RalGPS2 Interacts with Akt and PDK1 Promoting Tunneling Nanotubes Formation in Bladder Cancer and Kidney Cells Microenvironment

SIMPLE SUMMARY: Cell-to-cell communication in the tumor microenvironment is a crucial process to orchestrate the different components of the tumoral infrastructure. Among the mechanisms of cellular interplay in cancer cells, tunneling nanotubes (TNTs) are dynamic connections that play an important role. The mechanism of the formation of TNTs among cells and the molecules involved in the process remain to be elucidated. In this study, we analyze several bladder cancer cell lines, representative of tumors at different stages and grades. We demonstrate that TNTs are formed only by mid or high-stage cell lines that show muscle-invasive properties and that they actively transport mitochondria and proteins. The formation of TNTs is triggered by stressful conditions and starts with the assembly of a specific multimolecular complex. In this study, we characterize some of the protein components of the TNTs complex, as they are potential novel molecular targets for future therapies aimed at counteracting tumor progression. ABSTRACT: RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor for RalA GTPase that is involved in several cellular processes, including cytoskeletal organization. Previously, we demonstrated that RalGPS2 also plays a role in the formation of tunneling nanotubes (TNTs) in bladder cancer 5637 cells. In particular, TNTs are a novel mechanism of cell–cell communication in the tumor microenvironment, playing a central role in cancer progression and metastasis formation. However, the molecular mechanisms involved in TNTs formation still need to be fully elucidated. Here we demonstrate that mid and high-stage bladder cancer cell lines have functional TNTs, which can transfer mitochondria. Moreover, using confocal fluorescence time-lapse microscopy, we show in 5637 cells that TNTs mediate the trafficking of RalA protein and transmembrane MHC class III protein leukocyte-specific transcript 1 (LST1). Furthermore, we show that RalGPS2 is essential for nanotubes generation, and stress conditions boost its expression both in 5637 and HEK293 cell lines. Finally, we prove that RalGPS2 interacts with Akt and PDK1, in addition to LST1 and RalA, leading to the formation of a complex that promotes nanotubes formation. In conclusion, our findings suggest that in the tumor microenvironment, RalGPS2 orchestrates the assembly of multimolecular complexes that drive the formation of TNTs.