Ayse Elif Erson-Bensan

Matrix metalloprotease 16 expression is downregulated by microRNA-146a in spontaneously differentiating Caco-2 cells

Cellular differentiation in the gut is vital in maintaining the cellular and functional specialization of the epithelial layer. MicroRNAs (miRNAs) have recently emerged as one of the key players in orchestrating the differentiation process in the gut. Using the spontaneously differentiating Caco‐2 cell line, we observed an increased expression of miR‐146a but not miR‐146b in the course of differentiation. Bioinformatic analyses revealed that the membrane type matrix metalloprotease 16 (MMP16, MT3‐MMP) was a predicted target of miR‐146a and a decrease in the mRNA and protein expression of MMP16 was observed in the course of differentiation. Transfection of a luciferase reporter vector containing the 3′UTR of MMP16 showed decreased luciferase activity due to miR‐146a expression. With forced expression of miR‐146a in undifferentiated Caco‐2 cells, a decrease in the mRNA and protein levels of MMP16 and a lower gelatinase activity in a gelatin zymogram were observed. Additionally, forced expression of miR‐146a in HT‐29 colon cancer cells also resulted in decreased expression of MMP16, along with a decrease in the invasion through Matrigel. Taken together, we have shown here that MMP16 is regulated by miR‐146a in spontaneously differentiated Caco‐2 cells. As MMP16 activates the zymogen of MMP2, which is known to degrade extracellular matrix proteins, the regulation of MMP16 by miR‐146a may account, at least in part, for lower motility of well‐differentiated cells.

Alternative Polyadenylation: Another Foe in Cancer.

Advancements in sequencing and transcriptome analysis methods have led to seminal discoveries that have begun to unravel the complexity of cancer. These studies are paving the way toward the development of improved diagnostics, prognostic predictions, and targeted treatment options. However, it is clear that pieces of the cancer puzzle are still missing. In an effort to have a more comprehensive understanding of the development and progression of cancer, we have come to appreciate the value of the noncoding regions of our genomes, partly due to the discovery of miRNAs and their significance in gene regulation. Interestingly, the miRNA–mRNA interactions are not solely dependent on variations in miRNA levels. Instead, the majority of genes harbor multiple polyadenylation signals on their 3′ UTRs (untranslated regions) that can be differentially selected on the basis of the physiologic state of cells, resulting in alternative 3′ UTR isoforms. Deregulation of alternative polyadenylation (APA) has increasing interest in cancer research, because APA generates mRNA 3′ UTR isoforms with potentially different stabilities, subcellular localizations, translation efficiencies, and functions. This review focuses on the link between APA and cancer and discusses the mechanisms as well as the tools available for investigating APA events in cancer. Overall, detection of deregulated APA-generated isoforms in cancer may implicate some proto-oncogene activation cases of unknown causes and may help the discovery of novel cases; thus, contributing to a better understanding of molecular mechanisms of cancer. Mol Cancer Res; 14(6); 507–17. ©2016 AACR.

m6A Modification and Implications for microRNAs.

OBJECTIVE RNA is chemically modified with over 100 distinct reactions. Among these reactions, methylation is probably the most extensively studied modification on the RNA molecule. Studies suggest methylation of Adenine residues (m6A) to be widespread in the transcriptome with potentially important roles in biological processes. METHOD Here, we review recent literature on m6A modification and potential implications for microRNA (miRNA) mediated gene expression regulation. These implications involve miRNA biogenesis, mRNA-miRNA interactions and m6A target selection. CONCLUSION Understanding the extent and functions of m6A is likely to improve our understanding of the complexities of the transcriptome regulation in normal and in disease states.

ARID3B expression in primary breast cancers and breast cancer-derived cell lines

BackgroundARID3B (AT-rich interaction domain 3) is a member of the family of ARID proteins, which constitutes evolutionarily conserved transcription factors implicated in normal development, differentiation, cell cycle regulation and chromatin remodeling. In addition, ARID3B has been linked to cellular immortalization, epithelial-mesenchymal transition (EMT) and tumorigenesis. Given the emerging role of ARID3B in tumor development, we examined its expression in primary patient-derived breast cancer samples and breast cancer-derived cell lines.MethodsImmunohistochemistry (IHC) was used to detect ARID3B expression in 63 formalin-fixed paraffin-embedded (FFPE) invasive breast cancer samples. In addition, a panel of 6 (estrogen receptor-positive and -negative, ERBB2-positive and -negative) breast cancer-derived cell lines and immortalized non-tumorigenic epithelial breast cells were used for ARID3B expression analysis using RT-PCR. Specific primers and Western blotting were used to detect ARID3B isoforms.ResultsUsing IHC, nuclear, cytoplasmic and low levels of membranous ARID3B staining were detected in all 63 primary invasive breast tumors. Nuclear ARID3B staining positively correlated with estrogen receptor (ER) status and negatively correlated with tumor grade, mitotic index and ERBB2 status of the patients. Increased nuclear expression of ARID3B was confirmed in breast cancer-derived cell lines expressing ERα. In addition, two out of three ERBB2-positive breast cancer cell lines were found to lack full length ARID3B. Three ARID3B isoforms were found to be present in normal breast epithelial cells as well as in breast cancer cells.ConclusionWe report a positive correlation between ER positivity and nuclear ARID3B expression in primary breast cancers, along with a negative correlation with the ERBB2 status. Very similar correlations were noted in breast cancer-derived cell lines. Since in the recent past ARID3B expression has increasingly been related to cancer-associated proteins and microRNAs, knowledge on ARID3B expression and function may be instrumental for gaining further insight into potentially important cancer-related networks.

Alternative Polyadenylation Patterns for Novel Gene Discovery and Classification in Cancer

Certain aspects of diagnosis, prognosis, and treatment of cancer patients are still important challenges to be addressed. Therefore, we propose a pipeline to uncover patterns of alternative polyadenylation (APA), a hidden complexity in cancer transcriptomes, to further accelerate efforts to discover novel cancer genes and pathways. Here, we analyzed expression data for 1045 cancer patients and found a significant shift in usage of poly(A) signals in common tumor types (breast, colon, lung, prostate, gastric, and ovarian) compared to normal tissues. Using machine-learning techniques, we further defined specific subsets of APA events to efficiently classify cancer types. Furthermore, APA patterns were associated with altered protein levels in patients, revealed by antibody-based profiling data, suggesting functional significance. Overall, our study offers a computational approach for use of APA in novel gene discovery and classification in common tumor types, with important implications in basic research, biomarker discovery, and precision medicine approaches.

Abstract 3374: APA isoform diversity in triple negative breast cancers

Alternative polyadenylation (APA) plays a role in gene expression regulation generally by shortening of 39UTRs and relieving microRNA-mediated repression. Therefore, APA is gaining increased attention as a potential mechanism to activate oncogenes. Owing to high proliferative indices of triple negative breast cancers (TNBCs), we hypothesized APA to cause 39UTR length changes in this aggressive subgroup of breast cancers. Our probe-based meta-analysis approach identified 39UTR length alterations where the significant majority was shortening events (70%, 113 of 165) of mostly proliferation-related transcripts in over 500 TNBC patients compared with normal breast tissue. Representative shortening events correlated with increased protein levels and relapse free survival of patients, suggesting functional significance of isoform variability. To begin addressing the underlying mechanisms of 3’UTR shortening, we turned to APA machinery proteins. We detected variable expression of APA machinery proteins in different breast cancer subtypes but CSTF2 (cleavage stimulation factor 2) has the most prominent overexpression in breast cancer cells. Therefore, among potential regulators of 3’UTR shortening, we further investigated the role of CSTF2 in proximal polyA signal selection. Because some of the TNBC patients are EGFR positive, we found EGF treatment to cause increased CSTF2 levels. Higher CSTF2 levels indeed correlated with further shortening of the 39UTRs. Accordingly, RNAi-induced silencing of CSTF2 decreased the proliferative rate of cancer cells. Therefore, our integrated approach revealed a pattern of 39UTR length changes in TNBC patients and a potential link between APA and EGF signaling. Further studies are underway to investigate the mechanistic link between EGF signaling and regulation of 3’UTR lengths. (This work is funded by TUBITAK 112S478 and 114Z884) Citation Format: Ayse Elif Erson-Bensan, Begum H. Akman, Merve Oyken, Hizlan H. Agus, Esra Yavuz, Murat Erdem, Tolga Can. APA isoform diversity in triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3374. doi:10.1158/1538-7445.AM2017-3374

MIR106B (microRNA 106b)

Review on MIR106B (microRNA 106b), with data on DNA, on the protein encoded, and where the gene is implicated.

CHFR (checkpoint with fork-head associated and ring finger)

Review on CHFR (checkpoint with fork-head associated and ring finger), with data on DNA, on the protein encoded, and where the gene is implicated.