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Abstract
BackgroundAberrant activation of the Hedgehog (Hh) signaling pathway is frequently observed in hepatocellular carcinoma (HCC), nevertheless, the precise molecular mechanism remains unclear. Forkhead box M1 (FOXM1), a target of the Hh pathway, is a key oncofetal transcription factor and a master cell cycle regulator. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is an oncogene critical for mitosis. However, how these molecular events affect HCC progression remains unclear.MethodsRealtime PCR, immunohistochemistry, western blotting, and analyses of datasets TCGA and Gene Expression Omnibus (GEO) were conducted to assess the expression of TPX2 and FOXM1 at the mRNA and protein levels in HCC samples or HCC cells. Expression and knockdown of TPX2 and FOXM1 were performed to assess their role in regulating HCC cell proliferation in vitro and in vivo. Dual luciferase report assay and chromosome immunoprecipitation (ChIP) were investigated to seek the FOXM1 binding sites in the promoter of TPX2.ResultsSpecific antagonists (cyclopamine and GANT61) of the Hh pathway down-regulated TPX2, whereas activation of Hh signaling stimulated TPX2 expression. Furthermore, TPX2 over-expression accelerated HCC cell proliferation when upstream events of Hh signaling were inhibited, and TPX2 knockdown significantly alleviated Sonic Hh ligand (Shh)-induced HCC cell proliferation. Reporter assays and ChIP showed that FOXM1 bound to the TPX2 promoter, confirming that TPX2 is a direct downstream target of FOXM1. Xenograft model further verified the cell function and expression regulation of TPX2 and FOXM1 in vivo. Furthermore, FOXM1 regulated TPX2 activity to drive HCC proliferation. Immunohistochemical (IHC) analysis indicated that FOXM1 and TPX2 were highly-expressed in HCC samples and cohort study revealed that FOXM1 and TPX2 may act as negative predictors for the prognosis of patients with HCC.ConclusionsTPX2 acts as a novel downstream target and effector of the Hh pathway, and Hh signaling contributes to HCC proliferation via regulating the FOXM1-TPX2 cascade, suggesting that this signaling axis may be a novel therapeutic target for HCC.Graphical abstract
Highlights
Aberrant activation of the Hedgehog (Hh) signaling pathway is frequently observed in hepatocellular carcinoma (HCC), the precise molecular mechanism remains unclear
Targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression was regulated by the Hh signaling pathway To further investigate the effects of aberrant Hh signaling activation on the tumorigenesis or development of HCC, gene expression profiles of HCC cells were determined by RNA-Seq after GANT61, an antagonist of Gli transcriptional factors [26], treatment
Among these 203 genes, many had been reported as GLI target genes involved in cell proliferation, such as KIF20A, Forkhead box M1 (FOXM1), and CCNB1 (Fig. 1b), which may act as positive controls for confirming the authenticity of our screening results
Summary
Aberrant activation of the Hedgehog (Hh) signaling pathway is frequently observed in hepatocellular carcinoma (HCC), the precise molecular mechanism remains unclear. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is an oncogene critical for mitosis How these molecular events affect HCC progression remains unclear. GLI2 regulates the transcription of genes directly associated with cellular proliferation, such as MYC, CCND1/2, CCNB1, and CCNE [6], and those encoding transcription factors, such as FOXM1 [6, 7], which further activates more target genes. Despite that, it remains unclear whether there are other new target genes of Hh pathway and their roles in cell proliferation
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