Cancer-associated fibroblast-derived fibulin-5 promotes epithelial–mesenchymal transition in diffuse-type gastric cancer via cAMP response element-binding protein pathway, showing poor prognosis

Diffuse-type gastric cancer (DGC), characterized by poorly cohesive cells within fibrotic stroma, is associated with advanced disease and poor prognosis. Here, to identify distinct biomarkers for DGC compared with intestinal-type gastric cancer, we constructed a comprehensive large-scale signaling network using RNA-sequencing data from three genomic databases (The Cancer Genome Atlas, GSE62254 and GSE26253), developed a mathematical model and conducted simulation analyses. For validation, we used tissue microarray blocks of gastric cancers with immunohistochemical staining, single-cell RNA sequencing, primary cultures of cancer-associated fibroblasts (CAFs) and organoids, and a co-culture system involving CAFs and cancer cells. Signaling network analysis identified six differentially activated signaling components across the database, including BIRC5, TTK, NEK2, FHL1, NR2F1 and FBLN5. Among the differentially activated signaling components, high tumoral expression of fibulin-5 protein encoded by FBLN5 correlated with poor overall and disease-specific survival rates in patients with DGC, even after adjusting for the tumor, node, metastases (TNM) stage. Fibulin-5, derived from CAFs within DGC stroma, promoted organoid growth and epithelial-mesenchymal transition (EMT) in DGC cell lines via the cAMP response element-binding protein (CREB) pathway in a CAF co-culture system. FBLN5 knockdown in CAFs reduced the aggressive phenotype of co-cultured DGC cells, while CREB inhibitors reversed EMT. Furthermore, levels of secreted FBLN5 in patient blood samples correlated with its expression in primary tumors. In summary, fibulin-5 secreted by CAFs and interacted with DGC cells promotes EMT and is clinically associated with poor patient outcomes. These findings suggest fibulin-5 as a potential prognostic marker and therapeutic target in patients with DGC.