Cellular mechanisms of acquired drug resistance against regorafenib in colon cancer cells.

Colorectal cancer is the third most common cancer across the world. Acquired resistance to therapeutics is one of the major challenges in cancer cure. With the development of resistance to organometallic drugs there was switch for the usage of inhibitors of kinases which play a crucial role in cellular activities. Regorafenib is a multi-kinase inhibitor used as an oral anti-cancer drug for treating advanced colorectal cancer (CRC). With increasing reports of acquired resistance to regorafenib in long-term use it is inevitable to understand the mechanisms underlying in the development of resistance. To understand the molecular mechanism of acquired drug resistance towards regorafenib we have developed a regorafenib resistant HCT116 cell line (Reg-R-HCT116) and an integrated quantitative proteomic and phospho-proteomics approach is used to elucidate the molecular signaling mechanisms that help in drug tolerance. Proteome and Phosphoproteome analysis revealed an extensive remodelling of signaling pathways associated with metabolism, protein synthesis and stress adaptations. This also revealed a large set of phosphorylated proteins as well as proteins that might be associated with aberrant activation or differential alteration of PI3K-AKT-mTOR, EIF2, HIF-1, Apoptosis inhibition, Glucose metabolism, amino acid metabolism and DNA-repair-associated signaling. Differential phosphorylation of downstream molecules of the mentioned pathways NDRG1, ACINUS and RICTOR and enhanced cell survival further confirmed their role in drug tolerance. Targeted inhibition of both the mTOR complexes using Torin1 resensitized the cells to regorafenib. Combination treatment of regorafenib with torin1 showed a synergistic cytotoxicity and attenuated the expression of key survival proteins. These findings provide mechanistic insight into acquired resistance to regorafenib in colorectal cancer and identified mTOR/eIF2 signaling as one of the critical drivers of resistance phenotype. The results suggest combinatorial targeting of these pathways could be an effective strategy to overcome regorafenib resistance and improve clinical outcomes in CRC.