Horizontal transfer of miR-383 sensitise cells to cisplatin by targeting VEGFA-Akt signalling loop.

BACKGROUND: Cellular resistance to cisplatin has been one of the major obstacles in the success of combination therapy for many types of cancers. Emerging evidences suggest that exosomes released by drug resistant tumour cells play significant role in conferring resistance to drug sensitive cells by means of horizontal transfer of genetic materials such as miRNAs. Though exosomal miRNAs have been reported to confer drug resistance, the exact underlying mechanisms are still unclear. METHODS AND RESULTS: In the present study, mature miRNAs secreted differentially by cisplatin resistant and cisplatin sensitive HepG2 cells were profiled and the effect of most significantly lowered miRNA in conferring cisplatin resistance when horizontally transferred, was analysed. we report miR-383 to be present at the lowest levels among the differentially abundant miRNAs expressed in exosomes secreted by cisplatin resistant cells compared to that that of cisplatin sensitive cells. We therefore, checked the effect of ectopic expression of miR-383 in altering cisplatin sensitivity of Hela cells. Drug sensitivity assay and apoptotic assays revealed that miR-383 could sensitise cells to cisplatin by targeting VEGF and its downstream Akt mediated pathway. CONCLUSION: Results presented here provide evidence for the important role of miR-383 in regulating cisplatin sensitivity by modulating VEGF signalling loop upon horizontal transfer across different cell types.

Integrin ß4 induced epithelial-to-mesenchymal transition involves miR-383 mediated regulation of GATA6 levels.

BACKGROUND: The process of transdifferentiating epithelial cells to mesenchymal-like cells (EMT) involves cells gradually taking on an invasive and migratory phenotype. Many cell adhesion molecules are crucial for the management of EMT, integrin ß4 (ITGB4) being one among them. Although signaling downstream of ITGB4 has been reported to cause changes in the expression of several miRNAs, little is known about the role of such miRNAs in the process of EMT. METHODS AND RESULTS: The cytoplasmic domain of ITGB4 (ITGB4CD) was ectopically expressed in HeLa cells to induce ITGB4 signaling, and expression analysis of mesenchymal markers indicated the induction of EMT. ß-catenin and AKT signaling pathways were found to be activated downstream of ITGB4 signaling, as evidenced by the TOPFlash assay and the levels of phosphorylated AKT, respectively. Based on in silico and qRT-PCR analysis, miR-383 was selected for functional validation studies. miR-383 and Sponge were ectopically expressed in HeLa, thereafter, western blot and qRT-PCR analysis revealed that miR-383 regulates GATA binding protein 6 (GATA6) post-transcriptionally. The ectopic expression of shRNA targeting GATA6 caused the reversal of EMT and ß catenin activation downstream of ITGB4 signaling. Cell migration assays revealed significantly high cell migration upon ectopic expression ITGB4CD, which was reversed upon ectopic co-expression of miR-383 or GATA6 shRNA. Besides, ITGB4CD promoted EMT in in ovo xenograft model, which was reversed by ectopic expression of miR-383 or GATA6 shRNA. CONCLUSION: The induction of EMT downstream of ITGB4 involves a signaling axis encompassing AKT/miR-383/GATA6/ß-catenin.

Horizontal Transfer of miR-643 from Cisplatin-Resistant Cells Confers Chemoresistance to Recipient Drug-Sensitive Cells by Targeting APOL6.

Acquisition of resistance to cisplatin is a major impediment to the success of cisplatin-based combination therapies for cancer. Recent studies indicate that exosomal miRNAs derived from drug-resistant tumour cells can confer resistance properties to recipient cells by a horizontal transfer mechanism. Although the role of horizontal transfer of a few miRNAs has been described, little is known about the concerted action of horizontal transfer of miRNAs in conferring cisplatin resistance. The present study was designed to identify the role of miR-643, which is one of the most significantly increased miRNA in exosomes released from cisplatin-resistant Heptocarcinoma cells, in altering the cisplatin resistance properties of recipient cells. Drug-sensitivity assays involving miR-643 revealed that ectopic expression of miR-643 can desensitise the cells towards cisplatin. Furthermore, we identified APOL6 as a major target of miR-643. Further mechanistic studies showed that miR-643 can modulate APOL6 mRNA and protein levels, leading to a reversal of APOL6-mediated apoptosis. Altogether, our results suggest an APOL6-dependent mechanism for miR-643 mediated cisplatin resistance upon the horizontal transfer across cell types.

Role of Sirtuins in Tumor Angiogenesis.

Generally, changes in the metabolic status of cells under conditions like hypoxia and accumulation of lactate can be sensed by various sensing mechanisms, leading to modulation of a number of signal transduction pathways and transcription factors. Several of the proangiogenic cytokines like VEGF, FGF, PDGF, TGF-ß, Ang-2, ILs, etc. are secreted by cancer cells, under hypoxic microenvironment. These cytokines bind to their receptors on the endothelial cells and activates a number of signaling pathways including Akt/PIP3, Src, p38/MAPK, Smad2/3, etc., which ultimately results in the proliferation and migration of endothelial cells. Transcription factors that are activated in response to the metabolic status of tumors include HIFs, NF-κb, p53, El-2, and FOXO. Many of these transcription factors has been reported to be regulated by a class of histone deacetylase called sirtuins. Sirtuins are NAD+ dependent histone deacetylases that play pivotal role in the regulation of tumor cell metabolism, proliferation, migration and angiogenesis. The major function of sirtuins include, deacetylation of histones as well as some non-histone proteins like NF-κB, FOXOs, PPAR⋎, PGC1-α, enzymes like acetyl coenzymeA and structural proteins like α tubulin. In the cell, sirtuins are generally considered as the redox sensors and their activities are dependent on the metabolic status of the cell. Understanding the intricate regulatory mechanisms adopted by sirtuins, is crucial in devising effective therapeutic strategies against angiogenesis, metastasis and tumor progression. Keeping this in mind, the present review focuses on the role of sirtuins in the process of tumor angiogenesis and the regulatory mechanisms employed by them.

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.

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.

Horizontal transfer of miR-106a/b from cisplatin resistant hepatocarcinoma cells can alter the sensitivity of cervical cancer cells to cisplatin.

Recent studies indicate that horizontal transfer of genetic material can act as a communication tool between heterogenous populations of tumour cells, thus altering the chemosensitivity of tumour cells. The present study was designed to check whether the horizontal transfer of miRNAs released by cisplatin resistant (Cp-r) Hepatocarcinoma cells can alter the sensitivity of cervical cancer cells. For this exosomes secreted by cisplatin resistant and cisplatin sensitive HepG2 cells (EXres and EXsen) were isolated and characterised. Cytotoxicity analysis showed that EXres can make Hela cells resistant to cisplatin. Analysis of miR-106a/b levels in EXres and EXsen showed that their levels vary. Mechanistic studies showed that miR-106a/b play an important role in EXsen and EXres mediated change in chemosensitivity of Hela cells to cisplatin. Further SIRT1 was identified as a major target of miR-106a/b using in silico tools and this was proved by experimentation. Also the effect of miR-106a/b in chemosensitivity was seen to be dependent on regulation of SIRT1 by miR-106a/b. In brief, this study brings into light, the SIRT1 dependent mechanism of miR-106a/b mediated regulation of chemosensitivity upon the horizontal transfer from one cell type to another.