Siddharth Manvati

Despite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood. In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins. Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial–mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed. Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.

Background: Insulin is tightly associated with cancer progression; however, mechanistic insights into such observations are poorly understood. Recent studies show that metabolic transformation is critical to cancer cell proliferation. Here, we attempt to understand the role of insulin in promotion of cancer metabolism. To this end, the role of insulin in regulating glycolytic enzyme pyruvate kinase M2 (PKM2) was examined. Results: We observed that insulin up-regulated PKM2 expression, through PI3K/mTOR mediated HIF1α induction, but significantly reduced PKM2 activity independent of this pathway. Drop in PKM2 activity was attributed to subunit dissociation leading to formation of low activity PKM2 oligomers, as assessed by density gradient centrifugation. However, tyrosine 105 phosphorylation of PKM2, known for inhibiting PKM2 activity, remained unaffected on insulin treatment. Interestingly, insulin-induced ROS was found responsible for PKM2 activity reduction. The observed changes in PKM2 status led to augmented cancer metabolism. Insulin-induced PKM2 up-regulation resulted in enhanced aerobic glycolysis as confirmed by PKM2 knockdown studies. Further, PKM2 activity reduction led to characteristic pooling of glycolytic intermediates and increased accumulation of NADPH; suggesting diversion of glucose flux towards macromolecular synthesis, necessary for cancer cell growth. Conclusion: The study identifies new PKM2-mediated effects of insulin on cancer metabolism, thus, advancing the understanding of insulin’s role in cancer.

New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored.