A novel mTORC2 signaling pathway defines the pro-metastatic invadopodia network in urothelial carcinoma

Background Urothelial carcinoma (UC), the most common form of bladder cancer, remains a leading cause of cancer-related mortality worldwide. UC outcomes are driven primarily by stage and grade of the tumor, with highlyinvasive and metastatic tumors showing significantly reduced treatment outcomes. Despite ongoing research,mechanisms that define this behavior in UC remain poorly understood. Our laboratory has identifiedmammalian target of rapamycin complex 2 (mTORC2) as a key regulator of invasive behavior in UC and anassociation between increased mTORC2 activity and higher UC stage. Methods Gene and protein analyses were performed using Gene Set Enrichment Analysis (GSEA) andphosphoproteomic profiling. Immunohistochemistry and multi-Immunol fluorescence (mIF) assays were used todetermine expression and co-localization of proteins within invadopodia. Targeting of pathway componentswas performed using RNA silencing and inhibitor application, with functional outputs assayed using gelatindegradation assay, transwell invasion, and cell migration assessments. Results Our study identified that mTORC2 can interact with TKS5alpha, an invadopodial scaffold protein, through putativemTOR phosphorylation. Furthermore, mTORC2 and TKS5alpha appear co-localize within cells and to the invasiveedge of tumors. Silencing of either mTORC2 or TKS5alpha results in similar phenotypes in cell invasion and matrixdegradation. Analysis of the Cancer Genome Atlas UC cohort showed high TKS5alpha expression was associatedwith reduced survival, and GSEA analysis showed PI3K/AKT/mTOR signaling was significantly enriched intumors with high TKS5alpha expression. These findings suggest that mTORC2 may promote pro-invasive andpro-metastatic activities through invadopodia formation and via a novel, and actionable, signaling cascade thatincludes TKS5alpha. Summary Our studies support a critical role for mTORC2 in driving invasion and metastasis in UC, as demonstrated byboth in vitro and human bladder cancer sample analysis. The study introduces a previously unrecognizedsignaling axis involving mTORC2 and TKS5alpha in invadopodia formation and metastasis in UC. The results fromthese studies have the potential to define a novel signaling axis in UC invadopodia that broadens ourmechanistic understanding of bladder