The MEK inhibitors enhance the efficacy of sorafenib against hepatocellular carcinoma cells through reducing p-ERK rebound

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumours worldwide and has a poor prognosis. Sorafenib, the only targeted therapeutic agent for HCC, is a multiple kinase inhibitor with targets including RAF and VEGFR-2/3 that display a very limited ability to extend the survival of patients with advanced metastatic HCC for approximately three months. MEK inhibitors including trametinib and selumetinib have shown promising efficacy in combination with sorafenib in clinical trials. However, the mechanisms about the combined effect of these drugs remain unclear. METHODS: Two HCC cell lines (Bel7402 and SMMC7721) were used in the experiments. The protein expression of HCC cell lines was quantified via western blotting. Cell viability was examined by cell counting kit-8 and colony formation assays. Drug interactions between sorafenib and trametinib/selumetinib were determined by the combination index (CI) value. RESULTS: In this study, we found that short-term sorafenib treatment could inhibit the downstream RAF effectors phosphorylated (p)-MEK and p-ERK in Bel7402 and SMMC7721 cells, while long-term sorafenib treatment could induce a rebound of p-MEK and p-ERK expression in these two human HCC cell lines. We then tested the effect of sorafenib combined with two different FDA-approved MEK inhibitors, trametinib and selumetinib, in the two cell lines. Western blot analysis showed that trametinib/selumetinib could abolish the ERK activation caused by long-term sorafenib treatment. Cell counting kit-8 and colony formation assays indicated that the use of sorafenib or trametinib/selumetinib alone produced a minor effect on the proliferation of these HCC cell lines, while the combination therapies induced strong growth inhibition. CI assays using CompuSyn software indicated that the combined therapies could produce a synergistic effect in these two cell lines. In addition, mechanistic studies revealed that the combination therapies could synergistically reduce the expression of proliferation-related proteins, including cyclin D1 and c-Myc. CONCLUSIONS: Taken together, our study showed that the rebound of p-ERK induced by long-term sorafenib treatment might limit the benefit of sorafenib monotherapy, and the MEK inhibitors trametinib and selumetinib could enhance the efficacy of sorafenib in HCC cells.