Biomarkers Screeningof Sorafenib Resistance,and Its Overcoming By PPAR? Antagonists In Hepatocellular Carcinoma

Background:Primary liver cancer is one of the high incidence of malignant tumors in China,with limited treatment,lack of therapeutic efficacy and high mortality,which remains a major threat to public health in our country.About 383 thousand people die each year from liver cancer,accounting for about 51%of the total global cancer deaths.Surgery,transcatheter hepatic arterial chemoembolization and radiofrequency ablation are common approaches to the management of HCC,and chemotherapy is offered as a non-surgical option for the inoperable ones.Recent advances in molecular targeted therapy has brought new insight into studies of HCC treatment.Sorafenib,a molecular target agent,inhibits tumor angiogenesis by suppressing tyrosine kinase activity and directly inhibits tumor growth via Raf/MEK/ERK signaling pathway.Despite the favorable efficacy and safety in patients with advanced hepatocellular carcinoma,sorafenib induces drug resistance clinically,which has not been clearly understood and is usually related to the tumor cells,tumor stroma and individual genetic resistance.There are studies suggesting that changed expression of resistance-related molecules may indicate therapeutic efficacy and sensitivity of HCC treatment.Therefore,exploratory investigation of biomarker candidates for sorafenib efficacy will facilitate individual medication and further implementation of precision medicine.Moreover,interpretation of molecular mechanism based on key molecules can help overcome drug resistance and promote new drug development.Methods:Patients with postoperative recurrence after liver resection or liver transplantation received continuous sorafenib therapy,and were divided into sorafenib-resistant and sorafenib-sensitive based on results from imaging as well as serum markers(AFP).HCC and adjacent liver tissues were collected and used for iTRAQ analysis,with tissues from liver cirrhosis patients and healthy donors used as control.Protein profiles of sorafenib-resistant and sorafenib-sensitive groups were compared to identify the differentially expressed proteins,which were then annotated by GO and KEGG enrichment analysis to find specific biomarkers and biological pathways.We induced sorafenib resistance in HCC cells and verified the expression of identified biomarker candidates using western blot in these cells.The sorafenib-resistant cells,which were then treated with PPAR? antagonist and agonist,and phenotype alteration was detected,including cell proliferation analysis using CCK-8 assay,cell cycle and apoptosis analysis using cytometry,to investigate the potentially inhibitory effect of PPARy antagonist on sorafenib resistance.Results:1.By using iTRAQ analysis,we found 222 proteins with>1.5-fold change,of which 57 showed>2-fold change in expression levels,including 26 proteins upregulated and 31 downregulated.2.BP analysis demonstrated that the differentially expressed proteins function mostly in small molecule metabolism,degradation,carboxylic acid metabolism and oxoacid metabolism,and KEGG pathway analysis found carbon metabolism,fatty acid metabolism and glycolysis pathways involving these proteins.3.For proteins with>2-fold change,the alteration of ITGA6?FN1?ICAM1?ITPA?BAX?TXNDC17 and MSH2 in vitro expression was found consistent with that of in vivo expression,which made them important candidates in mechanism investigation.4.Based on the bioinformatic data,we found that the expression of PPARs,especially the PPARy,significantly changed in sorafenib-resistant cells.5.PPAR? antagonist T0070907 enhanced the sensitivity of HCC cells to sorafenib,whereas GW9662,another PPAR? antagonist,had no apparent effect.6.The combination of antagonist T0070907 and GW9662 did not result in a synergistic effect on sorafenib sensitivity.7.The enhanced sensitivity to sorafenib might be attributed to the cell cycle arrest induced by PPARy antagonists.Conclusions:1.The comparison of protein profiles between tissues from sorafenib-resistant and sorafenib-sensitive patients revealed multiple differentially expressed proteins that are involved in various biological processes and signaling pathways.2.PPAR? may play a pivotal role in the development of sorafenib resistance in HCC,and its antagonist T0070907 can enhance the sorafenib sensitivity of HCC cells,which suggests a potential value in clinical practice.