BACH1-mediated Upregulation Of IGF1R And PTK2 Promotes Hepatocellular Carcinoma Metastasis

Objective: Accumulating studies manifest that BTB and CNC homology 1(BACH1)is highly expressed in multiple malignant tumor tissues and promotes the occurrence,development,and metastasis of tumors.Targeting BACH1 or its upstream and downstream signal pathways significantly enhances the anti-tumor efficacy.Nevertheless,the exact role and therapeutic significance of BACH1 in hepatocellular carcinoma(HCC)remain unclear.Methods: The expression of BACH1 in human HCC specimens and adjacent nonneoplastic tissues was analyzed by Real-time Quantitative Polymerase Chain Reaction(RT-q PCR),western blot,and immunohistochemistry(IHC).The relationship between BACH1 expression and the clinicopathological characteristics of HCC patients was analyzed by correlation analysis in two independent HCC cohorts.The invasiveness and metastasis of HCC in vitro and in vivo were evaluated using transwell assays and orthotopic xenograft models.The Human Cell Motility PCR Array was used to screen the target genes regulated by BACH1.The luciferase reporter assays and chromatin immunoprecipitation(Ch IP)assays were performed to explore the transcriptional regulation of insulin-like growth factor 1 receptor(IGF1R)and protein tyrosine kinase 2(PTK2)by BACH1.The transwell assays and orthotopic xenograft models were used to explore the role of IGF1 R and PTK2 in BACH1-mediated HCC invasion and metastasis.To explore the signaling pathway of insulin-like growth factor 2(IGF2)-mediated BACH1 upregulation in HCC,the recombinant IGF2,related pathway inhibitors,luciferase reporter assays,and Ch IP assays were used.The role of BACH1-IGF1R/PTK2 regulatory pathway in IGF2-mediated invasion and metastasis of HCC was evaluated using transwell assays and orthotopic xenograft models.IHC and correlation analysis were used to evaluate the correlation of BACH1 expression with IGF1 R and PTK2 expression in human HCC specimens,as well as the correlation of IGF1 R and PTK2 expression with the clinicopathological characteristics of HCC patients.The expression of BACH1,IGF1 R,and PTK2 in human metastatic HCC tissues was tested by RT-q PCR and IHC.The transwell assays and orthotopic xenograft models were used to explore the therapeutic effect of IGF1 R inhibitor linsitinib combined with PTK2 inhibitor defactinib on BACH1-mediated HCC metastasis.Results: BACH1 was upregulated in human HCC samples.The elevated levels of BACH1 were related to increased tumor number,tumor encapsulation loss,microvascular invasion,less differentiation,and poor tumor-nodule-metastasis(TNM)staging in HCC patients.Overexpression of BACH1 significantly enhanced the invasiveness and metastasis of HCC,while knockdown of BACH1 led to a dramatic inhibition of invasiveness and metastasis of HCC.BACH1 regulated cell motility-associated genes,especially IGF1 R and PTK2.BACH1 directly bound to the promoter region of IGF1 R and PTK2 genes and promoted their transcriptional activation.IGF1 R and PTK2 mediated the pro-metastatic properties of BACH1.IGF2/IGF1 R upregulated BACH1 expression through ERK1/2-ETS1 signaling.Oncogenic IGF2 depended on BACH1-IGF1R/PTK2 regulatory pathway to promote HCC invasion and metastasis.The expression of IGF1 R and PTK2 positively correlated with the levels of BACH1 in HCC cohorts.Both elevated IGF1 R and PTK2 expression were related to increased tumor number,tumor encapsulation loss,microvascular invasion,less differentiation,and poor TNM staging in patients with HCC.The expression of BACH1,IGF1 R,and PTK2 in metastatic HCC tissues was significantly higher than that in primary HCC and paracancer tissues.IGF1 R inhibitor linsitinib combined with PTK2 inhibitor defactinib prominently suppressed BACH1-induced HCC metastasis.Conclusions: BACH1 is upregulated in human HCC samples and predicts poor prognosis.BACH1 promotes HCC metastasis by transcriptional regulation of the expression of cell motion-related genes IGF1 R and PTK2.IGF2 signaling transactivated BACH1 via IGF1R-ERK1/2-ETS1 cascades.IGF1 R blockade combined with PTK2 inhibition enhances HCC therapeutic efficacy.