ER stress mediated regulation of miR23a confer Hela cells better adaptability to utilize glycolytic pathway.

Cancer cells exhibit increased dependency on aerobic glycolysis, a phenomenon referred as the "Warburg effect" and therefore, blocking glycolysis by using non-metabolizable analogues of glucose, like 2-Deoxy glucose (2-DG), has been proposed to be of huge therapeutic importance. One of the major drawbacks of using 2-DG as a chemotherapeutic agent is that it can induce ER stress. ER stress is a hall mark in many solid tumors and the unfolded protein response (UPR) associated with it initiates many survival mechanisms in cancer cells. In the present study, we report a novel survival mechanism associated with ER stress, by which the cancer cells become more adapted to aerobic glycolysis. When ER stress was induced in Hela cells by treating them with 2-DG or Thapsigargin (TG) the expression and activity of LDH was significantly up regulated, conferring the cells a greater glycolytic potential. A simultaneous decrease was observed in the expression of miR-23a, which was predicted in silico to have target site on the 3'UTR of LDH A and B mRNAs. miRNA over expression studies and mRNA degradation assays suggest that miR-23a could target LDH A and LDH B mRNAs. Further on the basis of our results and previous scientific reports, we propose that "c-Myc," which is over expressed during ER stress, repress the expression of miR-23a, which in turn regulates the expression of its target genes viz., LDH A and LDH B, thereby making the cells more competent to survive in tumor microenvironment, which requires efficient use of aerobic glycolysis.

MicroRNA106a regulates matrix metalloprotease 9 in a sirtuin-1 dependent mechanism.

Cellular migration is important during many physiological as well as pathological conditions and is regulated very tightly by an intricate network of signaling and effector molecules. One of the important players during cellular migration are matrix metalloproteases and their levels have been reported to be important in determining the cellular migratory properties during metastasis. MMPs and regulators of MMPs therefore, present themselves as potent candidates for manipulation, to control conditions where they get dysregulated. Micro RNAs are a group of micro regulators that can modulate expression of a gene through transcriptional and post transcriptional regulations. Owing to the fact that many microRNAs have already been reported to regulate MMPs and that miR106a, a member of oncomir17 family has been implicated in metastatic conditions, the present study intended to analyze if miR106a can regulate levels of MMP9, an important inducible matrix metalloproteinase. The results of the in vitro experiments demonstrated that under conditions of migration cells showed elevated levels of miR106a, which could regulate the expression of major MMP9 regulator, SIRT-1. Decreased levels of SIRT1thus resulted in an increase in the expression and activity of MMP9. Over expression and mRNA stability studies carried out also suggested regulatory role of miR106a. The overall results thus suggested that the levels of miR106a gets modulated during cellular migration, causing a change in the levels of SIRT-1 mRNA by affecting its stability and the levels of SIRT-1 in turn can regulate the levels of MMP9.

Horizontal transfer of miR-23a from hypoxic tumor cell colonies can induce angiogenesis.

Neo vessel formation by angiogenesis is an important event during many pathological conditions including cancer, where it is indispensable for tumor growth and survival. Although, various pro-angiogenic cytokines and soluble factors, secreted by tumor cells, have been reported to promote angiogenesis, recent studies have shown regulatory role of exosomes, secreted by tumor cells in the process of angiogenesis. These exosomes are capable of carrying nucleic acids, proteins, etc., as their cargo. Under the light of these facts and considering the presence of miRNAs, the non-coding RNAs capable of regulating target gene expression, as one of the major cargos in the exosomes, we investigated, whether exosomes derived from normoxic and hypoxic tumor cell colonies exhibit difference in levels of miR-23∼27∼24 cluster members and if so, to check the significance of their horizontal transfer on the process of angiogenesis. Results of our study showed that exosomes secreted by hypoxic tumor cell colonies possess significantly higher levels of miR23a and can induce angiogenesis. Further, we have shown that exosomes secreted by cells that ectopically over express miR23a is capable of inducing angiogenesis in different angiogenic model systems such as CAM, in ovo Xenograft and HUVEC models systems. Further, mechanistic analysis revealed that miR23a driven regulation of angiogenesis is brought about by down regulation of SIRT1 in the recipient cells. Collectively, the results presented here suggest that exosomal transfer of miR23a from tumor cell colonies can induce the process of angiogenesis by targeting SIRT1 in the recipient endothelial cells.

2-Deoxy Glucose Modulates Expression and Biological Activity of VEGF in a SIRT-1 Dependent Mechanism.

Reprogramming of energy metabolism particularly switching over of cells to aerobic glycolysis leading to accumulation of lactate is a hallmark of cancer. Lactate can induce angiogenesis, an important process underlying tumor growth and metastasis. VEGF is one of the most important cytokines which regulate this process and the present study was designed to examine if blocking glycolytic pathway in tumor cells can affect its angiogenic potency with respect to VEGF. For this, the expression and biological activity of VEGF synthesized and secreted by tumor derived cell lines in the presence or absence of 2-deoxy glucose (2-DG), an inhibitor of glycolysis was determined. The results suggested that inhibition of glycolysis using sub-lethal doses of 2-DG down-regulated the expression of VEGF and also significantly reduced its biological activity. Further mechanistic studies revealed that the down regulation of VEGF gene expression by 2-DG was due to an increase in SIRT-1 activity and the reduced biological activity was found to be due to an increase in the PAR modification of VEGF. Activity of SIRT-1 and PAR modification of VEGF in turn, was found to be correlated to the cellular NAD+ levels. The results presented here therefore suggest that treatment of cancer cells with 2-DG can significantly reduce its overall angiogenic potency through transcriptional and post-translational mechanisms. J. Cell. Biochem. 118: 252-262, 2017. © 2016 Wiley Periodicals, Inc.