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

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.

Intercellular Communication in the Brain through Tunneling Nanotubes

“…RalGPS2 is a Ras-independent guanine nucleotide exchange factor for the RalA GTPase. It was demonstrated to allow TNT formation in bladder and kidney cancer cells through its interaction with Akt and PDK1, leading to activation of the Akt/PI3K/mTOR pathway, as well as with LST-1 and RalA [ 53 ]. The exocyst complex has been further involved in TNT-mediated cargo trafficking as Sec3 and Sec5 proteins, which are part of this complex, were identified at the site of actin cytoskeleton recruitment in TNTs between natural killer cells and MDA-MB-231 breast cancer cells and shown to promote mitochondria trafficking between these cells [ 54 ].…”

“…Other extracellular stimuli can support cellular metabolic stress. They include acidic microenvironment (pH 6.6), hypoxia (1% O 2 ), and serum starvation [ 32 , 53 , 55 ]. These metabolic stress induced TNT formation in human PC3 and LNCaP prostate cancer cell lines via the stress-induced chaperones clusterin (CLU) and YB-1 (Y-box binding protein-1) and PI3K/Akt activation [ 55 ].…”

“…As an example, oxidative stress (H 2 O 2 ) promoted TNT formation in HEK293 cells while it had no effect on 5637 bladder cancer cells. On the other hand, 5637 cells showed greater TNT formation under acidic conditions as well as under serum deprivation conditions [ 53 ]. Besides, as expected from the observed TNT stimulatory effect of PI3K/Akt/mTOR signaling, inhibition of mTOR activity, by rapamycin or by the rapamycin derivative everolimus, suppressed TNT formation in murine astrocytes and human mesothelioma MSTO-211H cells, respectively [ 32 , 58 ].…”

Tunneling Nanotubes: A New Target for Nanomedicine?

“…The exchange of misfolded proteins and damaged genetic material through TNTs in cancers is considered one of the major phenomena that contribute to the transformation of healthy cells into tumoral cells and increases in metastasis formation [5,27]. While TNTs have been linked to communication and spreading in several types of cancer [28], such as prostate [29], bladder [30][31][32], pancreatic [33], and breast cancer [34], as well as different types of leukemia [35][36][37]. Glioblastoma multiforme (GBM) is by far the most studied for the consequences of TNT activity [4,38].…”

Tunneling Nanotube-Mediated Communication: A Mechanism of Intercellular Nucleic Acid Transfer

“…The existing research on TNTs in physiological and pathological conditions demonstrates that TNT formation is highly sensitive to extracellular environmental stimuli [ 61 , 62 ]. Indeed, TNT formation is greatly augmented by cells exposed to oxidative stress, neurodegenerative oligomers, inflammation, radiation, or trauma, as well as in cells undergoing apoptosis [ 63 , 64 , 65 , 66 , 67 , 68 , 69 ]. In fact, the activation of several signaling pathways has been associated with the stress-induced increase in TNT formation [ 33 , 34 , 62 , 70 ].…”