Hepatic Stellate Cells Induce Hepatocellular Carcinoma Cell Resistance to Sorafenib Through the Laminin-332/α3 Integrin Axis Recovery of Focal Adhesion Kinase Ubiquitination

In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of alpha 3 beta 1 and alpha 6 beta 4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both alpha 3 beta 1 and alpha 6 beta 4. Inhibiting alpha 3 but not alpha 6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing alpha 6 beta 4 but lacking the alpha 3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B alpha 3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. Conclusion: This study unveils a novel mechanism of sorafenib resistance depending on the alpha 3 beta 1/Ln-332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC.