Abstract 226: Methylation specific silencing of miR-9 is associated with an epithelial-mesenchymal transition (EMT) phenotype in laryngeal squamous cell carcinoma

Abstract Background: Advanced stage laryngeal squamous cell carcinoma (LSCC) carries one of the highest mortality rates of all head and neck cancers. We previously demonstrated that low miR-9 expression impacts invasiveness and resistance to cisplatin in advanced stage LSCC. Here, we seek to further elucidate how low miR-9 influences LSCC pathogenesis. Methods: Gene expression and clinicopathologic data were obtained for 116 LSCC patients from The Cancer Genome Atlas (TCGA) and used to evaluate predictors of LSCC overall survival. A simple logistic regression combined with a Receiver Operating Characteristic (ROC) curve was used to determine a cutoff for categorizing LSCC patients into low and high miR-9 expression groups. A panel of epithelial-mesenchymal transition (EMT) related genes (MAP1B, DOCK10, FGFR1, FGF1, ZEB1, VCAN, WNT5A, SRGN, THEM30B, CDH1 and EGFR) and methylated miR-9 transcripts were assessed between low and high miR-9 expressing LSCC patients via Principal Component Analysis. In vitro validation of an EMT phenotype and miR-9 promoter hypermethylation was performed using four advanced stage LSCC cell lines with variable miR-9 expression. Cell line EMT gene expression was evaluated via RNA sequencing and validated by qPCR. Methylation and chromatin accessibility of the three miR-9 promoter regions were quantified in each LSCC cell line through PacBio sequencing. Low miR-9 expressing cell lines were treated with the global demethylating agent, 5-Azacytidine (0μM, 0.1μM, 0.5μM and 1μM), and differences in EMT-related gene expression were assessed via qPCR. Results: Low miR-9 and methylation of miR-9 were found to be significant predictors of poor LSCC survival (p < 0.05). Gene signatures representing a high EMT phenotype and high methylated miR-9 status were identified as unique classifiers of low compared with high miR-9 expressing LSCC patients. Differential expression of EMT related genes was detected in low compared with high miR-9 expressing cell lines; low miR-9 cell lines had significantly higher baseline expression of MAP1B, DOCK10, FGFR1, FGF1, and ZEB1 (>2-fold, p < 0.0001) and significantly lower baseline expression of CDH1 and EGFR (>2-fold, p < 0.0001). Low miR-9 expressing cell lines also had significantly greater methylation of miR-9 promoter regions (p < 0.0001) as well as significantly fewer chromatin accessibility sites open for miR-9 transcription (p < 0.0001). Global demethylation treatment of the low miR-9 expressing cell lines dose dependently restored miR-9 expression (>2-fold, p < 0.0001) and resulted in inverse expression of MAP1B, DOCK10, FGFR1, FGF1, ZEB1, CDH1 and EGFR at the 1μM dose (>2-fold, p < 0.0001). Conclusion: Our findings indicate that methylation specific silencing of miR-9 may be a mechanism that yields a high EMT phenotype, predictive of poor overall survival in LSCC. Citation Format: Christina Gobin, Matthew Chang, Marie Gauthier, Chayil C. Lattimore, Michael Kladde, Kristianna Fredenburg. Methylation specific silencing of miR-9 is associated with an epithelial-mesenchymal transition (EMT) phenotype in laryngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 226.