HOXA5 silencing via DNA methylation reveals the critical role of HOX gene deregulation in oral carcinogenesis

HOXA5 , a member of the HOX family, has been characterized as a tumor suppressor in several cancers. However, its regulation and role in oral cancer (OC) remain unclear. HOXA5 expression was assessed by transcriptome analysis and qRT-PCR, while methylation was evaluated using methyl-capture sequencing and bisulfite Sanger sequencing in a clinical cohort comprising OPMD, LN(-) and LN(+) groups and in vitro models. The effects of DNA methylation and retinoic acid were examined using DNMT inhibitors (DNMTi) and/or retinoic acid (RA). Promoter activity and RARB occupancy were assessed by dual-luciferase reporter and ChIP-qPCR assays, respectively. Effects of ectopic HOXA5 expression were evaluated using functional assays, whole-transcriptome profiling, and in-silico analyses. HOXA5 was upregulated and relatively hypomethylated in OPMD and the dysplastic DOK cell line, whereas its expression did not differ between LN(-) or LN(+) tumors and matched normal tissues despite increased promoter methylation. In SCC9 cells, HOXA5 was downregulated and promoter hypermethylated. Weak but significant inverse correlations between expression and promoter methylation were observed. DNMTi and/or RA treatment reversed HOXA5 expression. HOXA5- EPD2 region showed the highest promoter activity and RARB occupancy at the promoter regions was validated. HOXA5 overexpression reduced proliferation, migration, and invasion, while inducing S-phase arrest and apoptosis. Transcriptome and enrichment analyses identified pathways associated with cancer-related phenotypes, while protein interaction and in silico drug sensitivity analyses identified candidate drug-gene associations for further investigation in OC. Our findings support a role for DNA methylation in HOXA5 regulation and suggest tumor-suppressive properties of HOXA5 in OC.