Axon guidance molecule PLXNA3 promotes tumorigenesis and metastasis in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a highly heterogeneous and aggressive cancer with poor prognosis. Axon guidance molecules, such as Plexin A3 (PLXNA3), have been implicated in cancer growth and metastasis. Initial screening identified PLXNA3 as a candidate for in depth study, as it is highly expressed in tumor tissues compared to normal counterparts, associated with high alpha-fetoprotein levels, and identified as an oncofetal driver. This study aimed to investigate the involvement of PLXNA3 in HCC progression. Analysis of clinical datasets, TCGA-LIHC and GSE14520 revealed PLXNA3 expression to be significantly correlated with more aggressive tumor features, such as poor differentiation and advanced stage, as well as worse 5-year overall survival. PLXNA3 also positively correlated with progenitor markers and negatively with mature hepatocyte markers. To understand the functional role of PLXNA3, HCC cells were manipulated through CRISPR activation and CRISPR knockout of PLXNA3 in Huh1 cells and HepG2 cells respectively. Functional assays demonstrated that PLXNA3 enhanced migration, invasion, tumor-initiating potential, and foci formation abilities of HCC cells. In vivo orthotopic liver injection model and in vivo limiting dilution assay further validated the functional role of PLXNA3 in HCC. Using clinical datasets, hypoxia was found to modulate PLXNA3 expression, where high hypoxic signature scores were significantly correlated with high PLXNA3 expression. In vitro culture of HCC cells in hypoxic conditions confirmed that hypoxia drives increased transcriptional activity of PLXNA3, thereby elevated PLXNA3 gene and protein expression independent of the HIF1 system. Further analysis revealed histone modification, H3K4me3, to be significantly increased at the promoter region of PLXNA3 after hypoxia to drive expression of PLXNA3. To gain insights into the signaling pathway of PLXNA3, we coupled gene set enrichment analyses on RNA-seq and TCGA-LIHC datasets. We unraveled enrichment of TGF-β signaling and epithelial-mesenchymal transition (EMT) pathways in high PLXNA3-expressing cells and HCC patients, discovering the novel downstream signaling network of PLXNA3 in HCC. The findings from this study highlight the important role of PLXNA3 in HCC progression and provide valuable insights into the underlying molecular mechanisms. The discovery that PLXNA3 is upregulated under hypoxic conditions, together with the potential signaling pathways enriched, EMT, suggests that PLXNA3 may be a critical player in driving HCC aggressiveness and metastasis. These insights reveal PLXNA3 as a potential therapeutic target and novel prognostic biomarker for HCC, especially for metastatic HCC.