Context: The multityrosine kinase inhibitor sorafenib is the first and so far only drug to provide overall survival benefit in patients with advanced hepatocellular carcinoma (HCC). Sorafenib blocks the Raf pathway in tumor and endothelial cells as well as VEGFR and PDGFR activation in endothelial cells. Little is known about potential mechanisms that may modify tumor cell sensitivity towards sorafenib. The characterization of such mechanisms could help to alleviate primary or acquired resistance and improve this treatment. Here, we examined whether the activation of EGFR-dependent pathways modulate cell response to sorafenib in responsive or resistant human hepatoma cells.

Methods: Five HCC cell lines (HuH7, Hep3B, HepG2, HuH6, PLC/PRF5) were treated with sorafenib alone or in combination with gefitinib, an EGFR TK inhibitor, for up to 72 h. A pool of Hep3B cells resistant to sorafenib (Hep3B-SR) was generated in culture by growing cells in increasing concentrations of sorafenib over a period of several months. Cell viability, DNA synthesis, apoptosis marker expressions (PARP and caspase-3), and pERK/pAKT/cyclin A levels were evaluated. A panel of 42 phosphorylated RTK was analyzed in Hep3B-SR using an antibody array kit (R&D Systems).

Results: In the five HCC cell lines, the combination of gefitinib with sorafenib was more efficient in reducing cell viability than each drug alone. Supra-additive anti-proliferative and pro-apoptotic effects were observed in PLC/PRF5 and Hep3B cell lines. At the molecular level, the sorafenib/gefitinib combination markedly reduced pERK and to a lesser extent pAKT. In cell lines, which were the most responsive to combined therapy, sorafenib alone increased pERK, pAKT and cyclin A levels and that these paradoxical effects were abolished in the presence of gefitinib. Consistent with this result, the down-regulation of EGFR expression with a siRNA blunted the stimulatory effect of sorafenib on pERK. The activation of EGFR-dependent pathways under sorafenib treatment did not result from increased EGFR tyrosine phosphorylation. In addition, the analysis of a panel of 42 phosphorylated RTK (including EGFR) did not show any compensatory activation of receptors in the sorafenib-resistant pool Hep3B-SR compared to parental cells. However, Hep3B-SR cells displayed increased sensitivity to gefitinib associated with inhibition of pAKT and pERK levels although both their EGFR protein and mRNA levels were downregulated.

Conclusions: these findings indicate that EGFR blockade reinforces the anti-tumoral action of sorafenib in HCC cells. This effect appears to be optimal in cells showing ERK and AKT activation under sorafenib treatment through an EGFR-dependent mechanism. Therefore the combination of EGFR inhibitors to sorafenib might be a new therapeutic strategy able to reduce intrinsic and acquired resistance of HCC to sorafenib.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 623.