A novel role of hemochromatosis protein (HFE) in promoting cytokinesis in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC), one of the most prevalent malignancies, ranks second as the leading cause of cancer-related death in the globe. Cytokinesis refers to the last step of mitosis, a cell division process. HCC growth is highly dependent on correct cytokinesis, while healthy hepatocytes can tolerate cytokinesis defect well. As incomplete cytokinesis can induce chromosomal instability and influence viability in HCC cells, blocking cytokinesis could be an attractive targeted therapeutic strategy for HCC. Hemochromatosis protein (HFE) is an iron regulatory molecule highly expressed in HCC, while the function and molecular mechanism of its high expression are not known. This study addresses this knowledge gap by investigating a novel role of HFE in HCC development and to explore its potential therapeutic value. To evaluate the expression level of HFE, qRT-PCR, western blot and immunohistochemical staining assays were performed on HCC tumor tissues and adjacent non-tumor tissues. To explore the functional roles of HFE in HCC, in vitro (MTT assay, colony formation assay and wound healing assay) and in vivo (subcutaneous tumor xenograft model and orthotopic liver tumor xenograft model) experiments were performed. Time-lapse video microscopy and immunofluorescence were used to observe different cell cycle phases. To reveal the molecular mechanism of HFE, RNA sequencing analysis was employed and results were further validated with the use of various recombinant protein, inhibitors and siRNAs. HFE was markedly increased in HCC cell lines and tumor tissues. Up-regulation of HFE was significantly correlated with larger tumor size, presence of venous infiltration and advanced TNM tumor stage. Overexpression of HFE was significantly associated with poor prognosis of HCC patients, implicating its use as an independent prognostic marker for evaluating both overall and disease-free survival. Functionally, HFE significantly accelerated HCC cell growth in vitro and in vivo. Mechanistically, HFE depletion promoted HCC cell cycle arrest at G2/M phase, leading to an appearance of multi-nucleation and apoptosis induction. Centrosome amplification, multipolar spindle formation and cytokinetic abscission failure were observed in HCC cells after HFE knockdown. HFE inhibited Hippo/YAP signaling axis through triggering Smad2/3/4-mediated LIF expression. Verteporfin, a YAP inhibitor, significantly reversed the tumorigenic effect and affected the associated mechanism induced by HFE overexpression in HCC. Up-regulation of HFE expression in human HCC was correlated with worse prognosis. HFE remarkably stimulated HCC cell growth in vitro and in vivo via promoting cytokinesis through Hippo/YAP pathway inactivation. Targeting HFE/Smad/LIF/YAP cytokinetic axis may possess a therapeutic potential in HCC.