Roy Ehrnström

miR-34c is downregulated in prostate cancer and exerts tumor suppressive functions.

MicroRNAs (miRNAs) are small noncoding RNAs that post‐transcriptionally regulate gene expression. There have been several reports of miRNA deregulation in prostate cancer (PCa) and the biological evidence for an involvement of miRNAs in prostate tumorigenesis is increasing. In this study, we show that miR‐34c is downregulated in PCa (p = 0.0005) by performing qRT‐PCR on 49 TURPs from PCa patients compared to 25 from patients with benign prostatic hyperplasia. The miR‐34c expression was found to inversely correlate to aggressiveness of the tumor, WHO grade, PSA levels and occurrence of metastases. Furthermore, a Kaplan–Meier analysis of patient survival based on miR‐34c expression levels divided into low (< 50th percentile) and high (> 50th percentile) expression, significantly divides the patients into high risk and low risk patients (p = 0.0003, log‐rank test). The phenotypic effects of miR‐34c deregulation were studied in prostate cell lines, where ectopic expression of miR‐34c decreased cell growth, due to both a decrease in cellular proliferation rate and an increase in apoptosis. In concordance to this, miR‐34c was found to negatively regulate the oncogenes E2F3 and BCL‐2, which stimulates proliferation and suppress apoptosis in PCa cells, respectively. Reversely, we could also show that blocking miR‐34c in vitro increases cell growth. Further, ectopic expression of miR‐34c was found to suppress migration and invasion. Our findings provide new insight into the role of miR‐34c in the prostate, exhibiting tumor suppressing effects on proliferation, apoptosis and invasiveness.

Fruit and vegetable intake and the risk of gastric adenocarcinoma: a reanalysis of the European Prospective Investigation into Cancer and Nutrition (EPIC-EURGAST) study after a longer follow-up.

In a previous European prospective investigation into cancer and nutrition (EPIC) analysis, we found an inverse association between total intake of vegetables, onion and garlic, and risk of intestinal gastric cancer (GC) and between citrus fruit and risk of cardia GC. The aim of this study is to reanalyze the effect of fruit and vegetables (F&V), based on a longer follow‐up and twice the number of GC cases. Subjects are 477,312 men and women mostly aged 35 to 70 years participating in the EPIC cohort, including 683 gastric adenocarcinomas with 11 years of follow‐up. Information on diet and lifestyle was collected at baseline. A calibration study in a subsample was used to correct for dietary measurement errors. When comparing the highest vs. lowest quintile of intake, we found an inverse association between total intake of V&F and GC risk [hazard ratio (HR) 0.77; 95% confidence interval (CI) 0.57–1.04; p for trend 0.02], between fresh fruit and risk of the diffuse type (HR 0.59; 95% CI 0.36–0.97; p for trend 0.03) and an inverse association between citrus fruit and risk of cardia cancer (HR 0.61; 95% CI 0.38–1.00, p for trend 0.01). Although calibration revealed somewhat stronger inverse associations, none of the risks reached statistical significance. There was no association between total or specific vegetables intake and GC risk. The inverse association between fresh fruit and citrus fruits and risk of GC seems to be restricted to smokers and the Northern European countries. Fresh fruit and citrus fruit consumption may protect against diffuse and cardia GC, respectively.

Aberrant DNA methylation of cancer-associated genes in gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC–EURGAST)

Epigenetic events have emerged as key mechanisms in the regulation of critical biological processes and in the development of a wide variety of human malignancies, including gastric cancer (GC), however precise gene targets of aberrant DNA methylation in GC remain largely unknown. Here, we have combined pyrosequencing-based quantitative analysis of DNA methylation in 98 GC cases and 64 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort and in cancer tissue and non-tumorigenic adjacent tissue of an independent series of GC samples. A panel of 10 cancer-associated genes (CHRNA3, DOK1, MGMT, RASSF1A, p14ARF, CDH1, MLH1, ALDH2, GNMT and MTHFR) and LINE-1 repetitive elements were included in the analysis and their association with clinicopathological characteristics (sex, age at diagnosis, anatomical sub-site, histological sub-type) was examined. Three out of the 10 genes analyzed exhibited a marked hypermethylation, whereas two genes (ALDH2 and MTHFR) showed significant hypomethylation, in gastric tumors. Among differentially methylated genes, we identified new genes (CHRNA3 and DOK1) as targets of aberrant hypermethylation in GC, suggesting that epigenetic deregulation of these genes and their corresponding cellular pathways may promote the development and progression of GC. We also found that global demethylation of tumor cell genomes occurs in GC, consistent with the notion that abnormal hypermethylation of specific genes occurs concomitantly with genome-wide hypomethylation. Age and gender had no significant influence on methylation states, but an association was observed between LINE-1 and MLH1 methylation levels with histological sub-type and anatomical sub-site. This study identifies aberrant methylation patters in specific genes in GC thus providing information that could be exploited as novel biomarkers in clinics and molecular epidemiology of GC.

Fluid intake and the risk of urothelial cell carcinomas in the European Prospective Investigation into Cancer and Nutrition (EPIC)

Results from previous studies investigating the association between fluid intake and urothelial cell carcinomas (UCC) are inconsistent. We evaluated this association among 233,236 subjects in the European Prospective Investigation into Cancer and Nutrition (EPIC), who had adequate baseline information on water and total fluid intake. During a mean follow‐up of 9.3 years, 513 first primary UCC occurred. At recruitment, habitual fluid intake was assessed by a food frequency questionnaire. Multivariable hazard ratios were estimated using Cox regression stratified by age, sex and center and adjusted for energy intake, smoking status, duration of smoking and lifetime intensity of smoking. When using the lowest tertile of intake as reference, total fluid intake was not associated with risk of all UCC (HR 1.12; 95%CI 0.86–1.45, p‐trend = 0.42) or with risk of prognostically high‐risk UCC (HR 1.28; 95%CI 0.85–1.93, p‐trend = 0.27) or prognostically low‐risk UCC (HR 0.93; 95%CI 0.65–1.33, p‐trend = 0.74). No associations were observed between risk of UCC and intake of water, coffee, tea and herbal tea and milk and other dairy beverages. For prognostically low‐risk UCC suggestions of an inverse association with alcoholic beverages and of a positive association with soft drinks were seen. Increased risks were found for all UCC and prognostically low‐risk UCC with higher intake of fruit and vegetable juices. In conclusion, total usual fluid intake is not associated with UCC risk in EPIC. The relationships observed for some fluids may be due to chance, but further investigation of the role of all types of fluid is warranted.

Consumption of vegetables and fruit and the risk of bladder cancer in the European Prospective Investigation into Cancer and Nutrition

Previous epidemiologic studies found inconsistent associations between vegetables and fruit consumption and the risk of bladder cancer. We therefore investigated the association between vegetable and fruit consumption and the risk of bladder cancer among participants of the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Data on food consumption and complete follow‐up for cancer occurrence was available for a total of 478,533 participants, who were recruited in 10 European countries. Estimates of rate ratios were obtained by Cox proportional hazard models, stratified by age at recruitment, gender and study centre, and adjusted for total energy intake, smoking status, duration of smoking and lifetime intensity of smoking. A calibration study in a subsample was used to control for dietary measurement errors. After a mean follow‐up of 8.7 years, 1015 participants were newly diagnosed with bladder cancer. Increments of 100 g/day in fruit and vegetable consumption combined did not affect bladder cancer risk (i.e., calibrated HR = 0.98; 95%CI: 0.95–1.01). Borderline statistically significant lower bladder cancer risks were found among never smokers with increased consumption of fruit and vegetables combined (HR = 0.94 95%CI: 0.87–1.00 with increments of 100 g/day; calibrated HR = 0.92 95%CI 0.79–1.06) and increased consumption of apples and pears (hard fruit; calibrated HR = 0.90 95%CI: 0.82–0.98 with increments of 25 g/day). For none of the associations a statistically significant interaction with smoking status was found. Our findings do not support an effect of fruit and vegetable consumption, combined or separately, on bladder cancer risk.

Elevated level of wnt5a protein in localized prostate cancer tissue is associated with better outcome.

Background Wnt5a is a non-canonical secreted glycoprotein of the Wnt family that plays an important role in cancer development and progression. Previous studies report that Wnt5a is upregulated in prostate cancer and suggested that Wnt5a affects migration and invasion of prostate tumor cell. This study aimed to evaluate the prognostic value of Wnt5a protein expression in prostate cancer tissue and its potential to predict outcome after radical prostatectomy in patients with localized prostate cancer. Methodology and Results Immunohistochemical analysis of a tissue microarray containing prostate specimens of 503 patients with localized prostate cancer showed significantly higher Wnt5a protein expression in cancer compared to benign cores from the same patients (p<0.0001). Patients with high expression of Wnt5a protein had significantly better outcome in terms of time to biochemical recurrence compared to patients with low expression levels (p = 0.001, 95%CI 1.361–3.570, Hazards ratio 2.204). A combination of high Wnt5a expression with low levels of Ki-67 or androgen receptor expression had even better outcome compared to all other groups. Furthermore, we found that Wnt5a expression significantly correlated with VEGF and with Ki-67 and androgen receptor expression, although not highly significant. In vitro, we demonstrated that recombinant Wnt5a decreased invasion of 22Rv1 and DU145 cells and that siRNA knockdown of endogenous Wnt5a protein led to increased invasion of 22Rv1 and LNCaP cells. Conclusion We demonstrate that preserved overexpression of Wnt5a protein in patients with localized prostate cancer predicts a favorable outcome after surgery. This finding together with our in vitro data demonstrating the ability of Wnt5a to impair the invasive properties of prostate cancer cells, suggests a tumor suppressing effect of Wnt5a in localized prostate cancer. These results indicate that Wnt5a can be used as a predictive marker and that it also is a plausible therapeutic target for treatment of localized prostate cancer.

An increased expression of cysteinyl leukotriene 2 receptor in colorectal adenocarcinomas correlates with high differentiation

Increased levels of inflammatory mediators such as cysteinyl leukotrienes (CysLT) have been found in and around tumors. These data, along with our previous observation that the G-protein-coupled receptor CysLT(1)R, which signals survival and proliferation, is up-regulated in colon cancer, suggest an important role for CysLT(1)R in tumor development. The objective of this study was to examine the expression and function of the low-affinity CysLT2 receptor (CysLT2R) in colon cancer. We found lower expression levels of CysLT2R compared with CysLT(1)R in cancer cell lines as well as clinical tumor material. Interestingly, CysLT2R, like CysLT(1)R, was found to be one of few G-protein-coupled receptors that are located both at the plasma membrane and the nuclear membrane. No effect of CysLT2R signaling on cell proliferation was observed, nor was there a correlation between CysLT2R and different proliferation markers such as Ki-67 and cyclooxygenase-2 in the tumor material. Instead, we found that activation of this receptor in colon cancer cells led to cellular differentiation similar to the effects of butyrate treatment. In accordance with this finding, we found that reduced expression of CysLT2R in colon cancer was associated with poor prognosis. We report the novel finding that CysLT2R signaling leads to terminal differentiation of colon carcinoma cells and growth inhibition, and that its expression is relatively high in less malignant forms of colon cancer. These data suggest that the balance between these two receptors is important for tumor progression and disease outcome.

Red Meat, Dietary Nitrosamines, and Heme Iron and Risk of Bladder Cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC)

Background: Previous epidemiologic studies found inconsistent results for the association between red meat intake, nitrosamines [NDMA: N-nitrosodimethylamine, and ENOC (endogenous nitroso compounds)], and the risk of bladder cancer. We investigated the association between red meat consumption, dietary nitrosamines, and heme iron and the risk of bladder cancer among participants of the European Prospective Investigation into Cancer and Nutrition (EPIC). Methods: Data on food consumption and complete follow-up for cancer occurrence were available for a total of 481,419 participants, recruited in 10 European countries. Estimates of HRs were obtained by proportional hazard models, stratified by age at recruitment, gender, and study center and adjusted for total energy intake, smoking status, lifetime intensity of smoking, duration of smoking, educational level, and BMI. Results: After a mean follow-up of 8.7 years, 1,001 participants were diagnosed with bladder cancer. We found no overall association between intake of red meat (log2 HR: 1.06; 95% CI: 0.99–1.13), nitrosamines (log2 HR: 1.09; 95% CI: 0.92–1.30 and HR: 0.98; 95% CI: 0.92–1.05 for ENOC and NDMA, respectively) or heme iron (log2 HR: 1.05; 95 CI: 0.99–1.12) and bladder cancer risk. The associations did not vary by sex, high- versus low-risk bladder cancers, smoking status, or occupation (high vs. low risk). Conclusions: Our findings do not support an effect of red meat intake, nitrosamines (endogenous or exogenous), or heme iron intake on bladder cancer risk. Cancer Epidemiol Biomarkers Prev; 20(3); 555–9. ©2011 AACR.

Cysteinyl leukotriene receptor expression pattern affects migration of breast cancer cells and survival of breast cancer patients

The fact that breast cancer patients with local or distal dissemination exhibit decreased survival, promotes a search for novel mechanisms to suppress such tumor progression. Here, we have determined the expression of proinflammatory cysteinyl leukotriene receptors (CysLTRs) in breast tumor tissue and their signaling effect on breast cancer cell functions related to tumor progression. Patients with breast tumors characterized by high CysLT1R and low CysLT2R expression levels exhibited increased risk of cancer‐induced death in univariate analysis for both the total patient group (hazard ratio [HR] = 2.88, 95% confidence interval [CI] = 1.11–7.41), as well as patients with large (>20 mm) tumors (HR = 5.08, 95% CI = 1.39–18.5). Multivariate analysis revealed that patients with large tumors exhibiting high CysLT1R and low CysLT2R expression levels had a significantly reduced survival, also when adjusted for established prognostic parameters (HR = 7.51, 95% CI = 1.83–30.8). In patients with large (>20 mm) tumors, elevated CysLT2R expression predicted an improved 5‐year survival (log‐rank test p = 0.04). Surprisingly, for longer time periods, this prognostic value was lost. This disappearance coincided with the termination of hormonal treatment. Tamoxifen preserved and even induced transcription of CysLT2R, but not CysLT1R, in estrogene receptor‐positive MCF‐7 breast cancer cells. This elevated CysLT2R expression decreased, even below the level of untreated cells, when tamoxifen was withdrawn. CysLT2R signaling reduced MCF‐7 cell migration, but had no effect on either proliferation or apoptosis. Our data indicate that low CysLT1R together with high CysLT2R expression levels might be useful parameters in prognostication and treatment stratification of breast cancer patients.

Variety in vegetable and fruit consumption and risk of bladder cancer in the European Prospective Investigation into Cancer and Nutrition

Recent research does not show an association between fruit and vegetable consumption and bladder cancer risk. None of these studies investigated variety in fruit and vegetable consumption, which may capture different aspects of consumption. We investigated whether a varied consumption of vegetables and fruits is associated with bladder cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Detailed data on food consumption and complete follow‐up for cancer incidence were available for 452,185 participants, who were recruited from ten European countries. After a mean follow‐up of 8.7 years, 874 participants were diagnosed with bladder cancer. Diet diversity scores (DDSs) were used to quantify the variety in fruit and vegetable consumption. Multivariable Cox proportional hazard models were used to assess the effect of the DDSs on bladder cancer risk. There was no evidence of a statistically significant association between bladder cancer risk and any of the DDSs when these scores were considered as continuous covariates. However, the hazard ratio (HR) for the highest tertile of the DDS for combined fruit and vegetable consumption was marginally significant compared to the lowest (HR = 1.30, 95% confidence interval: 1.00–1.69, p‐trend = 0.05). In EPIC, there is no clear association between a varied fruit and vegetable consumption and bladder cancer risk. This finding provides further evidence for the absence of any strong association between fruit and vegetable consumption as measured by a food frequency questionnaire and bladder cancer risk.