder Cancer GWAS Signal: Association with Cyclin E Function and Aggressive Disease

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.

151: Interferon lambda 4 (IFN-λ4) induces a pro-inflammatory state in human hepatic cells which can be blocked by a monoclonal antibody

Interferon lambda 4 (IFN-λ4) is a novel human type-III interferon. An exonic genetic variant (rs368234815-ΔG), which creates the IFN-λ4 protein, is the strongest host factor predicting clearance of hepatitis C virus (HCV) infection. Individuals who carry the ΔG allele and thus can generate IFN-λ4, are less likely to clear HCV compared to individuals who do not carry this allele and are unable to generate IFN-λ4. We show that IFN-λ4 can be detected at a low level with a MesoScale ELISA assay in culture media of human hepatic cells (HepG2 and fresh primary human hepatocytes) transiently transfected with IFNL4-expressing construct. The secreted IFN-λ4 protein is able to induce strong expression of a panel of interferon stimulated genes (ISGs) in a transwell assay and this effect could be blocked by a monoclonal antibody against IFN-λ4 (α-IFN-λ4). Specifically, in primary hepatocytes secreted IFN-λ4 induced strong activation of the pro-inflammatory chemokine IP-10 (encoded by CXCL10) both on mRNA and protein level and this induction was blocked by the α-IFN-λ4 antibody. Increased expression of IP-10 is associated with hepatoinflammation and reduced response to HCV treatment. The α-IFN-λ4 antibody did not block the signalling of other interferons (IFN-α, IFN-β, IFN-γ, and IFN-λ3), suggesting a possible use of this antibody for targeted blocking of IFN-λ4 signalling. In conclusion, we propose a pathogenic mechanism of IFN-λ4 as a moderately secreted interferon stimulating ISGs and inducing a pro-inflammatory state in human hepatic cells; we also suggest a tool of blocking this induction with a monoclonal antibody.

The 19q12 bladder cancer GWAS signal

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.