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

Abstract

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-7 R 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.

Y-Box Binding Protein-1 Promotes Epithelial-Mesenchymal Transition in Sorafenib-Resistant Hepatocellular Carcinoma Cells

“…We performed quantitative phosphoproteomic analyses to analyze phosphoproteins in HuH-7 and HuH-7 R cells. The proteins showing >1.5-fold changes (including 533 phosphoproteins) were classified as significantly increased in phosphorylation in resistant HuH-7 R cells [ 16 ]. To obtain potential candidate genes correlated with poor OS of HCC patients, a total of 1056 genes were identified from the TCGA database ( p < 0.001).…”

“…In an earlier study, we have performed quantitative phosphoproteomic analyses to compare the proteomes of HuH-7 and HuH-7 R cells [ 16 ]. Phosphoproteins displaying >1.5-fold changes were considered significantly increased in phosphorylation in HuH-7 R cells.…”

“…For immunohistochemical experiments, sample slides prepared from 5 μm sections of paraffin-embedded xenograft tumors harvested from in vivo animal experiments were deparaffinized, and expression of p-YB-1 S102, YB-1, cleaved caspase-3, Ki-67, p-ERK, ERK, p-AKT, and AKT was examined. Immunoblotting and immunohistochemistry analyses were conducted as described previously [ 16 ]. For F-actin analysis, shCtrl and shYB-1 containing HuH-7 R cells were stained with Alexa 488-conjugated phalloidin (Thermo Fisher, Waltham, MA, USA), and for G-actin staining, Alexa 594-conjugated deoxyribonuclease I (Thermo Fisher, Waltham, MA, USA) was used in accordance with the manufacturer’s instructions.…”

“…Previously, we employed a quantitative phosphoproteomic approach to delineate the mechanisms underlying acquired resistance of HCC to sorafenib [ 16 ]. By integrating data on significantly upregulated phosphoproteins with genes implicated in poor prognosis in patients with liver cancer from the TCGA database and subjecting the intersecting 148 differentially expressed molecules to biological network analysis, Y-box binding protein-1 (YB-1) was identified as a potential regulatory molecule of this dysregulated downstream network in sorafenib-resistant cells.…”

Recent Advances of Functional Proteomics in Gastrointestinal Cancers- a Path towards the Identification of Candidate Diagnostic, Prognostic, and Therapeutic Molecular Biomarkers

“…Their results showed that sorafenib-mediated protein tyrosine kinase inhibition could lead to the activation of alternative cellular pathways such as the Akt, mTOR and FAK signaling pathways and showed that suppressing EphA2 increases its susceptibility to sorafenib. These observations suggested that targeting of EphA2 along with sorafenib treatment helps to overcome sorafenib resistance and resulted in better management of advanced hepatocellular carcinoma [ 73 ].…”