Abstract A57: Chondroitin sulfate proteoglycan 4(CSPG4) Mediates Melanoma Cell Survival During PI3K and mTOR Inhibition

Abstract

Abstract Background: Early development of resistance to therapy remains a major cause of cutaneous malignant melanoma treatment failure. Chondroitin sulfate proteoglycan 4 (CSPG4) is robustly expressed in a majority of metastatic melanomas. Previous work has shown CSPG4 to promote MAPK, PI3K and FAK activation, suggesting that CSPG4 represents a potential avenue for oncogenic growth and cell survival in response to chemotherapy. The PI3K-AKT-mTOR pathway regulates the cell growth and motility in the presence of nutrients. The inhibition of AKT and mTOR independently was shown to reduce the melanoma cell survival and enhanced the sensitivity of these cells to cisplatin. PF-05212384 (PKI-587) is a dual PI3K/mTOR inhibitor in clinical trials. We hereby report that the expression of CSPG4 on the melanoma cells caused them to become less responsive to the therapy with a dual PF-05212384. Material and methods: Melanoma cell lines that stably expressed CSPG4 were selected for this study. The expression of CSPG4 was knocked down by using lentivirus-transduced shRNA. Cell survival assays were performed to compare the inhibitory effects of the drug in CSPG4 expressing and non-expressing cell line. Wound healing migration assays were performed to examine the cell motility and migration. The activation of PI3K-mTOR pathway was analyzed by phosphorylation of downstream effectors for their activation using Western blotting. Results: Melanoma cells expressing active CSPG4 showed less sensitivity to the inhibitor. The average cell viability after treatment with maximum dose was 47.94% and 35.76% for CSPG4 positive 1205LU and WM1341D melanoma cell lines respectively, while the CSPG4 negative WM1552C showed an average cell viability of 19.07%. The difference in cell viability was statistically significant.(p<α=0.05)for higher doses. The suppression of CSPG4 using the shRNA sensitized the CSPG4 positive cell lines to the PI3K/mTOR inhibition. (p<α=0.05). The maximum effect was seen in the WM1341D where the cell viability was reduced at maximum dose to 26.30% as compared to 50% in non-targeting shRNA transduced control. The CSPG4 positive cell lines resisted the deactivation of p70Sk, a downstream effector of mTOR and had enhanced phosphorylation of transcription repressor 4E-BP1.At higher doses the cleavage of caspase to its activated forms, which is necessary for execution of apoptosis was reduced in the CSPG4 positive cell lines. The CSPG4 positive cells showed more motility as compared to the controls. Conclusion: This data supports the hypothesis that the CSPG4 is involved in inducing resistance in melanoma cells in response to the therapeutic challenge with the dual PI3k/mTOR inhibitor. The study highlights the need of using multiple treatment strategies to treat melanoma.