A role for RASSF1A in tunneling nanotube formation between cells through GEFH1/Rab11 pathway control

Background: By allowing intercellular communication between cells, tunneling nanotubes (TNTs) could play critical role in cancer progression. If TNT formation is known to require cytoskeleton remodeling, key mechanism controlling their formation remains poorly understood. Methods: The cells of human bronchial (HBEC-3, A549) or mesothelial (H2452, H28) lines are transfected with different siRNAs (inactive, anti-RASSF1A, anti-GEFH1 and / or anti-Rab11). At 48h post-transfection, i) the number and length of the nanotubes per cell are quantified, ii) the organelles, previously labeled with specific tracers, exchanged via these structures are monitored in real time between cells cultured in 2D or 3D and in normoxia, hypoxia or in serum deprivation condition. Results: We report that RASSF1A, a key-regulator of cytoskeleton encoded by a tumor-suppressor gene on 3p chromosome, is involved in TNTs formation in bronchial and pleural cells since controlling proper activity of RhoB guanine nucleotide exchange factor, GEF-H1. Indeed, the GEF-H1 inactivation induced by RASSF1A silencing, leads to Rab11 accumulation and subsequent exosome releasing, which in turn contribute to TNTs formation. Finally, we provide evidence involving TNT formation in bronchial carcinogenesis, by reporting that hypoxia or nutriment privation, two almost universal conditions in human cancers, fail to prevent TNTs induced by the oncogenic RASSF1A loss of expression. Conclusions: This finding suggests for the first time that loss of RASSF1A expression could be a potential biomarker for TNTs formation, such TNTs facilitating intercellular communication favoring multistep progression of bronchial epithelial cells toward overt malignancy.