Inactivation of the tumor suppressor gene RASSF1A by promoter hypermethylation represents a key event underlying the initiation and progression of lung cancer. RASSF1A inactivation is also associated with poor prognosis and may promote metastatic spread. In this study, we investigated how RASSF1A inactivation conferred invasive phenotypes to human bronchial cells. RNAi-mediated silencing of RASSF1A induced epithelial-to-mesenchymal transition (EMT), fomenting a motile and invasive cellular phenotype in vitro and increased metastatic prowess in vivo. Mechanistic investigations revealed that RASSF1A blocked tumor growth by stimulating cofilin/PP2A–mediated dephosphorylation of the guanine nucleotide exchange factor GEF-H1, thereby stimulating its ability to activate the antimetastatic small GTPase RhoB. Furthermore, RASSF1A reduced nuclear accumulation of the Hippo pathway transcriptional cofactor Yes-associated protein (YAP), which was reinforced by RhoB activation. Collectively, our results indicated that RASSF1 acts to restrict EMT and invasion by indirectly controlling YAP nuclear shuttling and activation through a RhoB-regulated cytoskeletal remodeling process, with potential implications to delay the progression of RASSF1-hypermethylated lung tumors. Cancer Res; 76(6); 1627–40. ©2016 AACR.

The Ras association domain family protein1 isoform A (RASSF1A) is a well-known tumor-suppressor protein frequently inactivated in various human cancers. Consistent with its function as a molecular scaffold protein, referred to in many studies, RASSF1A prevents initiation of tumorigenesis, growth, and dissemination through different biological functions, including cell cycle arrest, migration/metastasis inhibition, microtubular stabilization, and apoptosis promotion. As a regulator of key cancer pathways, namely Ras/Rho GTPases and Hippo signaling without ignoring strong interaction with microtubules, RASSF1A is indeed one of the guardians of cell homeostasis. To date, as we approach the two decade anniversary of RASSF1A’s discovery, this review will summarize our current knowledge on the RASSF1A key interactions as a tumor suppressor and discuss their impact on cell fate during carcinogenesis. This could facilitate a deeper understanding of tumor development and provide us with new strategies in cancer treatment by targeting the RASSF1A pathway.

By allowing insured communication between cancer cells themselves and with the neighboring stromal cells, tunneling nanotubes (TNTs) are involved in the multistep process of cancer development from tumorigenesis to the treatment resistance. However, despite their critical role in the biology of cancer, the study of the TNTs has been announced challenging due to not only the absence of a specific biomarker but also the fragile and transitory nature of their structure and the fact that they are hovering freely above the substratum. Here, we proposed to review guidelines to follow for studying the structure and functionality of TNTs in tumoral neuroendocrine cells (PC12) and nontumorigenic human bronchial epithelial cells (HBEC‐3, H28). In particular, we reported how crucial is it (i) to consider the culture conditions (culture surface, cell density), (ii) to visualize the formation of TNTs in living cells (mechanisms of formation, 3D representation), and (iii) to identify the cytoskeleton components and the associated elements (categories, origin, tip, and formation/transport) in the TNTs. We also focused on the input of high‐resolution cell imaging approaches including Stimulated Emission Depletion (STED) nanoscopy, Transmitted and Scanning Electron Microscopies (TEM and SEM). In addition, we underlined the important role of the organelles in the mechanisms of TNT formation and transfer between the cancer cells. Finally, new biological models for the identification of the TNTs between cancer cells and stromal cells (liquid air interface, ex vivo, in vivo) and the clinical considerations will also be discussed.

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. 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 48 h 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. 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. 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.

The focal adhesion proteins, Hic-5 and Paxillin have been previously identified as key regulators of MDA-MB-231 breast cancer cell migration and morphologic mesenchymal-amoeboid plasticity in three-dimensional extracellular matrices. However, their respective roles in other cancer cell types has not been evaluated. Herein, utilizing 3D cell-derived matrices and fibronectin-coated 1D substrates, we show that across a variety of cancer cell lines, the level of Hic-5 expression serves as the major indicator of the cells primary morphology, plasticity and in vitro invasiveness. Domain mapping studies reveal sites critical to the functions of both Hic-5 and Paxillin in regulating phenotype, while ectopic expression of Hic-5 in cell lines with low endogenous levels of the protein is sufficient to induce a Rac1-dependent mesenchymal phenotype and, in turn increase amoeboid-mesenchymal plasticity and invasion. We show that the activity of Vinculin, when coupled to the expression of Hic-5 is required for the mesenchymal morphology in 3D ECM. Taken together, our results identify Hic-5 as a critical modulator of tumor cell phenotype that could be utilized in predicting tumor cell migratory and invasive behavior in vivo.

Cell polarization and directed migration play pivotal roles in diverse physiological and pathological processes. Herein, we identify new roles for paxillin-mediated HDAC6 inhibition in regulating key aspects of cell polarization in both two-dimensional and one-dimensional matrix environments. Paxillin, by modulating microtubule acetylation through HDAC6 regulation, was shown to control centrosome and Golgi reorientation toward the leading edge, a hallmark of cell polarization to ensure directed trafficking of promigratory factors. Paxillin was also required for pericentrosomal Golgi localization and centrosome cohesion, independent of its localization to, and role in, focal adhesion signaling. In addition, we provide evidence of an accumulation of paxillin at the centrosome that is dependent on focal adhesion kinase (FAK) and identify an important collaboration between paxillin and FAK signaling in the modulation of microtubule acetylation, as well as centrosome and Golgi organization and polarization. Finally, paxillin was also shown to be required for optimal anterograde vesicular trafficking to the plasma membrane.