ETV4 Promotes Resistance To Sorafenib And Cisplatin In Hepatocellular Carcinoma

Background and ObjectiveHepatocellular carcinoma(HCC),the sixth most common tumor in the world and the third most common cancer death rate,is a serious threat to human health.For patients with advanced liver cancer,oral sorafenib is still the best treatment.As is known to all,sorafenib is a multi-target tyrosine kinase inhibitor(TKI)in cancer,which has been the first-line treatment of advanced liver cancer patients since 2007,and can significantly improve overall survival(OS)of patients.However,some patients developed resistance to sorafenib,which resulted in poor efficacy.The related pathways of sorafenib resistance mainly include Ras/Raf/MEK/ERK,PI3K/AKT and JAK/STAT pathways.There are no effective drugs to solve this problem.ETV4,ETS translocation variant 4,also known as PEA3 or ElAF,is a member of the ETS family.And the other members are ETV1,ETV5.ETV4 is a protein-coding gene that is highly expressed in many cancers,such as prostate cancer,breast cancer and liver cancer.ETV4 is a downstream factor of the K-Ras and MET pathways,which also strongly suggests that ETV4 may be associated with sorafenib resistance.In order to further explore the problem of sorafenib resistance in hepatocellular carcinoma,improving the curative effect of sorafenib is currently the most urgent clinical need.Methods and Content1.The expression of ETV4 in liver cancer and its influence on the prognosis of patients with hepatocellular carcinoma.TCGA data set and 11 pairs of hepatocellular carcinoma and normal tissue samples were used to determine the mRNA expression of ETV4 in hepatocellular carcinoma,and the relationship between ETV4 and the prognosis of hepatocellular carcinoma patients was analyzed.2.To explore the biological function of ETV4 in hepatocellular carcinoma.The biological processes in which ETV4 may be involved in hepatocellular carcinoma were analyzed by GSEA gene enrichment analysis,and the expression of ETV4 in hepatocellular carcinoma cells stimulated by sorafenib or cisplatin was detected by in vitro cell culture and western blot.3.To investigate the role of ETV4 in the drug resistance of sorafenib or cisplatin in hepatocellular carcinoma.Cell culture in vitro and western blot were used to detect the changes of apoptosis rate induced by ETV4 on sorafenib or cisplatin in hepatocellular carcinoma cells.The effect of ETV4 on the proliferation rate of hepatocellular carcinoma cells stimulated by sorafenib or cisplatin was detected by EdU cell proliferation assay.4.To explore the downstream factors of ETV4 that promote the drug resistance of hepatocellular carcinoma cells to sorafenib or cisplatin.Q-PCR fluorescence quantitative detection was used to detect the target genes of ETV4 in hepatocellular carcinoma cells under the stimulation of sorafenib or cisplatin.Results1.ETV4 is highly expressed in liver cancer tissues and is associated with poor prognosis of hepatocellular carcinoma patients.Both 50 pairs of TCGA data sets and 11 pairs of fresh hepatocellular carcinoma tissue samples showed that ETV4 was highly expressed in hepatocellular carcinoma,and the disease-free survival rate of patients with high ETV4 expression was significantly lower than that of patients with normal expression.2.ETV4 is associated with drug resistance in liver cancer.TCGA gene enrichment analysis showed that most of the enriched genes in the ETV4 high-expression group were genes regulating adriamycin tolerance,and the expression level of ETV4 protein was significantly increased after the stimulation of sorafenib or cisplatin.These results suggest that ETV4 may be involved in the biological process of regulating the resistance of hepatocellular carcinoma cells to sorafenib and cisplatin.3.ETV4 promoted the resistance of hepatocellular carcinoma cells to sorafenib or cisplatin.Western blot showed that the ability of sorafenib and cisplatin to induce PARP and caspase3 activation was significantly decreased after exogenous overexpression of ETV4.On the contrary,after specific interference with ETV4 expression,sorafenib and cisplatin can significantly increase the formation of cleaved PARP and caspase3.Meanwhile,apoptosis flow detection also showed that after specific interference with ETV4,sorafenib or cisplatin stimulation could significantly increase the proportion of apoptotic cells.At the same time,the proliferation of hepatocellular carcinoma cells significantly decreased after the specific interference of ETV4 expression was detected by EdU proliferation assay.This indicates that ETV4 is indeed involved in the biological process of hepatocellular carcinoma cell resistance to sorafenib or cisplatin.4.ETV4 may regulate the resistance of hepatocellular carcinoma cells to sorafenib or cisplatin through IER3.In order to find the downstream factors of ETV4,the GSE96793 data showed that IER3(Radiation-inducible immediate-early gene IEX-1)was among the 9 factors that were upregulated.After the specific interference of ETV4 in HCC cells,the q-PCR experiment results showed that IER3 was down-regulated after the stimulation of sorafenib or cisplatin,which was statistically significant.This suggests that ETV4 may play an important role in regulating the resistance of HCC cells to sorafenib and cisplatin through IER3.Conclusions1.The expression of ETV4 in liver cancer is significantly higher than that in normal tissue,and is significantly correlated with disease-free survival rate of hepatocellular carcinoma patients;2.ETV4 regulates the resistance of hepatocellular carcinoma cells to sorafenib or cisplatin;3.ETV4 may regulate the resistance of hepatocellular carcinoma cells to sorafenib or cisplatin through IER3.