NOTCH4 as a driver of epithelial-mesenchymal transition, angiogenesis, and stromal remodeling in lung adenocarcinoma: integrated transcriptomic and protein-level validation.

Although NOTCH4 plays an important role in tumor progression, its role in lung adenocarcinoma (LUAD) remains unclear. We examined the role of NOTCH4 in LUAD progression, its influence on the tumor microenvironment (TME), and its prognostic value, while isolating its specific contribution from those of NOTCH1 and NOTCH3. Gene set enrichment analysis (GSEA) and deconvolution-based TME analyses (xCell, ESTIMATE, and MCP-counter) were performed using three independent LUAD cohorts [The Cancer Genome Atlas (TCGA), OncoSG, and CPTAC]. Associations between NOTCH4 expression and epithelial-mesenchymal transition (EMT), angiogenesis, stromal remodeling, and survival were further validated using immunohistochemistry and histopathology in an independent LUAD cohort (n=347). GSEA revealed significant enrichment of the EMT pathway in NOTCH4-high tumors across all three mRNA datasets. Deconvolution analyses demonstrated that NOTCH4-high tumors exhibited increased numbers of endothelial cells, fibroblasts, pericytes, and higher stromal and microenvironment scores, particularly in TCGA and OncoSG cohorts. In CPTAC, NOTCH4-high tumors showed enrichment of endothelial and fibroblast signatures, although global stromal and immune scores were not significantly different. In the independent cohort, after adjusting for NOTCH1 and NOTCH3 levels as covariates, NOTCH4 remained significantly associated with vimentin (Rho =0.160, P=0.007), microvessel density (MVD) (Rho =0.279, P<0.001), and stromal proportion (Rho =0.274, P<0.001), confirming its independent correlation with these markers. High NOTCH4 expression at both the mRNA and protein levels was associated with poorer overall survival (P=0.009 and P=0.009, respectively). Our findings provide the first systematic evidence that NOTCH4 is associated with EMT, angiogenesis, and stromal remodeling in LUAD, independent of NOTCH1 and NOTCH3 expression. By validating these associations at the protein level in an independent cohort, we highlight the translational potential of NOTCH4 as a prognostic biomarker and therapeutic target.