SIX4 Promotes Hepatocellular Carcinoma Metastasis Through Upregulating YAP1 And C-MET

Background: Hepatocellular carcinoma(HCC)is the third most common cause of tumor-related death worldwide.Hepatic resection is still the major strategy for early-stage HCC.However,approximate ~75% of patients will develop recurrence and metastasis within 5 years after operation and most HCC patients are discovered in the advanced stage.Therefore,it is urgent to explore the mechanism under HCC metastasis and find the potential therapeutic strategies for advanced HCC.Mutated or dysfunctional transcription factors promote tumor progression by regulating the abnormal expression of oncogenes.The Sine Oculis Homeobox(SIX)belonging to the Homeobox superfamily,encodes six transcription factors,and has evolutionally conserved homologous and SIX domains.Several studies have evidenced that SIX family plays important roles in remodeling of embryo and development of tissue and organ.At present,a series of studies have shown that SIX family plays different roles in the tumor progression.For example,SIX1 and SIX2 are treated as oncogenes and promote tumor progression.In contrast,SIX3 inhibits tumor proliferation and metastasis.However,the role of SIX family in HCC is not well understood and deserves further study.Objectives: 1.Exploring the expression of SIX family in HCC and the role of SIX4 in HCC metastasis.2.To clarify the molecular mechanisms of SIX4 in promoting HCC metastasis.3.Finding the upstream molecular mechanisms of abnormal SIX4 expression in HCC.4.Revealing the potential interventional strategies of HCC with SIX4 overexpression.Methods:1.The RT-qPCR was used to detect the expression of SIX family members in normal liver tissues,adjacent nontumors and HCC tissues and screened the upregulated genes.The m RNA level of SIX4 in normal liver tissues and 50 paired HCC tissues was measured.Immunohistochemical staining(IHC)was performed for detecting the SIX4 expression in adjacent nontumor,HCC tissues and metastatic HCC tissues.The expression of SIX4 in two tissue arrays with HCC patient samples was shown by IHC.HCC cell lines PLC/PRF/5 and MHCC97 H were used to construct stable cell lines.The role of SIX4 in HCC metastasis was proved by a series of experiments in vitro and in vivo.2.The m RNA expression profiles between PLC/PRF/5-SIX4 and PLC/PRF/5-control cells were analyzed using a Liver cancer RT2 Profiler PCR Array.Truncated mutant,luciferase reporter and chromatin immunoprecipitation(Ch IP)assays were used to prove the interaction of YAP1 or MET promoter and SIX4.Western blotting was used to detect the nuclear YAP1 and the expression of the target genes of c-MET.In vitro and in vivo assays were performed to clarify the roles of YAP1 and c-MET in SIX4-induced HCC metastasis.3.PLC/PRF/5 cells were treated with HGF for 24 h and then SIX4 expression was tested.Luciferase reporter was performed to explore the roles of cis-regulatory elements in SIX4 promoter under the HGF treatment.Signaling pathway inhibitors of ERK,P38,JNK and PI3 K were used to identify which was responsive for SIX4 upregulation induced by HGF.The role of SIX4 in HGF-induced HCC metastasis was proved by transwell assay and liver orthotopic implantation model.4.YAP1 inhibitor Verteporfin and/or c-MET inhibitor Capmatinib were applied to show the migratory and invasive abilities of PLC/PRF/5-SIX4 cells in different groups.In vivo metastasis assay was performed to show effect of the combination of YAP1 and c-MET inhibitors on SIX4-induced HCC metastasis.Results: 1.SIX4 exhibited the largest fold change in HCC tissues.The m RNA level of SIX4 were dramatically upregulated in HCC tissues compared with that in adjacent nontumorous tissues and normal liver tissues.The SIX4 m RNA expression was higher in patients with recurrence or metastasis than in patients without recurrence or metastasis.IHC showed a higher protein level of SIX4 expression in metastatic HCC samples than in primary HCC samples.SIX4 protein expression was significantly up-regulated in HCC tissues compared to this in adjacent nontumorous tissues,and SIX4 expression was primarily localized in nucleus in two HCC cohorts.Overexpression of SIX4 increased the migrative and invasive abilities of PLC/PRF/5 cells,while SIX4 knockdown decreased migratory and invasive abilities of MHCC97 H cells.The in vivo metastatic assay showed that overexpression of SIX4 expedited the lung metastasis rate and increased the number of metastatic lung nodules and lowered the survival time of nude mice.In contrast,SIX4 downregulation largely impaired the lung metastasis rate and the number of metastatic lung nodules,and prolonged the survival time of nude mice.2.SIX4 overexpression induced the expression of several liver cancer-related genes,including MET,YAP1,TLR4,EGFR,HGF,MYC,CTNNB1,CXCR4,and ADAM17 by using a Liver cancer RT2 Profiler PCR Array.Both c-MET and YAP1 have been reported to be dramatically upregulated in HCC tissues and indicated poor prognosis.Overexpression of SIX4 upregulated YAP1 and c-MET expression,whereas knockdown of SIX4 reduced YAP1 and c-MET expression.Luciferase reporter assay showed that SIX4 upregulation transactivated YAP1 and MET expression.Deletion of the region between-940 and-238 bp significantly reduced YAP1 reporter activity medicated by SIX4 overexpression and mutation of putative binding site 1 in YAP1 promoter reduced luciferase reporter activity that was mediated by SIX4 overexpression.Similarly,deletion of the region between-1427 and-504 bp decreased MET reporter activity and the mutation of putative binding site 1 in MET promoter reduced luciferase reporter activity induced by SIX4 overexpression.Moreover,Ch IP assays showed that SIX4 directly bound to YAP1 and MET promoters in PLC/PRF/5-SIX4 cells and human HCC samples.SIX4 overexpression increased the expression of nuclear YAP1 and YAP1 target genes,whereas SIX4 knockdown decreased these genes expression.Western blotting analysis showed that SIX4 increased the phosphorylation of c-MET and activated downstream signaling pathways.Transwell assay showed that knockdown of YAP1 and c-MET significantly decreased SIX4-enhanced migratory and invasive abilities,whereas overexpression of YAP1 and c-MET rescued the reduced migratory and invasive abilities induced by SIX4 knockdown.The in vivo metastatic assay showed that knockdown of YAP1 and c-MET lowered the incidence of lung metastasis and the number of metastatic lung nodules and prolonged the overall survival of the PLC/PRF/5-SIX4 group.In contrast,overexpression of YAP1 and c-MET reversed the decreased lung metastasis and the number of metastatic lung nodules in MHCC97H-sh SIX4 group and decreased overall survival of mice.IHC staining showed that YAP1 was mainly localized in nucleus.SIX4 expression was positively correlated with nuclear YAP1 and c-MET expression in both cohorts.3.HGF treatment increased SIX4 expression in a dose dependent manner and HGF treatment transactivated SIX4 promoter activity.A significant reduction of SIX4 promoter activity was observed when PLC/PRF/5 cells were transfected with truncated(-365 to-43)SIX4 promoter construct.Mutation of second binding site significantly reduced SIX4 promoter activity induced by HGF.Pretreatment of cells with ERK inhibitor significantly inhibited HGF mediated SIX4 overexpression.A Ch IP assay demonstrated that ERK inhibitor treatment inhibited the binding of NF-kB to SIX4 promoter.HGF expression was upregulated in HCC tissues compared with that in adjacent nontumor tissues shown by IHC.SIX4 expression was positively correlated with HGF expression.HGF treatment significantly increased migratory and invasive abilities of PLC/PRF/5 cells,whereas knockdown of SIX4 largely lowered the increased migratory and invasive abilities induced by HGF.The in vivo metastasis experiment showed that HGF overexpression increased the incidence of lung metastasis and the number of metastatic lung nodules and decreased overall survival time in PLC/PRF/5-HGF group compared with that in control group.However,SIX4 knockdown decreased the incidence of lung metastasis and the number of metastatic lung nodules while extended the overall survival time in the PLC/PRF/5-HGF group.4.Treatment with VP or Capmatinib alone partially decreased the migratory and invasive abilities of PLC/PRF/5-SIX4 cells,whereas combination of the two agents dramatically lowered migratory and invasive abilities of PLC/PRF/5-SIX4 cells.The in vivo metastasis assay showed that VP or Capmatinib treatment alone partially decreased incidence of lung metastasis and the number of metastatic lung nodules while partially increased the overall survival time of the PLC/PRF/5-SIX4 group,whereas the combination of VP and Capmatinib dramatically inhibited lung metastasis and largely prolonged survival time compared with control or single agent treatment.Conclusion: SIX4 upregulation induced by HGF promoted HCC metastasis through transactivating YAP1 and c-MET expression.The combination treatment of YAP1 and c-MET inhibitors significantly suppressed SIX4-mediated HCC metastasis.Thus,SIX4 was a prognostic biomarker in HCC patients and targeting the HGF-SIX4-c-MET positive feedback loop may provide a promising strategy for treatment SIX4-driven HCC metastasis.