Quantitative phosphoproteomic analysis identifies the potential therapeutic target EphA2 for overcoming sorafenib resistance in hepatocellular carcinoma cells.

Limited therapeutic options are available for advanced-stage hepatocellular carcinoma owing to its poor diagnosis. Drug resistance to sorafenib, the only available targeted agent, is commonly reported. The comprehensive elucidation of the mechanisms underlying sorafenib resistance may thus aid in the development of more efficacious therapeutic agents. To clarify the signaling changes contributing to resistance, we applied quantitative phosphoproteomics to analyze the differential phosphorylation changes between parental and sorafenib-resistant HuH-7 cells. Consequently, an average of ~1500 differential phosphoproteins were identified and quantified, among which 533 were significantly upregulated in resistant cells. Further bioinformatic integration via functional categorization annotation, pathway enrichment and interaction linkage analysis led to the discovery of alterations in pathways associated with cell adhesion and motility, cell survival and cell growth and the identification of a novel target, EphA2, in resistant HuH-7R cells. In vitro functional analysis indicated that the suppression of EphA2 function impairs cell proliferation and motility and, most importantly, overcomes sorafenib resistance. The attenuation of sorafenib resistance may be achieved prior to its development through the modulation of EphA2 and the subsequent inhibition of Akt activity. Binding analyses and in silico modeling revealed a ligand mimic lead compound, prazosin, that could abate the ligand-independent oncogenic activity of EphA2. Finally, data obtained from in vivo animal models verified that the simultaneous inhibition of EphA2 with sorafenib treatment can effectively overcome sorafenib resistance and extend the projected survival of resistant tumor-bearing mice. Thus our findings regarding the targeting of EphA2 may provide an effective approach for overcoming sorafenib resistance and may contribute to the management of advanced hepatocellular carcinoma. Inhibiting the activity of a protein known as EphA2 may help overcome the development of drug resistance during treatment of hepatocellular carcinoma, the most common form of liver cancer. Resistance often develops to sorafenib, the main drug treatment option for the advanced disease. Researchers in Taiwan led by Lu-Ping Chow at the National Taiwan University in Taipei investigated the role of around 1500 phosphoproteins, which are regulated by the attachment and removal of small chemical groups containing phosphorus and oxygen. The activity of the phosphoprotein EphA2 was identified as a major contributor to drug resistance. This was linked to the ability of EphA2 to interfere with a molecular signaling pathway previously implicated in sorafenib activity. Suppressing EphA2 activity suppressed the development of sorafenib resistance in cultured cells and in mice with liver cancer, suggesting therapeutic possibilities.