HOXA9 orchestrates EMT and metastasis in oral cancer via transcriptional activation of vimentin and β-catenin signaling

Abstract Identifying biomarkers of epithelial‒mesenchymal transition (EMT) and metastasis play a decisive role in the prognosis and clinical management of Oral cancer (OC). Homeobox A9 (HOXA9), an imperative regulator of embryogenesis and endothelial cell proliferation, is aberrantly expressed in several malignancies, including OC. To date, HOXA9-mediated molecular mechanisms and their functional roles in EMT and OC metastasis remain poorly understood. In this study, we observed significant upregulation of HOXA9 in OC samples with lymph node-positive stage and higher histological grade. Notably, we demonstrated that retroviral-mediated knockdown of HOXA9 impairs proliferation, migration, and invasion while promoting apoptosis and cell cycle arrest. A substantial reduction in tumor volume and diminished lung metastasis was observed in nude mice receiving HOXA9-knockdown cells. Transcriptomic analysis of HOXA9-depleted cells revealed the downregulation of multiple pathways, with the most significant being “pathways in cancer”. We further demonstrated that HOXA9 transcriptionally activates VIM to promote EMT and it also facilitates β-catenin nuclear translocation, which subsequently activates downstream target genes driving Wnt/β-catenin cascade. Our study also unveils HOXA9-driven molecular interplay in which HOXA9-dependent transcriptional activation of VIM triggers Wnt/β-catenin cascade in OC. Furthermore, we demonstrated that hypomethylated CpGs at HOXA9 promoter (spanning −4201bp to −3574bp upstream of TSS) in stage III/IV tumors showed a significant inverse correlation between hypomethylation and gene upregulation. Consistently, analysis of the same distal promoter region revealed the enrichment of activating histone modifications, further supporting its transcriptional permissive state. Collectively, our study uncovers a novel mechanism by which epigenetically altered HOXA9 serves as a potential inducer of EMT through regulation of VIM/Wnt-β-catenin/EMT signaling axis. These findings signify HOXA9 as a promising biomarker in OC and targeting HOXA9 could be an effective strategy to improve clinical outcome of patients diagnosed with advanced stages.