The multikinase inhibitor sorafenib is currently the treatment of reference for advanced hepatocellular carcinoma (HCC). In our report, we examined the cytotoxic effects of sorafenib on HCC cells. We report that the depletion of the intracellular iron stores achieved by using the iron chelator deferoxamine (DFX) strikingly protects HCC cells from the cytotoxic effects of sorafenib. The protective effect of the depletion of intracellular iron stores could not be explained by an interference with conventional forms of programmed cell death, such as apoptosis or autophagic cell death. We also found that DFX did not prevent sorafenib from reaching its intracellular target kinases. Instead, the depletion of intracellular iron stores prevented sorafenib from inducing oxidative stress in HCC cells. We examined the possibility that sorafenib might exert a cytotoxic effect that resembles ferroptosis, a form of cell death in which iron-dependent oxidative mechanisms play a pivotal role. In agreement with this possibility, we found that pharmacological inhibitors (ferrostatin-1) and genetic procedures (RNA interference against IREB-2) previously reported to modulate ferroptosis, readily block the cytotoxic effects of sorafenib in HCC cells. Collectively, our findings identify ferroptosis as an effective mechanism for the induction of cell death in HCC. Ferroptosis could potentially become a goal for the medical treatment of HCC, thus opening new avenues for the optimization of the use of sorafenib in these tumors.

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Sorafenib is currently the medical treatment of reference for hepatocellular carcinoma (HCC), but it is not known whether sorafenib is equally active in all HCC. Here, our aim was to explore intrinsic differences in the response of HCC cells to sorafenib, to identify potential mechanisms leading to primary resistance to this treatment. We analyzed a panel of six human HCC cell lines and compared the activity of the main oncogenic kinase cascades, their clonogenic potential, proliferation and apoptosis upon exposure to sorafenib. We report that HCC cells present important differences in their response to sorafenib, and that some cell lines are more resistant to the actions of sorafenib than others. We identify the activated epidermal growth factor receptor (EGFR) as a parameter that promotes the resistance of HCC cells to sorafenib. In resistant cells, the efficacy of sorafenib was increased when EGFR was inhibited, as was demonstrated using two chemical inhibitors (erlotinib or gefitinib), a monoclonal antibody directed against EGFR (cetuximab), and RNA interference directed against EGFR. A combination of EGFR inhibitors and sorafenib affords a better control over HCC proliferation, most likely through an improved blockade of the RAF kinases. Our findings therefore confirm the importance of RAF kinases as therapeutic targets in HCC, and identify EGFR as a determinant of the sensitivity of HCC cells to sorafenib. Our findings bear possible implications for the improvement of the efficacy of sorafenib in HCC, and might be useful for the identification of predictive biomarkers in this context.

BackgroundSorafenib, a kinase inhibitor active against various solid tumours, induces oxidative stress and ferroptosis, a new form of oxidative necrosis, in some cancer cells. Clinically-applicable biomarkers that reflect the impact of sorafenib on the redox metabolism of cancer cells are lacking.MethodsWe used gene expression microarrays, real-time PCR, immunoblot, protein-specific ELISA, and gene reporter constructs encoding the enzyme luciferase to study the response of a panel of cancer cells to sorafenib. Tumour explants prepared from surgical hepatocellular carcinoma (HCC) samples and serum samples obtained from HCC patients receiving sorafenib were also used.ResultsWe observed that genes of the metallothionein-1 (MT1) family are induced in the HCC cell line Huh7 exposed to sorafenib. Sorafenib increased the expression of MT1G mRNA in a panel of human cancer cells, an effect that was not observed with eight other clinically-approved kinase inhibitors. We identified the minimal region of the MT1G promoter that confers inducibility by sorafenib to a 133 base pair region containing an Anti-oxidant Response Element (ARE) and showed the essential role of the transcription factor NRF2 (Nuclear factor erythroid 2-Related Factor 2). We examined the clinical relevance of our findings by analysing the regulation of MT1G in five tumour explants prepared from surgical HCC samples. Finally, we showed that the protein levels of MT1 increase in the serum of some HCC patients receiving sorafenib, and found an association with reduced overall survival.ConclusionThese findings indicate that MT1 constitute a biomarker adapted for exploring the impact of sorafenib on the redox metabolism of cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0526-2) contains supplementary material, which is available to authorized users.