CSMD3 loss drives aggressive behavior and tumor consolidation in oral squamous premalignant cells

Oral squamous cell carcinoma (OSCC) remains a significant global health burden. Genomic data identify Csmd3 as the third most frequently mutated gene in HNSCC (29% of cases) correlating with poor clinical outcome and prognosis. Despite this, the functional role of CSMD3 in oral premalignancy and malignant transformation remains largely unexplored. In this study, we utilized CRISPR-Cas9 system to inactivate CSMD3 in an oral premalignant cell line and characterize knockout in in vitro oral premalignant cell line and in vivo orthotopic model. Functional assays demonstrate that CSMD3 loss significantly enhances cell survival (P < 0.001), while decreasing migration (P = 0.005). Transcriptomic profiling and Gene Set Enrichment Analysis (GSEA) revealed multi-faceted reprogramming characterized by enriched expression of oncogenic survival and stress signaling genes, metabolic rewiring, inflammatory mediators, and structural remodeling driven by altered expression of contraction, actin cross-linking proteins, and polymerization genes. Orthotopic modeling further revealed that CSMD3 loss resulted in significantly increased tumor area fraction (P < 0.0001) and intratumoral cellular density (P < 0.0001). Our study emphasizes the role of CSMD3 loss in promoting aggressive behavior of oral premalignancy, which has a potential for risk stratification, a novel prognostic or diagnostic biomarker, and a target for therapeutic intervention.