ROBO1 Regulates the Junctional Actin Cytoskeleton of Pancreatic Tumor Cells Through the RhoA/ABI/Diaph1/NMII Pathway and Promotes Vasculogenic Mimicry

Abstract

SLIT ligand-ROBO receptor signaling, originally identified as a pathway involved in axon guidance, reportedly has a metastasis-regulating function in several solid tumors including pancreatic ductal adenocarcinoma (PDAC). However, its exact role and the mechanism of action are poorly understood. Immunofluorescent analysis of the subcellular distribution revealed that ROBO1 localized at filopodia, lamellipodia, ruffles, and adherens junctions of PDAC cells, overlapping with dynamic F-actin structures. ROBO1 expression levels impacted on the formation of actin-rich protrusions and ROBO1 loss resulted in disorganized cell junction–associated F-actin and disturbed adherens junctions with immature morphology. ROBO1 acted through the RhoA/ABI/Diaph1/Non-muscle Myosin II axis. Furthermore, ROBO1 supported adherens junctions and filopodial alignment accompanying vasculogenic mimicry (VM), a pro-metastatic competence of PDAC cells. Accordingly, expression of a VM gene signature (which includes RhoA) positively correlated with ROBO1 expression in tumor cells isolated from PDAC patients, validating its potential relevance in vivo. These results demonstrate that ROBO1 regulates actin cytoskeletal organization at tumor cell peripheries, thereby modulating cell-cell contacts and VM formation in pancreatic cancer.