An anoikis-related four-gene prognostic signature and functional characterization of ANXA5 in laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) remains one of the most lethal malignancies of the head and neck region. Tumor metastasis, the primary cause of treatment failure, is intimately linked to the evasion of anoikis. Yet the molecular drivers of this process in LSCC are incompletely understood. This study systematically investigates the prognostic and functional landscape of anoikis-related genes (ARGs) in LSCC, with an emphasis on their interplay with the tumor immune microenvironment and therapeutic vulnerability.

METHODS: RNA-sequencing profiles and clinical data from the TCGA-LSCC cohort were analyzed, and candidate anoikis-related genes (ARGs) were curated from the GeneCards and Harmonizome databases. Differentially expressed and prognosis-associated ARGs were screened using limma and univariate Cox regression. A four-gene prognostic signature was constructed in the training set using LASSO-Cox and multivariate Cox regression and internally evaluated in the testing set. The resulting anoikis-related risk score was assessed in relation to survival, clinicopathological variables, immune characteristics, pathway enrichment, and predicted drug response. Single-cell RNA-sequencing data were used to characterize the cellular distribution of the signature genes. ANXA5 was prioritized for experimental evaluation because it had the largest positive model coefficient among the risk-associated genes, showed tumor-associated dysregulation, and had biological relevance to anoikis-related processes. Its effects were examined in TU686 cells using expression, cell-viability, EMT-marker, apoptosis, and wound-healing assays.

RESULTS: The ARG-based signature effectively stratified LSCC patients into high- and low-risk groups with significantly different overall survival (p < 0.001). High anoikis-related risk score correlated with an immunosuppressive TME, suggesting a permissive milieu for immune evasion. Drug sensitivity analysis revealed that high-risk patients were more responsive to certain tyrosine kinase inhibitors but resistant to conventional chemotherapeutics, highlighting subtype-specific therapeutic opportunities. Among the ARGs, ANXA5 emerged as a hub gene with strong prognostic power. In vitro assays demonstrated that ANXA5 exerts oncogenic functions in LSCC. Following ANXA5 knockdown, we detected suppressed proliferative activity, differential expression of EMT signature genes, elevated apoptosis rate and weakened migratory ability in laryngeal squamous carcinoma cells. Collectively, these preliminary results suggest that ANXA5 is likely implicated in regulating proliferation, mesenchymal molecular traits, cell survival and migratory behaviors of laryngeal squamous carcinoma cells.

CONCLUSION: The four-gene anoikis-related signature showed prognostic stratification within the TCGA-LSCC cohort and was associated with differences in the tumor immune microenvironment. ANXA5 may represent a candidate prognostic biomarker and a biologically relevant gene for further mechanistic investigation; however, external validation and additional functional studies are required.