Inhibition of autophagy potentiates the antitumor effect of the multikinase inhibitor sorafenib in hepatocellular carcinoma

Abstract

Multikinase inhibitor sorafenib inhibits proliferation and angiogenesis of tumors by suppressing the Raf/MEK/ERK signaling pathway and VEGF receptor tyrosine kinase. It significantly prolongs median survival of patients with advanced hepatocellular carcinoma (HCC) but the response is disease-stabilizing and cytostatic rather than one of tumor regression. To examine the mechanisms underlying the relative resistance in HCC, we investigated the role of autophagy, an evolutionarily conserved selfdigestion pathway, in hepatoma cells in vitro and in vivo. Sorafenib treatment led to accumulation of autophagosomes as evidenced by conversion from LC3-I to LC3-II observed by immunoblot in Huh7, HLF and PLC/PRF/5 cells. This induction was due to activation of autophagic flux, as there was further increase in LC3-II expression upon treatment with lysosomal inhibitors, clear decline of the autophagy substrate p62, and an mRFP-GFP-LC3 fluorescence change in sorafenib-treated hepatoma cells. Sorafenib inhibited the mammalian target of rapamycin complex 1 and its inhibition led to accumulation of LC3-II. Pharmacological inhibition of autophagic flux by chloroquine increased apoptosis and decreased cell viability in hepatoma cells. siRNA-mediated knockdown of the ATG7 gene also sensitized hepatoma cells to sorafenib. Finally, sorafenib induced autophagy in Huh7 xenograft tumors in nude mice and coadministration with chloroquine significantly suppressed tumor growth compared with sorafenib alone. In conclusion, sorafenib administration induced autophagosome formation and enhanced autophagic activity, which conferred a survival advantage to hepatoma cells. Concomitant inhibition of autophagy may be an attractive strategy for unlocking the antitumor potential of sorafenib in HCC.Sorafenib is an orally available multikinase inhibitor recently approved as the first molecular targeting compound for hepatocellular carcinoma (HCC). 1 Sorafenib inhibits Raf kinases, including Raf-1 and B-Raf, which are members of the Raf/ MEK/ERK signaling pathway, and inhibits a number of receptor tyrosine kinases involved in neo-angiogenesis and tumor progression, such as vascular endothelial growth factor receptor (VEGFR) 2, platelet-derived growth factor receptor b and c-Kit. Two randomized, placebo-controlled trials revealed that sorafenib significantly prolongs the median survival of patients with advanced HCC but the response is disease-stabilizing and cytostatic rather than one of tumor regression. 2,3 Therefore, a more detailed understanding of the mechanisms underlying both the antitumor effect and the primary resistance to this compound may provide insights that can help to improve the therapeutic outcome in HCC.Macroautophagy (hereafter referred to as autophagy) is an evolutionally conserved catabolic process that transports cellular macromolecules and organelles to a lysosomal degradation pathway. 4 It is regulated by autophagy-related (atg) genes that control the formation and maturation of a doublemembrane vesicle, autophagosome, which seq...

Inhibition of Akt Reverses the Acquired Resistance to Sorafenib by Switching Protective Autophagy to Autophagic Cell Death in Hepatocellular Carcinoma

“…Even with the same concentration of sorafenib, another study showed that autophagy protected against sorafenib-induced cell death (20). Here, we have demonstrated that sorafenib at 10 mmol/L activated autophagy, which played a protective role against cell death of parental HCC cells, and is in agreement with a previous report (21); but exhibited a death-promoting role in sorafenib-resistant HCC cells. Autophagy induced by vorinostat, a histone deacetylase inhibitor that has been approved for treating cutaneous T-cell lymphoma in clinic (41), protected against cell death independent of mTOR in vorinostat-resistant lymphoma cells (42).…”

“…Autophagy induced by vorinostat, a histone deacetylase inhibitor that has been approved for treating cutaneous T-cell lymphoma in clinic (41), protected against cell death independent of mTOR in vorinostat-resistant lymphoma cells (42). The autophagic pathway crosstalks with both the caspase-dependent and -independent apoptotic pathways (13,21,43). Sustained drug exposure could induce imbalanced apoptotic pathways, leading to the resistance to apoptosis (44).…”

“…Activation of autophagy is involved in the resistance to cisplatin (19), whereas inhibition of autophagy enhances the effect of sorafenib (20,21) in HCC cells. However, the role of autophagy and its cross-talk with other pathways in mediating resistance to sorafenib of HCC have not yet been reported.…”

Autophagy Modulation As a Potential Therapeutic Target for Liver Diseases

“…82 Chloroquine has been used in combination with many chemotherapeutic agents and a large number of trials are ongoing on its effect on cancer therapy alone or in various combinations. 83 …”

Integration of Autophagy and Anoikis Resistance in Solid Tumors

“…Oridonin can also induce prostate cancer PC-3 cells ACD (Ye et al, 2012). In contrary, the administration of multikinase inhibitor sorafenib promotes the autophagosome formation and increases the probability of the hepatoma cells survival, suggesting that complementary strategies are urgently needed to expand the clinical application of sorafenib (Shimizu et al, 2012). For example, BO-1051, an N-mustard linked with a DNA-affinic molecule, has been reported to induce apoptosis and autophagy in hepatocellular carcinoma.…”