T-box Transcription Factor 19 Promotes Hepatocellular Carcinoma Metastasis Through Upregulating EGFR And RAC1

Objective: Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related death in the world.Metastasis is one of the main causes of recurrence and death after surgical resection of HCC.Despite the several advances in treatment strategies for HCC in recent years,the treatment effect of advanced HCC is still unsatisfactory.Therefore,it is necessary to explore the underlying mechanism of HCC progression and potential treatment strategies.Methods:1.Real-time quantitative polymerase chain reaction(RT-q PCR)was used to measure the expression of T-box family members in HCC tissues.Transwell assays were used to determine the effect of T-box family members on the migratory and invasive capability of HCC cells.T-box transcription factor 19(TBX19)expression in two independent HCC cohorts was evaluated by immunohistochemistry(IHC).Lentivirus was transfected into HCC cells in order to construct stable overexpression or knockdown cell lines.For exploring the effect of TBX19 on HCC metastasis,transwell assays and the orthotopic HCC mouse model were established.2.A Human Liver Cancer PCR array was applied to explore the potential downstream targets of TBX19-induced HCC metastasis.The regulation mechanism of TBX19 on downstream target genes was analyzed by luciferase reporter experiments and chromatin immunoprecipitation experiments.Transwell experiments and the orthotopic HCC mouse models were established for the purpose of defining the effect of epidermal growth factor receptor(EGFR)and Rac family small GTPase 1(RAC1)in TBX19-induced HCC metastasis.3.IHC staining was used to analyze the expression of TBX19,EGFR and RAC1 in two independent HCC cohorts.The expression levels of TBX19,EGFR,and RAC1 were measured in 20 pairs of adjacent nontumor tissues,primary and metastatic HCC tissues by RT-q PCR and IHC assays.4.PLC/PRF/5 and SNU398 cells were treated with several cytokines and growth factors involved in HCC metastasis to explore the underlying mechanism resulting in TBX19 upregulation.The effect of epidermal growth factor(EGF)treatment on the activity of TBX19 promoter was detected by luciferase reporter experiments.The role of TBX19 in EGF-mediated HCC metastasis was explored by transwell assays and the orthotopic HCC mouse model.5.The effect of EGFR inhibitor combined with RAC1 inhibitor on TBX19-mediated HCC metastasis was analyzed by transwell assays and orthotopic HCC mouse model.Results:1.The results of RT-qPCR showed that TBX19 was one of the most significantly upregulated genes of the T-box family in HCC.Compared with HCC patients without recurrence or metastasis,the TBX19 m RNA levels in HCC patients with recurrence or metastasis were significantly elevated.The results of IHC showed that the expression of TBX19 was significantly increased in HCC tissues.In vitro and in vivo experiments showed that overexpression of TBX19 enhanced HCC metastasis,and knockdown of TBX19 suppressed HCC metastasis.2.The Human Liver Cancer PCR array results exhibited that EGFR and RAC1 were the two genes with the most significant expression changes after TBX19 was upregulated or downregulated.TBX19 directly bound to the promoter of EGFR or RAC1 and upregulated the expression of EGFR and RAC1 through transcription activation.In vitro and in vivo metastatic experiments demonstrated that downregulating EGFR and RAC1 expression attenuated TBX19-induced HCC metastasis,and upregulation of EGFR and RAC1 restored the inhibition of HCC metastasis induced by TBX19 knockdown.3.In two independent HCC cohorts,TBX19 expression was positively correlated with the expression of EGFR and RAC1.Additionally,the expression of TBX19,EGFR and RAC1 was obviously upregulated in metastatic HCC tissues.4.EGF upregulated TBX19 expression in a dose-dependent way.EGF upregulated the expression of TBX19 via Nuclear Factor(NF)-κB.Extracellular signal-regulated kinase(ERK)inhibitor U0126 significantly inhibited EGF-mediated TBX19 expression.In vivo and in vitro experiments showed that overexpression of EGF promoted HCC metastasis,while knockdown of TBX19 significantly inhibited EGF-mediated HCC metastasis.5.In vitro and in vivo experiments showed that applying EGFR inhibitor erlotinib or RAC1 inhibitor NSC23766 alone moderately repressed TBX19-mediated HCC metastasis.Moreover,the combination treatment of EGFR inhibitor and RAC1 inhibitor obviously inhibited TBX19-mediated HCC metastasis.Conclusions: The study indicated that the expression of TBX19 was significantly upregulated in HCC.TBX19 accelerated HCC metastasis by enhancing EGFR and RAC1 expression through transcriptional activation.EGF upregulated the expression of TBX19 via the ERK/NF-κB pathway.Combination application of EGFR inhibitor and RAC1 inhibitor markedly suppressed TBX19-mediated HCC metastasis.Blocking this signaling pathway might offer a potential therapeutic way to restrain TBX19-mediated HCC metastasis.