ONECUT2 Facilitates Hepatocellular Carcinoma Metastasis By Transcriptionally Upregulating FGF2 And ACLY

Objective:Metastasis is the predominant cause of the low survival rate in hepatocellular carcinoma(HCC)patients,but its underlying mechanisms remain unclear.Liver-enriched transcription factors are essential for determining the hepatic phenotype.One cut homeobox 2(ONECUT2),a liver-enriched transcription factor,plays an important role in liver development.Previous studies showed that ONECUT2 is significantly upregulated in HCC.This study aimed to further investigated the clinical significance and functional role of ONECUT2 in HCC.Methods:1.The expression levels of ONECUT2 in clinical HCC samples were detected by RT-PCR and immunohistochemistry(IHC)staining;the correlation between clinicopathological features and prognosis of HCC patients and ONECUT2 expression were analyzed by SPSS software;Transwell assays and HCC orthotopic mouse model were ultilized to evaluate the effect of ONECUT2 on the migration,invasion and metastasis of HCC.2.The potential target genes of ONECUT2 were identified by the human Cancer Pathway Finder RT~2 Profiler PCR Array;chromatin immunoprecipitation(Ch IP)and luciferase reporter assays were conducted to determine the regulation of target genes by ONECUT2;Transwell assays and HCC orthotopic mouse model were ultilized to evaluate the role of FGF2 and ACLY in ONECUT2-mediated HCC metastasis.3.The expression levels of ONECUT2 in HCC cells were detected after FGF2 treatment by RT-PCR and western blot assays;the upstream regulator responsible for ONECUT2expression was predicted by JASPAR website and determined by luciferase reporter assays;HCC cells were treated with ERK,JNK,P38,PI3K and PKC inhibitors respectively,then western blot and Ch IP assays were ultilized to analyze the influences of these inhibitors on FGF2-induced ONECUT2 expression and the binding of ELK1 to ONECUT2 promoter under FGF2 stimulation.4.ONECUT2-overexpressing HCC cells and HCC orthotopic mice were treated with FGFR1 inhibitor PD173074 and ACLY inhibitor ETC-1002 alone or in combination,then evaluated the effects of different strategies on ONECUT2-mediated HCC metastasis.Results:1.Compared with adjacent nontumor tissues,primary HCC tissues exhibited higher ONECUT2 levels.Positive ONECUT2 expression was statistically correlated with multiple clinicopathological features of HCC patients,including increased tumor number,tumor encapsulation loss,microvascular invasion,poor tumor differentiation,and advanced tumor-node-metastasis(TNM)stage.HCC patients with high ONECUT2 expression exhibited decreased overall survival and increased recurrence probability.ONECUT2overexpression promoted migration,invasion and metastasis in HCC,while ONECUT2knockdown exhibited the opposite effect.2.FGF2 and ACLY were identified as the most significantly upregulated molecules following ONECUT2 overexpression.ONECUT2 transcriptionally upregulated the expression of FGF2 and ACLY by binding to the promoters of FGF2 and ACLY.The downregulation of FGF2 or ACLY could inhibit ONECUT2-mediated HCC metastasis,while the upregulation of FGF2 or ACLY could partially reverse the decrease in HCC metastasis induced by ONECUT2 knockdown.3.The expression levels of ONECUT2 in HCC cells increased following FGF2stimulation.The ELK1 binding site on the ONECUT2 promoter was essential for the transactivation of ONECUT2 by FGF2.The FGF2-induced ONECUT2 expression and the binding of ELK1 to the ONECUT2 promoter under FGF2 stimulation were significantly inhibited by ERK inhibitor.4.FGFR1 inhibitor PD173074 in combination with ALCY inhibitor ETC-1002 could more markedly inhibit ONECUT2-mediated HCC metastasis compared with PD173074 and ETC-1002 alone.Conclusion:Our study revealed the clinical value and pro-metastatic function of ONECUT2 in HCC.ONECUT2 promoted HCC metastasis by transcriptionally upregulating FGF2 and ACLY.FGF2/ERK/ELK1 signaling pathway induced the aberrant expression of ONECUT2 in HCC.FGFR1 inhibitor PD173074 in combination with ACLY inhibitor ETC-1002 could significantly inhibit ONECUT2-mediated HCC metastasis.