MP46-10 UTILIZING INSULIN IN THE TREATMENT OF PROSTATE CANCER WITH BKM120 ABROGATES THE THERAPEUTIC EFFECT OF PI3K PATHWAY INHIBITION

Abstract

INTRODUCTION AND OBJECTIVES: The anti-tumor role of metformin has been widely reported, however, the molecular mechanism of this biguanide agent in the inhibition of tumor progression remains unclear. Increasing evidence supported that biguanides impede nucleotide synthesis but how metformin regulates microRNA (miRNA) expression profiles has not been thoroughly investigated in prostate cancer. METHODS: We performed serial experiments to address these questions: firstly, through a microRNA array screening, we identified the miRNA expression profile of metformin treated prostate cancer cells. Secondly, we determined the target of metformin regulated miR-708-5p by luciferase reporter assay, realtime PCR and western blots. Thirdly, cells were trasfected with miR-708-5p or negative control. We measured the intracellular calcium levels using a colormetric assay. Transmission electron microscopy and flow cytometry were applied to determine ER stress dependent pro-apoptosis in prostate cancer cells. RESULTS: Here, we identified miR-708-5p as a novel target of metformin in prostate cancer cells. Metformin promotes increased expression of miR-708-5p, leading to suppression of endoplasmic reticulum (ER) membrane protein neuronatin (NNAT) expression and subsequently induces apoptosis of prostate cancer cells through the ER stress pathway. Further,miR-708-5p induces knockdownofNNAT is associated with down-regulated intracellular calcium levelsand inducedmalformation of ER-ribosome structure revealed by electronic microscopy. Meanwhile, the unfolded-protein response (UPR) proteins CHOP, p-eIF2a, Calreticulin, GRP78 and ATP2A1, all of which are also considered as ER stress markers, are up-regulated by metformin and miR-708-5p. CONCLUSIONS: Together, our findings clearly demonstrate that metformin stimulates increased expression of miR-708-5p to target the NNAT mediated response to ER stress and apoptosis. This novel regulatory mechanism of metformin in prostate cancer cells not only advances our knowledge on the molecular mechanism of metformin but also provides a promising therapeutic strategy by targeting miR-708-5p and NNAT for prostate cancer treatment.