Elise D. Bowman

MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma.

CONTEXT MicroRNAs have potential as diagnostic biomarkers and therapeutic targets in cancer. No study has evaluated the association between microRNA expression patterns and colon cancer prognosis or therapeutic outcome. OBJECTIVE To identify microRNA expression patterns associated with colon adenocarcinomas, prognosis, or therapeutic outcome. DESIGN, SETTING, AND PATIENTS MicroRNA microarray expression profiling of tumors and paired nontumorous tissues was performed on a US test cohort of 84 patients with incident colon adenocarcinoma, recruited between 1993 and 2002. We evaluated associations with tumor status, TNM staging, survival prognosis, and response to adjuvant chemotherapy. Associations were validated in a second, independent Chinese cohort of 113 patients recruited between 1991 and 2000, using quantitative reverse transcription polymerase chain reaction assays. The final date of follow-up was December 31, 2005, for the Maryland cohort and August 16, 2004, for the Hong Kong cohort. MAIN OUTCOME MEASURES MicroRNAs that were differentially expressed in tumors and microRNA expression patterns associated with survival using cancer-specific death as the end point. RESULTS Thirty-seven microRNAs were differentially expressed in tumors from the test cohort. Selected for validation were miR-20a, miR-21, miR-106a, miR-181b, and miR-203, and all 5 were enriched in tumors from the validation cohort (P < .001). Higher miR-21 expression was present in adenomas (P = .006) and in tumors with more advanced TNM staging (P < .001). In situ hybridization demonstrated miR-21 to be expressed at high levels in colonic carcinoma cells. The 5-year cancer-specific survival rate was 57.5% for the Maryland cohort and was 49.5% for the Hong Kong cohort. High miR-21 expression was associated with poor survival in both the training (hazard ratio, 2.5; 95% confidence interval, 1.2-5.2) and validation cohorts (hazard ratio, 2.4; 95% confidence interval, 1.4-3.9), independent of clinical covariates, including TNM staging, and was associated with a poor therapeutic outcome. CONCLUSIONS Expression patterns of microRNAs are systematically altered in colon adenocarcinomas. High miR-21 expression is associated with poor survival and poor therapeutic outcome.

MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers

Fifteen percent of lung cancer cases occur in never-smokers and show characteristics that are molecularly and clinically distinct from those in smokers. Epidermal growth factor receptor (EGFR) gene mutations, which are correlated with sensitivity to EGFR-tyrosine kinase inhibitors (EGFR-TKIs), are more frequent in never-smoker lung cancers. In this study, microRNA (miRNA) expression profiling of 28 cases of never-smoker lung cancer identified aberrantly expressed miRNAs, which were much fewer than in lung cancers of smokers and included miRNAs previously identified (e.g., up-regulated miR-21) and unidentified (e.g., down-regulated miR-138) in those smoker cases. The changes in expression of some of these miRNAs, including miR-21, were more remarkable in cases with EGFR mutations than in those without these mutations. A significant correlation between phosphorylated-EGFR (p-EGFR) and miR-21 levels in lung carcinoma cell lines and the suppression of miR-21 by an EGFR-TKI, AG1478, suggest that the EGFR signaling is a pathway positively regulating miR-21 expression. In the never-smoker–derived lung adenocarcinoma cell line H3255 with mutant EGFR and high levels of p-EGFR and miR-21, antisense inhibition of miR-21 enhanced AG1478-induced apoptosis. In a never-smoker–derived adenocarcinoma cell line H441 with wild-type EGFR, the antisense miR-21 not only showed the additive effect with AG1478 but also induced apoptosis by itself. These results suggest that aberrantly increased expression of miR-21, which is enhanced further by the activated EGFR signaling pathway, plays a significant role in lung carcinogenesis in never-smokers, as well as in smokers, and is a potential therapeutic target in both EGFR-mutant and wild-type cases.

MicroRNA expression in squamous cell carcinoma and adenocarcinoma of the esophagus: Associations with survival

Purpose: The dismal outcome of esophageal cancer patients highlights the need for novel prognostic biomarkers, such as microRNAs (miRNA). Although recent studies have established the role of miRNAs in esophageal carcinoma, a comprehensive multicenter study investigating different histologic types, including squamous cell carcinoma (SCC) and adenocarcinoma with or without Barretts, is still lacking. Experimental Design: miRNA expression was measured in cancerous and adjacent noncancerous tissue pairs collected from 100 adenocarcinoma and 70 SCC patients enrolled at four clinical centers from the United States, Canada, and Japan. Microarray-based expression was measured in a subset of samples in two cohorts and was validated in all available samples. Results: In adenocarcinoma patients, miR-21, miR-223, miR-192, and miR-194 expression was elevated, whereas miR-203 expression was reduced in cancerous compared with noncancerous tissue. In SCC patients, we found elevated miR-21 and reduced miR-375 expression levels in cancerous compared with noncancerous tissue. When comparing cancerous tissue expression between adenocarcinoma and SCC patients, miR-194 and miR-375 were elevated in adenocarcinoma patients. Significantly, elevated miR-21 expression in noncancerous tissue of SCC patients and reduced levels of miR-375 in cancerous tissue of adenocarcinoma patients with Barretts were strongly associated with worse prognosis. Associations with prognosis were independent of tumor stage or nodal status, cohort type, and chemoradiation therapy. Conclusions: Our multicenter-based results highlight miRNAs involved in major histologic types of esophageal carcinoma and uncover significant associations with prognosis. Elucidating miRNAs relevant to esophageal carcinogenesis is potentially clinically useful for developing prognostic biomarkers and identifying novel drug targets and therapies. (Clin Cancer Res 2009;15(19):6192–200)

Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer.

Circulating micro‐RNA (miR) profiles have been proposed as promising diagnostic and prognostic biomarkers for cancer, including lung cancer. We have developed methods to accurately and reproducibly measure micro‐RNA levels in serum and plasma. Here, we study paired serum and plasma samples from 220 patients with early stage nonsmall cell lung cancer (NSCLC) and 220 matched controls. We use qRT‐PCR to measure the circulating levels of 30 different miRs that have previously been reported to be differently expressed in lung cancer tissue. Duplicate RNA extractions were performed for 10% of all samples, and micro‐RNA measurements were highly correlated among those duplicates. This demonstrates high reproducibility of our assay. The expressions of miR‐146b, miR‐221, let‐7a, miR‐155, miR‐17‐5p, miR‐27a and miR‐106a were significantly reduced in the serum of NSCLC cases, while miR‐29c was significantly increased. No significant differences were observed in plasma of patients compared with controls. Overall, expression levels in serum did not correlate well with levels in plasma. In secondary analyses, reduced plasma expression of let‐7b was modestly associated with worse cancer‐specific mortality in all patients, and reduced serum expression of miR‐223 was modestly associated with cancer‐specific mortality in stage IA/B patients. MiR profiles also showed considerable differences comparing African American and European Americans. In summary, we found significant differences in miR expression when comparing cases and controls and find evidence that expression of let‐7b is associated with prognosis in NSCLC.

Increased Levels of Circulating Interleukin 6, Interleukin 8, C-Reactive Protein, and Risk of Lung Cancer

BACKGROUND Previous studies that were based primarily on small numbers of patients suggested that certain circulating proinflammatory cytokines may be associated with lung cancer; however, large independent studies are lacking. METHODS Associations between serum interleukin 6 (IL-6) and interleukin 8 (IL-8) levels and lung cancer were analyzed among 270 case patients and 296 control subjects participating in the National Cancer Institute-Maryland (NCI-MD) case-control study. Results were validated in 532 case patients and 595 control subjects in a nested case-control study within the prospective Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Association with C-reactive protein (CRP), a systemic inflammation biomarker, was also analyzed. Associations between biomarkers and lung cancer were estimated using logistic regression models adjusted for smoking, stage, histology, age, and sex. The 10-year standardized absolute risks of lung cancer were estimated using a weighted Cox regression model. RESULTS Serum IL-6 and IL-8 levels in the highest quartile were associated with lung cancer in the NCI-MD study (IL-6, odds ratio [OR] = 3.29, 95% confidence interval [CI] = 1.88 to 5.77; IL-8, OR = 2.06, 95% CI = 1.19 to 3.57) and with lung cancer risk in the PLCO study (IL-6, OR = 1.48, 95% CI = 1.04 to 2.10; IL-8, OR = 1.57, 95% CI = 1.10 to 2.24), compared with the lowest quartile. In the PLCO study, increased IL-6 levels were only associated with lung cancer diagnosed within 2 years of blood collection, whereas increased IL-8 levels were associated with lung cancer diagnosed more than 2 years after blood collection (OR = 1.57, 95% CI = 1.15 to 2.13). The 10-year standardized absolute risks of lung cancer in the PLCO study were highest among current smokers with high IL-8 and CRP levels (absolute risk = 8.01%, 95% CI = 5.77% to 11.05%). CONCLUSIONS Although increased levels of both serum IL-6 and IL-8 are associated with lung cancer, only IL-8 levels are associated with lung cancer risk several years before diagnosis. Combination of IL-8 and CRP are more robust biomarkers than either marker alone in predicting subsequent lung cancer.

p53 isoforms Δ133p53 and p53β are endogenous regulators of replicative cellular senescence

The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumour progression in vivo. We show that the human p53 isoforms Δ133p53 and p53β function in an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53β and diminished Δ133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA, the antisense inhibition of which delayed the onset of replicative senescence. The siRNA (short interfering RNA)-mediated knockdown of endogenous Δ133p53 induced cellular senescence, which was attributed to the regulation of p21WAF1 and other p53 transcriptional target genes. In overexpression experiments, whereas p53β cooperated with full-length p53 to accelerate cellular senescence, Δ133p53 repressed miR-34a expression and extended the cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (that is, elevated p53β and reduced Δ133p53) was observed in vivo in colon adenomas with senescent phenotypes. The increased Δ133p53 and decreased p53β isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma.

DEPRESSION AND SELF-MEDICATION WITH NICOTINE : THE MODIFYING INFLUENCE OF THE DOPAMINE D4 RECEPTOR GENE

This study evaluated whether there are genetic subgroups of depressed individuals who are more or less predisposed to engage in self-medication smoking practices. Smokers (N = 231) completed self-report questionnaires of depression and smoking practices and were genotyped for the dopamine D4 receptor (DRD4) gene. A significant interaction (DRD4 Genotype x Depression) was found for stimulation smoking and negative-affect reduction smoking. Specifically, these smoking practices were significantly heightened in depressed smokers homozygous for the short alleles of DRD4 but not in those heterozygous or homozygous for the long alleles of DRD4. These preliminary results suggest that the rewarding effects of smoking and the beneficial effects of nicotine replacement therapy for depressed smokers may depend, in part, on genetic factors involved in dopamine transmission.

Cruciferous Vegetables, Genetic Polymorphisms in Glutathione S-Transferases M1 and T1, and Prostate Cancer Risk

Abstract: Cruciferous vegetables contain anticarcinogenic isothiocyanates (ITCs), particularly the potent sulforaphane, which may decrease risk of prostate cancer through induction of phase II enzymes, including glutathione S-transferases (GSTs). We evaluated this hypothesis in a population-based, case-control study of prostate cancer, including 428 men with incident prostate cancer and 537 community controls. An in-person interview included an extensive food-frequency questionnaire. Genotyping for deletions in GSTM1 and GSTT1 was performed in a subset of men who provided blood. Intakes of cruciferous vegetables and of broccoli, the greatest source of sulforaphane, were associated with decreased prostate cancer risk at all levels above the lowest consumers [adjusted 4th quartile odds ratio (OR) = 0.58; 95% confidence interval (CI) = 0.38, 0.89, and 0.72 (95% CI = 0.49, 1.06)], respectively. In relation to genotypes, there was a nonsignificant increase in risk with the GSTT1 null genotype (OR = 1.51; 95% CI = 0.98, 2.31) but no effects of GSTM1 genotype. However, men with GSTM1-present genotype and high broccoli intake had the greatest reduction in risk (OR = 0.49; 95% CI = 0.27, 0.89). Our findings provide evidence that two or more servings per month of cruciferous vegetables may reduce risk of prostate cancer, especially among men with GSTM1-present alleles, and are consistent with a role of dietary ITCs as chemopreventive agents against prostate cancer.

The Association of MicroRNA Expression with Prognosis and Progression in Early-Stage, Non–Small Cell Lung Adenocarcinoma: A Retrospective Analysis of Three Cohorts

Purpose: There is increasing evidence that altered microRNA expression is associated with tumor progression and survival in cancer patients. We tested if the expression of specific microRNAs was associated with prognosis and disease progression in early-stage lung adenocarcinoma. Experimental Design: The expression of miR-21, miR-17, and miR-155 was measured by quantitative RT-PCR in tissues from 317 non–small cell lung cancer (NSCLC) patients that originated from Maryland, Norway, and Japan. Kaplan-Meier and Cox regression analysis evaluated associations of microRNA expression with cancer-specific mortality and disease-free survival. Results: Elevated miR-21 (HR 2.06, 1.13–3.75), miR-17 (HR 2.00, 1.10–3.61), and miR-155 (HR 2.37, 1.27–4.42) was associated with worse cancer-specific mortality in the Maryland cohort. These were evaluated in two additional cohorts and only miR-21 was associated with worse cancer-specific mortality in the Norwegian cohort (HR 2.78, 1.22–6.31) and worse relapse-free survival in the Japanese cohort (HR 2.82, 1.57–5.07). More advanced stage tumors expressed significantly higher levels of miR-21 compared with TNM stage I tumors. TNM stage I patients were evaluated separately and high levels of miR-21 was associated with worse cancer-specific mortality (HR 2.16, 1.11–4.21) and relapse-free survival (3.40, 1.57–7.36) independent of other clinical factors. Conclusions: This is the first study to report that increased miR-21 expression is associated with disease progression and survival in stage I lung cancer. This suggests that expression of miR-21 may contribute to lung carcinogenesis and serve as a therapeutic target or early-stage prognostic biomarker for lung adenocarcinoma. Clin Cancer Res; 17(7); 1875–82. ©2011 AACR.

Association of Inflammation-Related and microRNA Gene Expression with Cancer-Specific Mortality of Colon Adenocarcinoma

Purpose: Inflammatory genes and microRNAs have roles in colon carcinogenesis; therefore, they may provide useful biomarkers for colon cancer. This study examines the potential clinical utility of an inflammatory gene expression signature as a prognostic biomarker for colon cancer in addition to previously examined miR-21 expression. Experimental Design: Quantitative reverse transcriptase-PCR. was used to measure the expression of 23 inflammatory genes in colon adenocarcinomas and adjacent noncancerous tissues from 196 patients. These data were used to develop models for cancer-specific mortality on a training cohort (n = 57), and this model was tested in both a test (n = 56) and a validation (n = 83) cohort. Expression data for miR-21 were available for these patients and were compared and combined with inflammatory gene expression. Results: PRG1, IL-10, CD68, IL-23a, and IL-12a expression in noncancerous tissue, and PRG1, ANXA1, IL-23a, IL-17a, FOXP3, and HLA-DRA expression in tumor tissues were associated with poor prognosis based on Cox regression (|Z-score| >1.5) and were used to generate the inflammatory risk score (IRS). IRS was associated with cancer-specific mortality in the training, test (P = 0.01), and validation (P = 0.02) cohorts. This association was strong for stage II cases (P = 0.002). Expression of miR-21 was associated with IL-6, IL-8, IL-10, IL-12a, and NOS2a, providing evidence that the function of this microRNA and these inflammatory genes are linked. Both IRS and miR-21 expression were independently associated with cancer-specific mortality, including stage II patients alone. Conclusion: IRS and miR-21 expression are independent predictors of colon cancer prognosis and may provide a clinically useful tool to identify high-risk patients. (Clin Cancer Res 2009;15(18):5878–87)