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. 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. 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. 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.

中文翻译：

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失调的 Hh-FOXM1-TPX2 信号在人肝细胞癌细胞增殖中的关键作用。

Hedgehog (Hh) 信号通路的异常激活在肝细胞癌 (HCC) 中经常观察到，然而，确切的分子机制仍不清楚。Forkhead box M1 (FOXM1) 是 Hh 通路的目标，是关键的癌胚转录因子和主细胞周期调节剂。非洲爪蟾驱动蛋白样蛋白 2 (TPX2) 的靶向蛋白是对有丝分裂至关重要的致癌基因。然而，这些分子事件如何影响 HCC 进展仍不清楚。进行实时 PCR、免疫组织化学、蛋白质印迹以及数据集 TCGA 和基因表达综合 (GEO) 的分析，以评估 TPX2 和 FOXM1 在 HCC 样本或 HCC 细胞中的 mRNA 和蛋白质水平的表达。进行 TPX2 和 FOXM1 的表达和敲低以评估它们在体外和体内调节 HCC 细胞增殖中的作用。研究了双荧光素酶报告测定和染色体免疫沉淀 (ChIP) 以寻找 TPX2 启动子中的 FOXM1 结合位点。Hh 通路的特定拮抗剂（环巴胺和 GANT61）下调 TPX2，而 Hh 信号的激活刺激 TPX2 表达。此外，当 Hh 信号的上游事件被抑制时，TPX2 过表达加速了 HCC 细胞增殖，并且 TPX2 敲低显着减轻了 Sonic Hh 配体 (Shh) 诱导的 HCC 细胞增殖。报告基因检测和 ChIP 显示 FOXM1 与 TPX2 启动子结合，证实 TPX2 是 FOXM1 的直接下游靶标。异种移植模型进一步验证了TPX2和FOXM1在体内的细胞功能和表达调控。此外，FOXM1 调节 TPX2 活性以驱动 HCC 增殖。免疫组织化学（IHC）分析表明 FOXM1 和 TPX2 在 HCC 样本中高表达，队列研究显示 FOXM1 和 TPX2 可能作为 HCC 患者预后的阴性预测因子。TPX2 作为 Hh 通路的新下游靶点和效应子，Hh 信号通过调节 FOXM1-TPX2 级联反应促进 HCC 增殖，表明该信号轴可能是 HCC 的新治疗靶点。