Abstract 1196: Enhancement of anti-melanoma effect of BRAF and MEK inhibition by metformin
Authors: Zhang Yanping , Peng Guangyong , Hsueh Eddy C. 
Published: August 2015

Abstract
Introduction: Metformin has been shown to inhibit human cancer cell growth via activation of MAP kinase with resultant inhibition of mTOR signaling pathway. Recent clinical evidence demonstrated the anti-melanoma effect of combined mutant BRAF inhibition and MEK inhibition. We determined the effect of metformin in combination with mutant BRAF and MEK inhibition on melanoma proliferation and the possible mechanisms.
Methods: Human melanoma cells (SK-23, Mel888, Mel1861) were treated with Metformin (1-20 mM), PLX4032 (a mutant BRAF inhibitor, 1-20 uM), AZD6244 (a MEK inhibitor, 1-20 uM), or combination. Cell proliferation assay was performed using Cell Titer Blue assay. Western blotting was performed to determine the expression of PARP, caspases-9, LC3A/B,Beclin1, pERK, pMEK, AMPKa, p70S6, and p4E-BP1. GAPDH was used as internal control. Autophagy was further analyzed with immunofluorescence with LC-3 and NECN1 anti-antibody for visualization of lysosomal morphology and evaluated under the Nikon Eclipse Ti microscope (20×). DAPI was used to counter stain the nucleus. Apoptosis was further analyzed with flow cytometry using FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p&lt; 0.05 was considered to be statically significant.
Results: There was dose-dependent inhibition of melanoma cell proliferation with either metformin(M), PLX4032(P), and AZD6244(A). Addition of M to PA combination had synergistic effect on the anti-melanoma proliferative effect of PA. Significantly increased expression (p&lt;0.05) of apoptosis markers (PARP and caspases-9) and autophagy markers (LC3B and Beclin1) were observed in cells treated with PAM compared with PA. Enhanced induction of autophagy with the triple drugs was confirmed by visualization of autophagosome and autolysosome formation on immunofluorescence. Synergistic effect on apoptosis was confirmed on Annexin V assay. The PAM combination treatment also lead to significant enhancement of AMPK activation and increased phosphorylation of AMPKα at Thr-172 compared with metformin alone (p&lt;0.01). The addition of M to PA combination resulted in inhibition of mTOR signaling with decreased phosphorylation of p70S6K and 4E-BP1 in treated cancer cells, when compared with PA or M alone.
Conclusion: We have observed a synergistic effect on suppressing human melanoma proliferation with the addition of metformin to combined inhibition of mutant BRAF and MEK. The synergism is due to enhanced inhibition of mTOR signaling pathways.
Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Enhancement of anti-melanoma effect of BRAF and MEK inhibition by metformin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2015-1196