The critical role of dysregulated Hh-FOXM1-TPX2 signaling in human hepatocellular carcinoma cell proliferation

Abstract

Background: Aberrant 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. Methods: Realtime 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. Results: Specific 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. Conclusions: TPX2 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.

TPX2 Induces Mitotic Survival via BCL2L1 Induction Through YAP1 Protein Stabilization in Human Embryonic Stem Cells

“…Therefore, we propose that other cues to activate BCL2L1 promoter activity such as YAP1 activation would be required to confer the cells with the survival advantage trait (e.g., YAP1 stabilization by Aurora-A activity due to TPX2 induction). A recent study demonstrated that the Hedgehog signal is responsible for TPX2 induction through the FOXM1 transcription factor in cancer cells [30]. Considering the complexity of the YAP1 activation mechanism, we could not rule out the role of other events (e.g., Factin stabilization [24]) in YAP1 activation for culture adaptation of hESCs other than the TPX2-Aurora-A axis.…”

“…2D). Considering the roles of TPX2 in cancer malignancy [30] and survival/chemoresistance [31], it is readily presumed that TPX2 expression may be associated with BCL2L1 induction and consequent survival traits. As predicted, depletion of TPX2 in P4 hESCs signi cantly attenuated BCL2L1 expression (Fig.…”

Hedgehog−Gli2 Signaling Promotes Chemoresistance in Ovarian Cancer Cells by Regulating MDR1

“…Tumor tissues collected from mice were fixed in 10% neutral-buffered formalin solution, dried, and embedded in paraffin blocks, which were subsequently cut into 3-μm-thick sections for IHC staining ( 28 ).…”

TPX2 Amplification-Driven Aberrant Mitosis in Culture Adapted Human Embryonic Stem Cells With Gain of 20q11.21

“…The genetic abrogation of TPX2 in mouse embryos leads to arrest at the morula stage and early embryonic lethality due to the incorrect formation of bipolar spindles [31] and the missegregation of chromosomes, implying that critical roles of TPX2 in early embryonic development [32]. On the other hand, the ectopic expression TPX2 is su cient to induce polyploidy [33] and elevates malignancy in cancers [34]. Considering the strong association of TPX2 expression with CIN in cancer [35], and with poor patient survival in various forms of cancers [28,34], the deregulation of TPX2 is a putative driver of CIN by accelerating aberrant mitotic spindle dynamics and improper chromosome segregation.…”

“…On the other hand, the ectopic expression TPX2 is su cient to induce polyploidy [33] and elevates malignancy in cancers [34]. Considering the strong association of TPX2 expression with CIN in cancer [35], and with poor patient survival in various forms of cancers [28,34], the deregulation of TPX2 is a putative driver of CIN by accelerating aberrant mitotic spindle dynamics and improper chromosome segregation.…”