Effect of mTOR inhibitors on sorafenib-induced endoplasmic reticulum stress and apoptosis in melanoma cells.

Abstract

e19027 Background: In melanoma, the RAF-MEK-ERK (MAPK) and the PI3K-AKT-mTOR (AKT) signaling pathways are constitutively activated and have key functions in tumor progression. In our previous studies, combination of the nonselective RAF inhibitor sorafenib with the mTOR inhibitor sirolimus significantly potentiated growth inhibition and led to an approximately 2-fold increase of apoptosis of melanoma cells in monolayer culture compared with sorafenib monotreatment (p<0.05). Moreover, sorafenib in combination with sirolimus completely suppressed invasive melanoma growth in organotypic culture. Methods: To investigate the molecular mechanisms involved in the antitumoral action of this drug combination we analyzed the gene expression profile of melanoma cells in response to sorafenib or/and sirolimus. Results: Data analysis showed a series of genes (p8, ATF4/3, CHOP, TRB3) that were upregulated after treatment with sorafenib and sirolimus and have been proposed to be responsible for the execution of endoplasmic reticulum (ER) stress-induced apoptosis. Using real-time quantitative PCR, we confirmed that sorafenib and sirolimus upregulate p8, ATF4/3, CHOP and TRB3 mRNA levels in melanoma cells. Likewise, western blot analysis showed that sorafenib and sirolimus increase p8 and CHOP protein levels. Furthermore, the farnesyl transferase inhibitor lonafarnib inhibiting mTOR signaling also augmented sorafenib-induced apoptosis and upregulation of the ER stress-related transcription factor p8. Of note, growth inhibitory effects and upregulation of ER stress-related genes by sorafenib and sirolimus or lonafarnib did not depend on BRAF or NRAS mutation status. By contrast, the BRAFV600E kinase inhibitor PLX4032 potently inhibited growth and upregulated the ER stress-related transcription factor p8 preferentially in BRAFV600E melanoma cells. Conclusions: These data suggest that mTOR inhibitors enhance sorafenib-induced endoplasmic reticulum stress and apoptosis in melanoma cells. No significant financial relationships to disclose.