Abstract INTRODUCTION MicroRNAs (miRs) have been implicated in various human malignancies and are being developed as diagnostic, predictive, and prognostic biomarkers. Our previous global profiling studies found miR-449a to be over-expressed in archival lymph node-negative invasive ductal breast carcinoma samples (n=74) when compared to normal tissues. Furthermore, miR-449a expression level was significantly associated with relapse. The mechanism(s) by which miR-449a functions remains unknown, and the aim of the current study was therefore to elucidate the roles and mRNA targets of miR-449a in breast cancer. MATERIAL AND METHODS MiR-449a expression was evaluated in six human breast cancer cell lines (T47D, MDA-MB-468, MDA-MB-231, MCF-7, MDA-MB-453, and ZR75-1). Three of these cell lines, T47D, MMDA-MB-468, and MDA-MB-231, were selected and used to assess the biological effects of miR-449a on cell viability, clonogenicity, cell migration, and invasion. Downstream target genes were identified by combining in silico miRWalk prediction and cell line/patient sample microarray data. Targets were validated using qRT-PCR and luciferase reporter assays. RESULTS Consistent with our global profiling work, several (4/6) of the tested breast cancer cell lines over-expressed miR-449a. CDC20B, the miR-449a host gene and an essential regulator of cell division, was also over-expressed. Knockdown of miR-449a resulted in significantly decreased cell viability, clonogenic survival, migration, and invasion. MiR-449a candidate targets were then identified by integrating in silico prediction algorithms, cell line mRNA expression profiling (Affymetrix Human Genome U133 Plus 2.0), and clinical specimen mRNA expression data. Two genes, CRIP2 (Cysteine rich protein 2, a transcription factor) and PRKAG1 (Protein Kinase, AMP-Activated, Gamma 1 Non-Catalytic), were verified as miR-449a targets using qRT-PCR. Moreover, direct interactions between miR-449a and the 3′-UTRs of both CRIP2 and PRKAG1 were confirmed using luciferase reporter assays. Inhibition of CRIP2 by miR-449a allowed for NF-κB-induced transcription of survival, proliferation, and growth-related genes, providing further evidence for the role of miR-449a in breast cancer progression. CONCLUSIONS MiR-449a is over-expressed in breast cancer cells, promoting cellular proliferation, migration, and invasion. One mechanism by which miR-449a functions is to down-regulate CRIP2, leading to NF-kB activation and possible cancer progression. Further investigations into gene regulation by miR-449a may reveal other promising targets for the management of breast cancer. Citation Format: Wei Shi, Matthew Lee, Ryunosuke Kogo, Jeff Bruce, Christine How, Kenneth W. Yip, Fei-Fei Liu. MiR-449a promotes breast cancer progression by activating the NF-κB pathway. [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 4372. doi:10.1158/1538-7445.AM2014-4372
This chapter contains sections titled: Introduction Lifestyle Risk Factors for CRC Oxidative Stress Relates to Lifestyle and CRC Risk Energy Excess Relates Lifestyle and CRC Risk Interaction of Toxicity of Energy Excess and Oxidative Stress Future Research References
Abstract MicroRNA (miR)-218 down-regulation has been reported in numerous human malignancies. In cervical cancer, we identified that lower miR-218 expression was significantly associated with poorer overall survival, disease-free survival, and pelvic/para-aortic lymph node recurrence. Further analyses of cervical cancer data from The Cancer Genome Atlas (TCGA) identified that this down-regulation was associated with a genomic locus loss (hsa-mir-218-1:4p15.31, hsa-mir-218-2:5q34, n=105). The objective of the current study was to elucidate the cellular and molecular functions of miR-218. MiR-218 transfection into cervical cancer cells (SiHa and ME-180) significantly reduced cell migration (by 66% and 89%, respectively), invasion (by 49% and 67%, respectively), and clonogenic capacity (by 42% and 53%, respectively), relative to control-transfected cells (P<0.05). In order to identify clinically relevant miR-218 target genes, we used an integrated trimodal approach, incorporating DNA microarray (Affymetrix Human Genome U133 Plus 2.0) analyses of 79 clinical samples, miR-218 transfection, and miRDB target prediction. The most significant target was survivin (BIRC5); miR-218 transfection confirmed a reduction in survivin mRNA and protein expression in both SiHa and ME-180 cells. Furthermore, a direct interaction between the survivin-3′UTR and miR-218 was validated using a luciferase reporter assay. siRNA knockdown of survivin in SiHa and ME-180 significantly reduced cell migration (by 76% and 83%, respectively), invasion (by 79% and 88%, respectively), and clonogenic capacity (by 98% and 97%, respectively), relative to control cells (P<0.05). YM155, a small-molecule survivin suppressant, effectively reduced survivin mRNA and protein levels in a concentration- and time-dependent manner. This compound correspondingly decreased cervical cancer cell proliferation and clonogenic survival. Our results suggest that the miR-218-survivin axis plays an important role in cervical cancer progression. Citation Format: Ryunosuke Kogo, Christine How, Jeff Bruce, Willa Shi, Kenneth W. Yip, Laurie Ailles, Fei-Fei Liu. The microRNA-218-survivin axis regulates cervical cancer cell migration and invasion. [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 4369. doi:10.1158/1538-7445.AM2014-4369
Insulin resistance and hepatotoxicity induced in high fructose fed rats may involve fructose derived endogenous toxins formed by inflammation. Thus fructose was seventy-fold more toxic if hepatocytes were exposed to non-toxic levels of hydrogen peroxide (H2O2) released by inflammatory cells. This was prevented by iron (Fe) chelators, hydroxyl radical scavengers, and increased by Fe, copper (Cu) or catalase inhibition. Fructose or glyceraldehyde/dihydroxyacetone metabolites were oxidized by Fenton radicals to glyoxal. Glyoxal (15μM) cytotoxicity was increased about 200-fold by H2O2. Glycolaldehyde was enzymically formed from glyceraldehyde, the fructokinase/aldolase B product of fructose. Glycolaldehyde cytotoxicity was increased 20-fold by H2O2. The oxidative stress cytotoxicity induced was attributed to the Fenton oxidation of glycolaldehyde forming glycolaldehyde radicals and glyoxal, since cytotoxicity was prevented by aminoguanidine (glyoxal trap) or Fenton inhibitors. Glyoxal was also the Fenton product responsible for glycolaldehyde protein carbonylation as carbonylation was prevented by aminoguanidine or Fenton inhibitors.
This chapter contains sections titled: Introduction Recent Increased Consumption of Fructose Health Concerns Associated with High Chronic Consumption of Fructose Sugars as a Source of Endogenous Reactive Carbonyl Formation and AGEs Rat Hepatocyte Studies on Endogenous Toxins Formed by Fructose Metabolism and/or Oxidation Cancer Risk and Genotoxicity of Fructose or Carbonyl Metabolites Disease Prevention by Fruits and Vegetables versus Fructose Concern Conclusions References
There is increasing evidence supporting the role of members of the polycomb group (PcG) gene family in tumor development and progression. However, their precise role in tumorigenesis and mechanisms of their regulation remain to be elucidated. Using nasopharyngeal carcinoma (NPC) as a disease model, a comprehensive analysis was undertaken on the clinical significance of EZH2 expression, identification of the cellular processes regulated by EZH2, and the mechanisms of its deregulated expression. Herein, we report EZH2 as being associated with a higher risk of relapse in NPC patients (P=0.002). Genome-wide microarray and bioinformatics identified several vital cellular processes (such as differentiation, development, and apoptosis) to be regulated by EZH2, corroborated by in vitro lethality, and delayed tumor formation in vivo upon EZH2 depletion. The combination of global microRNA (miR) profiling in primary NPC specimens, and in silico analyses provided several candidate miRs that could regulate EZH2. Using a luciferase-based assay, miR-26a, miR-101, and miR-98 were validated as bona fide regulators of EZH2 expression. In particular, miR-98 was underexpressed in relapsed patient samples, strongly suggesting an important role for the miR-98 and EZH2 axis in NPC biology.
20021 Background: There are conflicting reports on the relationship between tumour blood flow and patient survival. Poor flow is associated with tumour hypoxia and has been associated with a poor outcome (Lehtio et al 2004;Int J Radiat Oncol Biol Phys:59, 971–982). However, good flow is associated with angiogenesis which drives tumorigenesis, and has also been linked with a poor prognosis (Hermans 200 Int J Radiat Oncol Biol Phys:57, 1351–6). In this study, we examined the relationship between PET measurements of blood flow in liver metastases, and survival, in patients with colorectal cancer. Methods: Thirty seven patients with metastatic colorectal cancer, with a range of previous treatments, were studied. Regional perfusion was measured in liver metastases using H 2 [ 15 O] or C[ 15 O] 2 PET. Blood flow was correlated with overall survival from the time of PET scan. Relationship between outcome and flow was analysed separately for patients with 1 or >1 metastatic sites; a recent meta-analysis of patient with metastatic colorectal cancer showed number of metastatic sites to be an independent prognostic factor (Kohne et al 2002; Ann Oncol. 13, 308–317). Results: There was no significant relationship between survival and metastasis blood flow when the data were taken as a whole. However, when the data were corrected for number of metastatic sites, there was a trend for high flow to be associated with shorter survival (p = 0.063). Specifically, for patients with multiple metastatic sites, median survival times for patients with high flow (n = 9) was 171 days [95% CI 25–317] compared with 441 days [95% CI 12–870] for patients with low flow (n = 7) (p = 0.026). Conclusions: In patients with multiple sites of metastasis, high tumour blood flow is a poor prognostic factor. This may be due to angiogenesis, which promotes tumorigenesis. No significant financial relationships to disclose.
MicroRNAs (miRs) are involved in the regulation of many processes that contribute to malignancy, including cell proliferation, radiation resistance, invasion and metastasis. The role of miR-330-3p, an miR upregulated in breast cancer, remains unclear.We examine the association of miR-330-3p with distant relapse-free survival in the Oxford cohort of breast cancer patients. We also study miR-330-3p function using in vitro invasion and ex ovo metastasis assays. Using in vitro luciferase assays, we validate a novel target gene for miR-330-3p, Collagen And Calcium Binding EGF Domains 1 (CCBE1). We assess functional consequences of CCBE1 loss by using siRNA-mediated knockdown followed by in vitro invasion assays. Lastly, we examine the expression profile of CCBE1 in breast carcinomas in the Curtis and TCGA Breast Cancer data sets using Oncomine Platform as well as distant relapse-free and overall survival of patients in the Helsinki University breast cancer data set according to CCBE1 expression status.miR-330-3p is enriched in breast cancer, and higher levels of miR-330-3p expression are associated with lower distant relapse-free survival in a cohort of breast cancer patients. Consistent with these observations, overexpression of miR-330-3p in breast cancer cell lines results in greater invasiveness in vitro, and miR-330-3p-overexpressing cells also metastasise more aggressively ex ovo. We identify CCBE1 as a direct target of miR-330-3p, and show that knockdown of CCBE1 results in a greater invasive capacity. Accordingly, in breast cancer patients CCBE1 is frequently downregulated, and its loss is associated with reduced distant relapse-free and overall survival.We show for the first time that miR-330-3p targets CCBE1 to promote invasion and metastasis. miR-330-3p and CCBE1 may represent promising biomarkers in breast cancer.
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