Mechanism Investigation Of HGF/c-Met Signaling Pathway Regulating Sorafenib Resistance In Hepatocellular Carcinoma

Background:Sorafenib,a multi-kinase inhibitor,was approved by the FDA in 2007 for the treatment of patients with advanced hepatocellular carcinoma(HCC).The resistance to sorafenib has been a major impediment to improving survival in HCC patients.Therefore,clarifying the mechanism of resistance to sorafenib and developing effective strategies to delay or overcome resistance is a great challenge and urgent requirement.Acquired sorafenib resistance is often associated with abnormal expression of certain molecules,most of which are also closely correlated with the development of HCC.The increased expression of c-Met and/or HGF in human tumor cells is usually associated with the development of HCC and resistance to sorafenib.c-Met,the hepatocyte growth factor(HGF)receptor,is a tyrosine kinase receptor.Activation of c-Met results in phosphorylation of receptors,recruitment of junctional proteins and subsequent activation of various signal pathways,including phosphatidylinositol 3-kinase(PI3K)and extracellular regulated kinase(ERK)1/2,ultimately affecting proliferation,survival,migration and invasion in certain tumor cell types.Therefore,elucidation of the regulated expression mechanism of c-Met and the important mechanisms regulating sorafenib resistance in hepatocellular carcinoma after overexpression and activation provides an experimental and theoretical basis for the development of novel therapeutic strategies based on hepatocellular carcinoma with sorafenib in combination with c-Met activity inhibitors.Objective:To evaluate the expression levels of c-Met in hepatocellular carcinoma tumor tissues and cells after sorafenib resistance,and to elucidate the molecular transcriptional regulatory mechanisms of c-Met and the molecular mechanisms and biological effects that drive HCC progression and sorafenib resistance after c-Met activation.Methods:1.30 tumor histopathological sections of hepatocellular carcinoma sorafenib-resistant patients and 30 hepatocellular patients not treated with sorafenib were collected and HE staining was performed to observe histomorphological changes;Differences in c-Met expression levels were analyzed by immunohistochemistry.A Huh-7sorafenib-resistant cell line of hepatocellular carcinoma was constructed(Huh-7~R),and CCK-8,Ed U,cell scratching assay,colony-forming experiment,and Acridine orange/ethidium bromide(AO/EB)assays were used to evaluate the differences in cell viability,proliferation,migration and anti-apoptotic biological effects between the resistant cell line and the parental cell line.Western blotting,indirect immunofluorescence and immunocytochemistry assays were performed to analyze the expression levels of c-Met in sorafenib-resistant cell lines.2.Bioinformatics was performed to predict the transcription factors and binding sites involved in the regulation of the c-Met gene promoter.Binding sites were verified by Ch IP experiments.Treatment of resistant cells with signaling pathway inhibitors,Western blotting and RT-PCR were performed to analyze the molecular levels of c-Met and signaling pathways regulating transcription factors.3.c-Met overexpression adenovirus and c-Met si RNA lentivirus were transfected with Huh-7 parental and sorafenib-resistant cells,respectively.c-Met expression levels and key molecular levels of signaling pathway were analyzed by western blotting to reveal the influence mechanism of c-Met expression and activation levels on sorafenib resistance.CCK-8,Ed U,colony formation assay,JC-1,Annexin V-FITC/PI double staining,AO/EB double staining method,cell scratching assay,Transwell assay were conducted to analyze the effect of biological effects.4.A human hepatocellular carcinoma xenograft tumor model was established in BALB/c nude mouse,the tumor-bearing nude mice were random Ly divided into six groups:control group,sorafenib group,PHA-665752 group,c-Met si RNA control lentivirus cell with sorafenib treatment group,c-Met si RNA lentivirus with sorafenib treatment group and PHA-665752 combined with sorafenib group,and tumor growth was calculated by monitoring tumor volume.To assess the effect of inhibition of c-Met expression and activation on tumor growth in vivo in a liver cancer xenograft model,immunohistochemical staining was performed to detect changes in the levels of relevant kinases,Ki67 staining was implemented to detect differences in proliferation levels between treatment groups,HE staining was performed to analyze histopathological changes,and Western blotting was performed to analyze the expression activation levels of relevant signaling pathways.Results:1.Patients with sorafenib-resistant HCC statistically overexpressed c-Met significantly compared to patients not treated with sorafenib,and sorafenib-resistant cells(Huh-7~R)significantly overexpressed c-Met and had significantly increased pro-proliferation,migration and anti-apoptotic capacity compared to parental cells(Huh-7).2.MAPK signaling pathway promoted the binding to c-Met promoter region and upregulated c-Met expression through activation of transcription factor ETS-1.3.Huh-7 parental cells lost sensitivity to sorafenib after c-Met overexpression.c-Met activation was followed by activation of the downstream PI3K/Akt and MAPK signaling pathways.The c-Met si RNA and c-Met inhibitor PHA-665752 restored the sensitivity of drug-resistant cells to sorafenib by blocking the activation of downstream signaling pathways after decreasing the expression and activation levels of c-Met,respectively.4.In the in vivo experiment,c-Met expression and activation were blocked,and the activation level of downstream signaling pathway was subsequently suppressed,which significantly improved the anti-tumor efficacy of sorafenib.Conclusion:Aberrant activation of the MAPK signaling pathway in HCC-resistant cells promotes the binding of the transcription factor ETS-1 to the c-Met gene promoter,upregulating the transcription and expression of c-Met.Overexpression and activation of c-Met enhances the pro-proliferation,migration and anti-apoptotic ability of HCC cells through activation of PI3K/Akt and MAPK signaling pathways which constitute an important mechanism of sorafenib resistance in hepatocellular carcinoma.Combinations of sorafenib with silencing of c-Met expression or with c-Met inhibitors improved HCC resistance to sorafenib and may restore the efficacy of sorafenib in the treatment of HCC.Figure[32]table[8]reference[74]...