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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.

Background: MicroRNAs (miRNAs) are important regulators of molecular pathways in psychiatric disease. Here, we examine differential miRNAs expression in lymphoblastoid cell lines (LCLs) derived from 10 individuals with autism spectrum disorder (ASD) and compare them to seven typically developing unrelated age- and gender-matched controls and 10 typically developing siblings. Small RNAseq analysis identified miRNAs, and selected miRNAs were validated using quantitative real-time polymerase reaction (qRT-PCR). KEGG analysis identified target pathways, and selected predicted mRNAs were validated using qRT-PCR. Results: Small RNAseq analysis identified that multiple miRNAs differentiated ASD from unrelated controls and ASD from typically developing siblings, with only one, hsa-miR-451a_R-1, being in common. Verification with qRT-PCR showed that miR-320a differentiated ASD from both sibling and unrelated controls and that several members of the miR-181 family differentiated ASD from unrelated controls. Differential expression of AKT2, AKT3, TNF α and CamKinase II predicted by KEGG analysis was verified by qRT-PCR. Expression of CamKinase II βwas found to be correlated with the severity of stereotyped behavior of the ASD participants. Conclusions: This study provides insight into the mechanisms regulating molecular pathways in individuals with ASD and identifies differentiated regulated genes involved in both the central nervous system and the immune system.

Autism Spectrum Disorder (ASD) comprises a heterogeneous group of neurodevelopmental disorders with a strong heritable genetic component. At present, ASD is diagnosed solely by behavioral criteria. Advances in genomic analysis have contributed to numerous candidate genes for the risk of ASD, where rare mutations and s common variants contribute to its susceptibility. Moreover, studies show rare de novo variants, copy number variation and single nucleotide polymorphisms (SNPs) also impact neurodevelopment signaling. Exploration of rare and common variants involved in common dysregulated pathways can provide new diagnostic and therapeutic strategies for ASD. Contributions of current innovative molecular strategies to understand etiology of ASD will be explored which are focused on whole exome sequencing (WES), whole genome sequencing (WGS), microRNA, long non-coding RNAs and CRISPR/Cas9 models. Some promising areas of pharmacogenomic and endophenotype directed therapies as novel personalized treatment and prevention will be discussed.

Pharmacogenomics (PGx) is a growing field within precision medicine. Testing can help predict adverse events and sub-therapeutic response risks of certain medications. To date, the US FDA lists over 280 drugs which provide biomarker-based dosing guidance for adults and children. At Arkansas Children’s Hospital (ACH), a clinical PGx laboratory-based test was developed and implemented to provide guidance on 66 pediatric medications for genotype-guided dosing. This PGx test consists of 174 single nucleotide polymorphisms (SNPs) targeting 23 clinically actionable PGx genes or gene variants. Individual genotypes are processed to provide per-gene discrete results in star-allele and phenotype format. These results are then integrated into EPIC- EHR. Genomic indicators built into EPIC-EHR provide the source for clinical decision support (CDS) for clinicians, providing genotype-guided dosing.

We showed previously that a 28-day combined dietary exposure to melamine and cyanuric acid (MEL&CYA) induced kidney lesions in NCTR Fisher 344 (F344) rats. Histopathological changes were significant in females dosed with ≥240ppm MEL&CYA and in males dosed with ≥180ppm MEL&CYA; however, the nephrotoxicity biomarkers blood urea nitrogen (BUN) and serum creatinine (SCr) were increased only by ≥240ppm MEL&CYA. The serum miRNome has been reported to reflect toxicity of several organs, including the kidney. Here, we compared the dose-response of alterations in serum miRNAs to those of BUN, SCr, and kidney histopathology in rats co-exposed to MEL&CYA. The serum miRNome of male F344 rats dosed with 0, 180, or 240ppm MEL&CYA was screened using quantitative real-time RT-PCR (qRT-PCR) and the levels of selected serum miRNAs were analyzed further in both sexes over the full dose range. The levels of several miRNAs were significantly reduced in rats treated with 240ppm MEL&CYA versus control. In addition, miR-128-3p and miR-210-3p were decreased in males treated with 180pm MEL&CYA, a dose at which the levels of BUN and SCr were not yet affected by treatment. These data suggest that the serum miRNome is affected by nephrotoxic doses of MEL&CYA in male and female rats.

Data are presented on the number and levels of 384 microRNAs (miRNAs) quantified by reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR) in human serum analyzed under different experimental conditions. The technical variables tested were 1) heating of the serum samples at 60 °C for 120 minutes prior to RNA extraction versus no heating; 2) RNA extraction using an Exiqon miRCURY RNA Isolation kit for Biofluids versus a Systems Biosciences SeraMir Exosome RNA Purification kit; 3) miRNA quantitation by RT-qPCR using an Exiqon SYBR Green Human Panel I versus an Applied Biosystems TaqMan Human microRNA Array A.

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.

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.

Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designated IFNL4, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.

A monoclonal antibody against prostate stem cell antigen (PSCA) has emerged as a novel cancer therapy currently being tested in clinical trials for prostate and pancreatic cancers, but this treatment is likely to be efficient only in patients with PSCA-expressing tumors. The present study demonstrates that a genetic variant (rs2294008) discovered by bladder cancer genome-wide association studies is a strong predictor of PSCA protein expression in bladder tumors, as measured by two-sided multivariable linear regression (P = 6.46×10(-11); n = 278). The association pattern is similar in non-muscle-invasive tumors, stages Ta (P = 3.10×10(-5); n = 173) and T1 (P = 2.64×10(-5); n = 60), and muscle-invasive tumors, stages T2 (P =.01; n = 23) and T3/4 (P =.03; n = 22). The study suggests that anti-PSCA immunotherapy might be beneficial for bladder cancer patients with high tumor PSCA expression, which is statistically significantly associated with the presence of CT and TT genotypes of a common genetic variant, rs2294008. Future clinical studies will be needed to validate PSCA as a therapeutic target for bladder cancer.

Purpose The nucleoside analog 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine) is a potent inhibitor of DNA methylation in vitro. Cellular treatment with this agent induces the re-expression of methylation-silenced genes. It remains unclear to what extent this compound inhibits DNA methylation in vivo. A clinical study was designed to examine the molecular effects and toxicity of a continuous 1-week intravenous infusion of decitabine in solid tumor patients. Methods Ten patients with refractory solid tumors were included in this study. Decitabine was administered at 2 mg/m2/d via continuous infusion for 168 hours. Quantitative polymerase chain reaction and high performance liquid chromatography were utilized to measure promoter-specific and global DNA methylation in peripheral-blood cells before and after treatment. Results Transient grade III/IV neutropenia (two patients) and grade II thrombocytopenia (one patient) was observed at the lowest planned dose step (2 mg/m2/d for 7 days). Nonhematologic toxicities were not observed. Quantitative polymerase chain reaction demonstrated significant MAGE-1 promoter hypomethylation by 14 days after the start of treatment in all 13 treatment cycles examined. Significant genomic DNA hypomethylation was also seen by day 14 in 11 of 13 treatment cycles analyzed. Genomic DNA methylation reverted to baseline levels by 28 to 35 days after the start of treatment, demonstrating that inhibition of DNA methylation by decitabine is transient. Conclusion A 168-hour continuous infusion of decitabine is well tolerated and results in the inhibition of promoter-specific and genomic DNA methylation in vivo. This treatment schedule is suitable for evaluation of decitabine in combination with agents whose activity may be enhanced by the reversal of DNA methylation–mediated gene silencing.

Proopiomelanocortin (POMC) is a precursor polypeptide for various bioactive peptides, including adrenocorticotropic hormone, alpha-, beta-, and gamma-melanotropin, beta-endorphin, and beta-lipotropin. Although the classical source of POMC is the pituitary, various studies indicate the expression of POMC in several nonpituitary tissues. In this study, in situ hybridization with anti-sense cRNA riboprobe was used to show expression of POMC mRNA in human epidermis and cultured human epidermal cells (melanocytes and keratinocytes). POMC mRNA was amplified by reverse transcriptase-polymerase chain reaction using anti-sense and sense primers designed from Exons 2 and 3 of POMC gene. A approximately 300 bp product was present in normal human skin, grafted human skin, and cultured normal human melanocytes and keratinocytes. By Southern analysis this product was hybridized specifically to the POMC cDNA. Sequence analysis of the reverse transcriptase polymerase chain reaction product from tissues or cells showed 85% homology to POMC cDNA from human, bovine, pig, and monkey sources. This suggests the existence of a putative isoform or variant of POMC mRNA in human epidermis.

Genome-wide association studies have identified a SNP, rs2294008, on 8q24.3 within the prostate stem cell antigen ( PSCA ) gene, as a risk factor for bladder cancer. To fine-map this region, we imputed 642 SNPs within 100 Kb of rs2294008 in addition to 33 markers genotyped in one of the reported genome-wide association study in 8,652 subjects. A multivariable logistic regression model adjusted for rs2294008 revealed a unique signal, rs2978974 ( r 2 = 0.02, D′ = 0.19 with rs2294008). In the combined analysis of 5,393 cases and 7,324 controls, we detected a per-allele odds ratio (OR) = 1.11 [95% confidence interval (CI) = 1.06–1.17, P = 5.8 × 10 −5 ] for rs2294008 and OR = 1.07 (95% CI = 1.02–1.13, P = 9.7 × 10 −3 ) for rs2978974. The effect was stronger in carriers of both risk variants (OR = 1.24, 95% CI = 1.08–1.41, P = 1.8 × 10 −3 ) and there was a significant multiplicative interaction ( P = 0.035) between these two SNPs, which requires replication in future studies. The T risk allele of rs2294008 was associated with increased PSCA mRNA expression in two sets of bladder tumor samples ( n = 36, P = 0.0007 and n = 34, P = 0.0054) and in normal bladder samples ( n = 35, P = 0.0155), but rs2978974 was not associated with PSCA expression. SNP rs2978974 is located 10 Kb upstream of rs2294008, within an alternative untranslated first exon of PSCA . The non-risk allele G of rs2978974 showed strong interaction with nuclear proteins from five cell lines tested, implying a regulatory function. In conclusion, a joint effect of two PSCA SNPs, rs2294008 and rs2978974, suggests that both variants may be important for bladder cancer susceptibility, possibly through different mechanisms that influence the control of mRNA expression and interaction with regulatory factors.

A recent genome-wide association study of bladder cancer identified the UGT1A gene cluster on chromosome 2q37.1 as a novel susceptibility locus. The UGT1A cluster encodes a family of UDP-glucuronosyltransferases (UGTs), which facilitate cellular detoxification and removal of aromatic amines. Bioactivated forms of aromatic amines found in tobacco smoke and industrial chemicals are the main risk factors for bladder cancer. The association within the UGT1A locus was detected by a single nucleotide polymorphism (SNP) rs11892031. Now, we performed detailed resequencing, imputation and genotyping in this region. We clarified the original genetic association detected by rs11892031 and identified an uncommon SNP rs17863783 that explained and strengthened the association in this region (allele frequency 0.014 in 4035 cases and 0.025 in 5284 controls, OR = 0.55, 95%CI = 0.44-0.69, P = 3.3 × 10(-7)). Rs17863783 is a synonymous coding variant Val209Val within the functional UGT1A6.1 splicing form, strongly expressed in the liver, kidney and bladder. We found the protective T allele of rs17863783 to be associated with increased mRNA expression of UGT1A6.1 in in-vitro exontrap assays and in human liver tissue samples. We suggest that rs17863783 may protect from bladder cancer by increasing the removal of carcinogens from bladder epithelium by the UGT1A6.1 protein. Our study shows an example of genetic and functional role of an uncommon protective genetic variant in a complex human disease, such as bladder cancer.

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer.

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 in patients with estrogen receptor (ER)+ versus ER- cancer. Results We found association between SNP rs11249433 and expression of the NOTCH2 gene located in the 1p11.2 region. Examined in 180 breast tumors, the expression of NOTCH2 was found to be lowest in tumors with TP53 mutations and highest in TP53 wild-type/ER+ tumors (p = 0.0059). In the latter group, the NOTCH2 expression was particularly increased in carriers of the risk genotypes (AG/GG) of rs11249433 when compared to the non-risk AA genotype (p = 0.0062). Similar association between NOTCH2 expression and rs11249433 was observed in 60 samples of purified monocytes from healthy controls (p = 0.015), but not in total blood samples from 302 breast cancer patients and 76 normal breast tissue samples. We also identified the first possible dominant-negative form of NOTCH2, a truncated version of NOTCH2 consisting of only the extracellular domain. Conclusion This is the first study to show that the expression of NOTCH2 differs in subgroups of breast tumors and by genotypes of the breast cancer-associated SNP rs11249433. The NOTCH pathway has key functions in stem cell differentiation of ER+ luminal cells in the breast. Therefore, increased expression of NOTCH2 in carriers of rs11249433 may promote development of ER+ luminal tumors. Further studies are needed to investigate possible mechanisms of regulation of NOTCH2 expression by rs11249433 and the role of NOTCH2 splicing forms in breast cancer development.

A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.

Phenotypic characteristics of members of a melanoma prone kindred with a V126D CDKN2A gene mutation were monitored over approximately 15 y. Thirty-eight previously studied subjects were recruited. Participants underwent a complete skin examination by the same dermatologist who examined them initially. The size and location of all nevi were recorded on a body map diagram. Total nevus number (TNN) and total nevus density (TND) were determined. CDKN2A sequencing verified 13 mutation carriers and 16 non-carriers. Nine participants were spouse controls without a history of melanoma and did not carry a CDKN2A mutation. Mutation carriers demonstrated a greater mean TNN and TND at initial and follow-up examinations compared with non-carriers and continued to develop nevi rather than show nevus regression seen in non-carriers and spouse controls. Non-carriers showed an intermediate nevus phenotype between mutation carriers and spouse controls. Four of the 13 mutation carriers and one non-carrier have developed invasive melanoma. Over a 15-y interval, TNN and TND were increased in mutation carriers compared with non-carriers and spouse controls. Continued accumulation of nevi in mutation carriers supports a nevogenic role for this CDKN2A mutation. An intermediate nevus phenotype in non-carrier family members suggests the presence of additional modifier genes.

Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells.

Genome-wide association studies (GWAS) identified a single nucleotide polymorphism (SNP) rs12979860 located upstream of the IFNL3 ( IL28B ) gene as a genetic variant strongly predictive of spontaneous and treatment-induced HCV clearance. Further genetic and genomic studies showed that rs12979860 is located in the intronic region of a novel gene, IFNL4 , and is in linkage disequilibrium (LD) with an IFNL4 exonic frame-shift polymorphism rs368234815-TT/ΔG. The rs368234815-ΔG allele, which creates an open reading frame for a novel human interferon, IFN-λ4, is strongly associated with decreased HCV clearance. The rs368234815-ΔG allele is a major allele in individuals of African ancestry (70%), while it is less common in Europeans (30%) and Asians (0–5%). Primary human hepatocytes are the only currently known cell type where IFN-λ4 expression can be induced by stimulation with Poly(I:C) or by infection with HCV. In contrast with other interferons, IFN-λ4 is poorly secreted. Using live quantitative fluorescence microscopy we show that a significant proportion of IFN-λ4 transiently overexpressed in HepG2 cells remains intracellularly retained, consistent with its poor secretion. We show that intracellular retention of IFN-λ4 is associated with increased cell death. Treatment with the pan-caspase inhibitor Z-VAD but not with the caspase-1 inhibitor Y-VAD decreased the IFN-λ4-induced cell death. We also observed increased caspase 3 activation in both primary human hepatocytes and HepG2 cells, transiently overexpressing IFN-λ4, suggesting apoptosis as a mechanism of cell death. In conclusion, we propose a novel mechanism associated with pathogenic effect of IFN-λ4 in human hepatic cells through induction of apoptotic cell death. Induction of IFN-λ4 in HCV-infected hepatocytes leading to cell death and tissue repair in the liver, may result in hepatic fibrosis and cirrhosis, which are relevant factors affecting clearance of HCV and, possibly progression to hepatocellular carcinoma.

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.

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

The NPLC-KC human hepatoma cell line expresses corticotropin-releasing factor (CRF) and it has been demonstrated that CRF secretion and synthesis in this cell line increases in response to activators of the protein kinase A (PKA) and C (PKC) pathways as well as interleukin-1 (IL1). CRF expression with all three agents can be inhibited with the synthetic steroid-dexamethasone (DEX). In this report, we have examined the effect of IL1 (beta form) in the presence and absence of DEX on CRF mRNA (mRNA) expression as well as the expression of human glucocorticoid receptor (GR) mRNA and the mRNA of the proto-oncogenes (c-jun and c-fos) that have been implicated in CRF regulation. NPLC-KC cells were incubated with picomolar concentrations of IL1. Following this total RNA was extracted from the cells and Northern Blots were probed with 32P-labelled human DNA probes for the CRF, GR, c-jun and c-fos genes. Levels of mRNA expression were measured using a PhosphoImager and were normalized to mRNA levels of control probe glyceraldehyde-3-phosphate dehydrogenase (GAPD). CRF mRNA was significantly increased with IL1 treatment in a time and concentration dependent manner. CRF mRNA expression increased with increasing concentrations of IL1 over the range of 1-100 pM; expression of CRF mRNA also peaked after 24 h of 100 pM IL1 treatment and reached a level of expression approximately seven times higher than control. This pattern of expression was significantly inhibited in the presence of 100 nM DEX. Levels of the GR, c-fos and c-jun mRNAs were also significantly increased in the presence of IL1 and inhibited when DEX was co-incubated with IL1. The results reveal that IL1 stimulation of CRF mRNA expression by IL1 in the NPLC-KC cell line is accompanied by activation of GR mRNA as well as the mRNA of the immediate early genes--c-fos and c-jun. The results also demonstrate that this cell line may serve as a model system for the molecular mechanisms by which IL1 regulates CRF in central nervous system (CNS) neurons.

We have identified a novel human interferon, designated as interferon-λ4 (IFN-λ4) (Prokunina-Olsson et al., Nature Genetics, 2013). Inducible expression of IFNL4 , along with three related genes ( IFNL1 , IFNL2 and IFNL3 ) located on chromosome 19q13.13 and encoding other interferon-λ proteins, IFN-λ1, IFN-λ2 and IFN-λ3, was revealed by RNA-sequencing in primary human hepatocytes treated with polyI:C. In humans, IFN-λ4 is fully genetically controlled – it is produced only by mRNA transcripts with a frameshift dG allele of a genetic variant ss469415590 (TT/dG) within the first exon of the gene, while transcripts with the TT allele can generate only unrelated proteins or protein fragments. We characterized IFN-λ4 as a class-2 cytokine based on its protein homology with IFN-λ3 (29% amino acid identity), the ability to cause STAT1 and STAT2 phosphorylation, activation of an ISRE-Luc reporter, induction of ISGs and antiviral response in HepG2 cells transiently transfected with an IFNL4-expressing construct. IFN-λ4 signaling is decreased by a JAK inhibitor, siRNA-silencing of IFN-λR1, and blocking of IL10R2. However, HepG2 cells did not respond to treatment with purified recombinant IFN-λ4 protein. Protein expression of IFN-λ4 is detectable in cells transfected with an IFNL4-expressing construct but not in the culture supernatants. It remains unclear whether IFN-λ4 is an unusual intracellular interferon, which signals through yet unknown intracellular partners or it is released at low levels through secretion or other mechanisms. Individuals homozygous for the derived human-specific ss469415590-TT allele (90% of Asians, 50% of Europeans and 10% of individuals of African ancestry) are genetically unable to produce IFN-λ4. This pattern suggests strong positive selection for elimination of IFN-λ4, which might be caused by current or historic infectious diseases. Individuals who are unable to produce IFN-λ4 are more likely to clear hepatitis C virus (HCV) either spontaneously or after treatment with IFN-α and ribavirin.

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

Genetic mapping studies have identified multiple cancer susceptibility regions at chromosome 8q24, upstream of the MYC oncogene. MYC has been widely presumed as the regulated target gene, but definitive evidence functionally linking these cancer regions with MYC has been difficult to obtain. Here we examined candidate functional variants of a haplotype block at 8q24 encompassing the two independent risk alleles for prostate and breast cancer, rs620861 and rs13281615. We used the mapping of DNase I hypersensitive sites as a tool to prioritise regions for further functional analysis. This approach identified rs378854, which is in complete linkage disequilibrium (LD) with rs620861, as a novel functional prostate cancer-specific genetic variant. We demonstrate that the risk allele (G) of rs378854 reduces binding of the transcription factor YY1 in vitro. This factor is known to repress global transcription in prostate cancer and is a candidate tumour suppressor. Additional experiments showed that the YY1 binding site is occupied in vivo in prostate cancer, but not breast cancer cells, consistent with the observed cancer-specific effects of this single nucleotide polymorphism (SNP). Using chromatin conformation capture (3C) experiments, we found that the region surrounding rs378854 interacts with the MYC and PVT1 promoters. Moreover, expression of the PVT1 oncogene in normal prostate tissue increased with the presence of the risk allele of rs378854, while expression of MYC was not affected. In conclusion, we identified a new functional prostate cancer risk variant at the 8q24 locus, rs378854 allele G, that reduces binding of the YY1 protein and is associated with increased expression of PVT1 located 0.5 Mb downstream., Author Summary Genome-wide association studies have been very successful at identifying gene loci that are associated with common diseases. However for many of these it has been very difficult to elucidate the identity and function of the risk variant and how altered function predisposes to disease. A 1.2-Mb gene-poor region upstream of the MYC oncogene has been shown to contain at least 12 risk loci associated with different types of cancer. In this study, we use the analysis of chromatin conformation as a tool to identify sub-regions within this 1.2 Mb region that are involved in the control of gene expression, and we identify molecular mechanisms which may confer risk of different specific cancer types. In particular, we examine a risk locus that is associated with predisposition to prostate cancer and identify a DNA sequence variation that results in a change in the binding site for the nuclear factor YY1 as a potential causative mechanism. We show that the risk locus is able to interact with two downstream genes, MYC and PVT1, and present evidence that PVT1 is a candidate new target gene regulated by the 8q24 risk region.

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.

(et al.)