| PurposeAdvanced hepatocellular carcinoma(HCC)with various pathogenic factors has strong heterogeneity and often develops with basic diseases such as chronic inflammation,which makes the targeted drugs for the treatment of advanced HCC particularly limited.Sorafenib is the only systemic drug effective for advanced HCC in recent ten years(2007～2017).Its derivative regorafenib was approved as a second-line drug in 2017 for patients with advanced HCC who are resistant to sorafenib treatment.Although both drugs can improve the overall survival rate of patients,drug resistance is still the main obstacle to their efficacy.Therefore,finding the mechanism of drug resistance of sorafenib and regorafenib to HCC and how to overcome this mechanism to improve the anti-HCC efficacy of these drugs are important questions that need to be answered urgently.Liver inflammation(hepatitis)could secrete a variety of inflammation related factors to affect the tumor microenvironment,among which Interleukin-6(IL-6)is a particularly important inflammatory factor.IL-6 is a multi-functional cytokine,which plays an important role in liver regeneration,the occurrence and development of hepatitis and HCC.Several studies have shown that IL-6 mediates the drug resistance of HCC to sorafenib by activating STAT3(signal transducer and activator of transcription 3).However,the receptor of IL-6 is IL-6Rα(IL-6 receptor alpha,also known as gp80)is indispensable for transmitting IL-6 ligand signal.Its expression and function under the treatment of sorafenib or regorafenib in HCC have not been reported in detail.Therefore,this topic aim is to explore IL-6Rαexpression’s fine molecular mechanism layer by layer,and then explore the targeted inhibition of IL-6Rα to enhance the efficacy of sorafenib or regorafenib as a potential scheme for the treatment of HCC,and to use IL-6Rα monoclonal antibody synergistically enhance the efficacy of sorafenib or regorafenib.Method1.Deep sequencing of Huh7 cells treated with different concentrations of sorafenib by transcriptome method to analyze the up-regulated genes of sorafenib;the sequencing results were verified by quantitative real-time PCR(qRT-PCR);IL-6Rα was detected by fluorescence activated cell sorting(FACS)and enzyme linked immunosorbent assay(ELISA)in HCC cells.2.Set up a control group and three treatment groups(sorafenib single drug group,IL-6 single drug group and sorafenib+IL-6 combined group respectively),and verify whether IL-6 will affect the sensitivity of HCC cells to sorafenib in vitro through colony formation assays;then a control group and two treatment groups(sorafenib monotherapy group and sorafenib+IL-6combination group)were established to verify whether IL-6 can increase the drug resistance of HCC to sorafenib in vivo through cell line derived xenograft model(CDX).3.Construct IL-6Rα gene overexpression or deletion cell lines which were confirmed by FACS,ELISA,first generation sequencing or Western blotting,respectively;phosphorylation of STAT3 at tyrosine-705 was determined by immunoblotting;investigate whether IL-6Rα is essential for IL-6 mediated STAT3 activation and sorafenib resistance in HCC.4.Analyze the above transcriptome sequencing data and reference eukaryotic promoter database to screen potential transcription factors which may regulate IL-6Rα’s expression;the overexpression plasmids of these transcription factors were constructed and IL-6Rα promoter sequence were constructed into luciferase reporter plasmid;Hela cells were used for luciferase reporting experiment and Huh7 cell lines with ATF3(activating transcription factor 3)deletion were used to study IL-6Rα induction mechanism;the effect of ATF3 on IL-6-mediated sorafenib resistance was investigated by colony formation assays.5.Select qRT-PCR,Western blotting,ELISA,FACS,colony formation assays to explore whether regorafenib,an analog of sorafenib,could also act on ATF3-IL-6Rα pathway.6.Whether Tocilizumab and Sarilumab block IL-6Rα signal;construct PDX(patient-derived xenograft model);the expression of human IL-6 mRNA in CDX and PDX tissues was observed with a new highly sensitive in situ hybridization technique(RNAscope);the efficacy of antibody drug Sarilumab combined with sorafenib or regorafenib in the treatment of PDX;immunohistochemical analysis of STAT3(Y705),ATF3,CD31,CD68 andα-SMA.7.The expression of IL-6Rα was explored by using the data of the Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)in hepatocellular carcinoma and its adjacent tissues;the overall survival rate of Kaplan-Meier plotter was analyzed by using the HCC cohort with high macrophages infiltration in TCGA database to evaluate IL-6Rα relationship between expression and survival rate of patients with HCC.Result1.Sorafenib induces IL-6Rα expression in HCC cells.2.Both IL-6Rα knockout in Huh7 and IL-6Rα knockdown in Hep3 B cells abolished the synergistic effect of sorafenib on IL-6 induced STAT3 activation.Colony formation assay also showed that IL-6Rα depletion largely attenuated IL-6 mediated sorafenib resistance ex vivo.Intraperitoneal injection of human IL-6 enhanced tumor growth either in the presence or absence of sorafenib treatment,which was abolished by IL-6Rαdepletion.3.ATF3 binds to the IL-6Rα promoter and is involved in IL-6Rα induction.4.Regorafenib,an analogue of sorafenib,also activates ATF3-IL-6Rα axis.5.Human IL-6 mRNA was found in HCC PDX samples by RNAscope technology.On this basis,the PDX model was used to confirm effect of Sarilumab on IL-6Rα which can significantly enhance the efficacy of sorafenib or regorafenib in HCC.6.The expression of IL-6Rα in HCC was higher than that in adjacent tissues,and the high expression of IL-6Rα was positively correlated with poor prognosis.ConclusionOur data reveal that ATF3-mediated IL-6Rα up-regulation promotes both sorafenib and regorafenib resistance in HCC,and targeting IL-6Rαrepresents a novel therapeutic strategy to enhance sorafenib/regorafenib efficacy for advanced HCC treatment. |
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