Angeline S. Andrew

MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31-mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.

Genetic variation in the prostate stem cell antigen gene PSCA confers susceptibility to urinary bladder cancer

We conducted a genome-wide association study on 969 bladder cancer cases and 957 controls from Texas. For fast-track validation, we evaluated 60 SNPs in three additional US populations and validated the top SNP in nine European populations. A missense variant (rs2294008) in the PSCA gene showed consistent association with bladder cancer in US and European populations. Combining all subjects (6,667 cases, 39,590 controls), the overall P-value was 2.14 × 10−10 and the allelic odds ratio was 1.15 (95% confidence interval 1.10–1.20). rs2294008 alters the start codon and is predicted to cause truncation of nine amino acids from the N-terminal signal sequence of the primary PSCA translation product. In vitro reporter gene assay showed that the variant allele significantly reduced promoter activity. Resequencing of the PSCA genomic region showed that rs2294008 is the only common missense SNP in PSCA. Our data identify rs2294008 as a new bladder cancer susceptibility locus.

Replication of Lung Cancer Susceptibility Loci at Chromosomes 15q25, 5p15, and 6p21: A Pooled Analysis From the International Lung Cancer Consortium

BACKGROUND Genome-wide association studies have identified three chromosomal regions at 15q25, 5p15, and 6p21 as being associated with the risk of lung cancer. To confirm these associations in independent studies and investigate heterogeneity of these associations within specific subgroups, we conducted a coordinated genotyping study within the International Lung Cancer Consortium based on independent studies that were not included in previous genome-wide association studies. METHODS Genotype data for single-nucleotide polymorphisms at chromosomes 15q25 (rs16969968, rs8034191), 5p15 (rs2736100, rs402710), and 6p21 (rs2256543, rs4324798) from 21 case-control studies for 11 645 lung cancer case patients and 14 954 control subjects, of whom 85% were white and 15% were Asian, were pooled. Associations between the variants and the risk of lung cancer were estimated by logistic regression models. All statistical tests were two-sided. RESULTS Associations between 15q25 and the risk of lung cancer were replicated in white ever-smokers (rs16969968: odds ratio [OR] = 1.26, 95% confidence interval [CI] = 1.21 to 1.32, P(trend) = 2 x 10(-26)), and this association was stronger for those diagnosed at younger ages. There was no association in never-smokers or in Asians between either of the 15q25 variants and the risk of lung cancer. For the chromosome 5p15 region, we confirmed statistically significant associations in whites for both rs2736100 (OR = 1.15, 95% CI = 1.10 to 1.20, P(trend) = 1 x 10(-10)) and rs402710 (OR = 1.14, 95% CI = 1.09 to 1.19, P(trend) = 5 x 10(-8)) and identified similar associations in Asians (rs2736100: OR = 1.23, 95% CI = 1.12 to 1.35, P(trend) = 2 x 10(-5); rs402710: OR = 1.15, 95% CI = 1.04 to 1.27, P(trend) = .007). The associations between the 5p15 variants and lung cancer differed by histology; odds ratios for rs2736100 were highest in adenocarcinoma and for rs402710 were highest in adenocarcinoma and squamous cell carcinomas. This pattern was observed in both ethnic groups. Neither of the two variants on chromosome 6p21 was associated with the risk of lung cancer. CONCLUSIONS In this international genetic association study of lung cancer, previous associations found in white populations were replicated and new associations were identified in Asian populations. Future genetic studies of lung cancer should include detailed stratification by histology.

Arsenic Exposure Is Associated with Decreased DNA Repair in Vitro and in Individuals Exposed to Drinking Water Arsenic

The mechanism(s) by which arsenic exposure contributes to human cancer risk is unknown; however, several indirect cocarcinogenesis mechanisms have been proposed. Many studies support the role of As in altering one or more DNA repair processes. In the present study we used individual-level exposure data and biologic samples to investigate the effects of As exposure on nucleotide excision repair in two study populations, focusing on the excision repair cross-complement 1 (ERCC1) component. We measured drinking water, urinary, or toenail As levels and obtained cryopreserved lymphocytes of a subset of individuals enrolled in epidemiologic studies in New Hampshire (USA) and Sonora (Mexico). Additionally, in corroborative laboratory studies, we examined the effects of As on DNA repair in a cultured human cell model. Arsenic exposure was associated with decreased expression of ERCC1 in isolated lymphocytes at the mRNA and protein levels. In addition, lymphocytes from As-exposed individuals showed higher levels of DNA damage, as measured by a comet assay, both at baseline and after a 2-acetoxyacetylaminofluorene (2-AAAF) challenge. In support of the in vivo data, As exposure decreased ERCC1 mRNA expression and enhanced levels of DNA damage after a 2-AAAF challenge in cell culture. These data provide further evidence to support the ability of As to inhibit the DNA repair machinery, which is likely to enhance the genotoxicity and mutagenicity of other directly genotoxic compounds, as part of a cocarcinogenic mechanism of action.

Implications of LINE1 Methylation for Bladder Cancer Risk in Women

Purpose: Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of the disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood–derived DNA is associated with increased risk of bladder cancer. Experimental Design: LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case-control study of bladder cancer in New Hampshire. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation. Results: Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer [95% confidence interval (95% CI), 1.12-2.90] in models controlling for gender, age, and smoking, and the association was stronger in women than in men (odds ratio, 2.48; 95% CI, 1.19-5.17 in women; and odds ratio, 1.47; 95% CI, 0.79-2.74 in men). Among controls, women were more likely to have lower LINE1 methylation than men (P = 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (P = 0.04). Conclusions: LINE1 hypomethylation may be an important biomarker of bladder cancer risk, especially among women. Clin Cancer Res; 16(5); 1682–9

Decreased DNA repair gene expression among individuals exposed to arsenic in United States drinking water.

Arsenic is well established as a human carcinogen, but its precise mechanism of action remains unknown. Arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA repair mechanisms, leading indirectly to increased mutations from other DNA damaging agents. The molecular mechanism underlying arsenic inhibition of nucleotide excision repair after UV irradiation (Hartwig et al., Carcinogenesis 1997;18:399–405) is unknown, but could be due to decreased expression of critical genes involved in nucleotide excision repair of damaged DNA. This hypothesis was tested by analyzing expression of repair genes and arsenic exposure in a subset of 16 individuals enrolled in a population based case‐control study investigating arsenic exposure and cancer risk in New Hampshire. Toenail arsenic levels were inversely correlated with expression of critical members of the nucleotide excision repair complex, ERCC1 (r2 = 0.82, p < 0.0001), XPF (r2 = 0.56, p < 0.002), and XPB (r2 = 0.75, p < 0.0001). The internal dose marker, toenail arsenic level, was more strongly associated with changes in expression of these genes than drinking water arsenic concentration. Our findings, based on human exposure to arsenic in a US population, show an association between biomarkers of arsenic exposure and expression of DNA repair genes. Although our findings need verification in a larger study group, they are consistent with the hypothesis that inhibition of DNA repair capacity is a potential mechanism for the co‐carcinogenic activity of arsenic.

Epigenetic Inactivation of SFRP Genes and TP53 Alteration Act Jointly as Markers of Invasive Bladder Cancer

In the United States each year, almost 13,000 deaths are attributable to bladder cancer, with the majority of these deaths related to higher stage, muscle-invasive solid tumors. Epigenetic silencing of the secreted frizzled receptor proteins (SFRP), antagonists of the WNT pathway, leads to constitutive WNT signaling, altering cell morphology and motility. Identifying alterations in this pathway in bladder cancer may prove useful for defining the invasive phenotype and provide targets for guiding therapy. Using a population-based study of bladder cancer (n = 355), we examined epigenetic alterations, specifically gene promoter hypermethylation, of four SFRP genes in addition to immunohistochemical staining of TP53, which has been previously shown to be a predictor of invasive disease. We observed a significant linear trend (P < 0.0004) in the magnitude of the risk of invasive disease with the number of SFRP genes methylated. Both TP53 alteration and SFRP gene methylation showed significant independent associations with invasive bladder cancer. Strikingly, in examining the joint effect of these alterations, we observed a >30-fold risk of invasive disease for patients with both altered SFRP gene methylation and intense TP53 staining (odds ratio, 32.1; P < 10(-13)). Overall patient survival was significantly poorer in patients with any SFRP genes methylated (P < 0.0003) and in proportional hazards modeling, patients with methylation of any SFRP gene had significantly poorer overall survival (hazard ratio, 1.78; P < 0.02) controlled for TP53 staining intensity and other survival-associated factors. Classifying tumors based on SFRP methylation status and TP53 protein staining intensity may be a clinically powerful predictor of invasive, deadly disease.

A Case–Control Study of Smoking and Bladder Cancer Risk: Emergent Patterns Over Time

BACKGROUND Cigarette smoking is a well-established risk factor for bladder cancer. The effects of smoking duration, intensity (cigarettes per day), and total exposure (pack-years); smoking cessation; exposure to environmental tobacco smoke; and changes in the composition of tobacco and cigarette design over time on risk of bladder cancer are unclear. METHODS We examined bladder cancer risk in relation to smoking practices based on interview data from a large, population-based case-control study conducted in Maine, New Hampshire, and Vermont from 2001 to 2004 (N = 1170 urothelial carcinoma case patients and 1413 control subjects). We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. To examine changes in smoking-induced bladder cancer risk over time, we compared odds ratios from New Hampshire residents in this study (305 case patients and 335 control subjects) with those from two case-control studies conducted in New Hampshire in 1994-1998 and in 1998-2001 (843 case patients and 1183 control subjects). RESULTS Regular and current cigarette smokers had higher risks of bladder cancer than never-smokers (for regular smokers, OR = 3.0, 95% CI = 2.4 to 3.6; for current smokers, OR = 5.2, 95% CI = 4.0 to 6.6). In New Hampshire, there was a statistically significant increasing trend in smoking-related bladder cancer risk over three consecutive periods (1994-1998, 1998-2001, and 2002-2004) among former smokers (OR = 1.4, 95% CI = 1.0 to 2.0; OR = 2.0, 95% CI = 1.4 to 2.9; and OR = 2.6, 95% CI = 1.7 to 4.0, respectively) and current smokers (OR = 2.9, 95% CI = 2.0 to 4.2; OR = 4.2, 95% CI = 2.8 to 6.3; OR = 5.5, 95% CI = 3.5 to 8.9, respectively) (P for homogeneity of trends over time periods = .04). We also observed that within categories of intensity, odds ratios increased approximately linearly with increasing pack-years smoked, but the slope of the increasing trend declined with increasing intensity. CONCLUSIONS Smoking-related risks of bladder cancer appear to have increased in New Hampshire since the mid-1990s. Based on our modeling of pack-years and intensity, smoking fewer cigarettes over a long time appears more harmful than smoking more cigarettes over a shorter time, for equal total pack-years of cigarettes smoked.

Lung Cancer in a U.S. Population with Low to Moderate Arsenic Exposure

Background Little is known about the carcinogenic potential of arsenic in areas with low to moderate concentrations of arsenic (< 100 μg/L) in drinking water. Objectives We examined associations between arsenic and lung cancer. Methods A population-based case–control study of primary incident lung cancer was conducted in 10 counties in two U.S. states, New Hampshire and Vermont. The study included 223 lung cancer cases and 238 controls, each of whom provided toenail clippings for arsenic exposure measurement by inductively coupled–plasma mass spectrometry. We estimated odds ratios (ORs) of the association between arsenic exposure and lung cancer using unconditional logistic regression with adjustment for potential confounders (age, sex, race/ethnicity, smoking pack-years, education, body mass index, fish servings per week, and toenail selenium level). Results Arsenic exposure was associated with small-cell and squamous-cell carcinoma of the lung [OR = 2.75; 95% confidence interval (CI), 1.00–7.57] for toenail arsenic concentration ≥ 0.114 μg/g, versus < 0.05 μg/g. A history of lung disease (bronchitis, chronic obstructive pulmonary disease, or fibrosis) was positively associated with lung cancer (OR = 2.86; 95% CI, 1.39–5.91). We also observed an elevated risk of lung cancer among participants with a history of lung disease and toenail arsenic ≥ 0.05 μg/g (OR = 4.78; 95% CI, 1.87–12.2) than among individuals with low toenail arsenic and no history of lung disease. Conclusion Although this study supports the possibility of an increased risk of specific lung cancer histologic types at lower levels of arsenic exposure, we recommend large-scale population-based studies.

DNA Repair Polymorphisms Modify Bladder Cancer Risk: A Multi-factor Analytic Strategy

Objectives: A number of common non-synonymous single nucleotide polymorphisms (SNPs) in DNA repair genes have been reported to modify bladder cancer risk. These include: APE1-Asn148Gln, XRCC1-Arg399Gln and XRCC1-Arg194Trp in the BER pathway, XPD-Gln751Lys in the NER pathway and XRCC3-Thr241Met in the DSB repair pathway. Methods: To examine the independent and interacting effects of these SNPs in a large study group, we analyzed these genotypes in 1,029 cases and 1,281 controls enrolled in two case-control studies of incident bladder cancer, one conducted in New Hampshire, USA and the other in Turin, Italy. Results: The odds ratio among current smokers with the variant XRCC3-241 (TT) genotype was 1.7 (95% CI 1.0–2.7) compared to wild-type. We evaluated gene-environment and gene-gene interactions using four analytic approaches: logistic regression, Multifactor Dimensionality Reduction (MDR), hierarchical interaction graphs, classification and regression trees (CART), and logic regression analyses. All five methods supported a gene-gene interaction between XRCC1-399/XRCC3-241 (p = 0.001) (adjusted OR for XRCC1-399 GG, XRCC3-241 TT vs. wild-type 2.0 (95% CI 1.4–3.0)). Three methods predicted an interaction between XRCC1-399/XPD-751 (p = 0.008) (adjusted OR for XRCC1-399 GA or AA, XRCC3-241 AA vs. wild-type 1.4 (95% CI 1.1–2.0)). Conclusions: These results support the hypothesis that common polymorphisms in DNA repair genes modify bladder cancer risk and highlight the value of using multiple complementary analytic approaches to identify multi-factor interactions.