Event Abstract Back to Event NaV1.5 sodium channels increase breast cancer cell invadopodial activity by both controlling Src kinase-dependent F-actin polymerization and promoting NHE-1-dependent proton efflux and extracellular matrix digestion Lucie Brisson1, Virginie Driffort1, Lauriane Benoist1, Mallorie Poet2, Laurent Counillon2, Ester Antelmi3, Rosa Rubino3, Pierre Besson1, Fabien Labbal2, Stépan Chevalier1, Stephan J. Reshkin1, 3, Jacques Goré1 and Sébastien Roger1* 1 UMR inserm 1069 Université François-Rabelais de Tours, France 2 CNRS FRE3472; Université de Nice-Sophia Antipolis, France 3 Department of Biosciences, Biotechnology and Pharmacological Sciences, University of Bari, Italy Background and aim. The degradation of the extracellular matrix (ECM) by cancer cells is a critical essential step in metastatic progression. NaV1.5 sodium channels are overexpressed in breast tumours and associated with metastatic occurrence. NaV1.5 activity in breast cancer cells promotes ECM degradation through the perimembrane acidification and the subsequent activation of cysteine cathepsins [1,2]. The aim of this study was to identify cellular pathways involved in the NaV1.5-dependent H+ efflux and invasiveness of highly aggressive human MDA-MB-231 breast cancer cells. Methods. Highly aggressive MDA-MB-231 breast cancer cells express functional NaV1.5 channels. By using cell fractionation and microscopy analyses, we studied the presence of NaV1.5 channels in invadopodia which are key cellular structures for cancer cells invasiveness. In MDA-MB-231-derived cell lines expressing or not NaV1.5 channels (shRNA) we examined the ability of cancer cells to form invadopodia, to invade through an ECM composed of Matrigel™, and to regulate the efflux of protons. Results. We showed that NaV1.5 was co-localised with NHE-1, and caveolin-1 in MDA-MB-231 breast cancer cells invadopodia, at sites of ECM remodelling. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicated a close association between these proteins. The expression of NaV1.5 was responsible for the allosteric modulation of NHE-1 rendering it more active at intracellular pH range 6.4 to 7, thus potentially extruding more protons in the extracellular space. Furthermore, NaV1.5 increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, controlled F-actin polymerization and the acquisition of an invasive morphology. Conclusions. This study suggests that NaV1.5 is an important regulator of invadopodia formation (F-actin polymerization) and degradative activity (NHE-1 over-activation resulting in extracellular acidic proteases activation) in breast cancer cells [3]. Acknowledgements This work was supported by the "Ministère de la Recherche et des Technologies", the Inserm, the "Ligue Nationale Contre le Cancer", by the “Programme Hubert Curien – Galileo” (Campus France) and the "Associazione Italiana per la Ricerca sul Cancro" (AIRC) grant #11348 to SJR. SJR was recipient of a visiting professorship position at the University of Tours in 2013-2014. References 1. Gillet, L., Roger, S., Besson, P., Lecaille, F., Gore, J., Bougnoux, P., Lalmanach, G. and Le Guennec, J.Y. (2009) Voltage-gated Sodium Channel Activity Promotes Cysteine Cathepsin-dependent Invasiveness and Colony Growth of Human Cancer Cells. J Biol Chem, 284, 8680-91. 2. Brisson, L., Gillet, L., Calaghan, S., Besson, P., Le Guennec, J.Y., Roger, S. and Gore, J. (2011) Na(V)1.5 enhances breast cancer cell invasiveness by increasing NHE1-dependent H(+) efflux in caveolae. Oncogene, 30, 2070-6. 3. Brisson, L., Driffort, V., Benoist, L., Poet, M., Counillon, L., Antelmi, E., Rubino, R., Besson, P., Labbal, F., Chevalier, S., Reshkin, S.J., Gore, J. and Roger, S. (2013) NaV1.5 sodium channels allosterically regulate the NHE-1 exchanger and promote breast cancer cell invadopodial activity. J Cell Sci. Nov 1;126(Pt 21):4835-42. doi: 10.1242/jcs.123901. Epub 2013 Jul 31 Keywords: voltage-gated sodium channels, NHE-1, pH, cancer cell invasiveness, invadopodia Conference: 4th Annual Meeting of the International Society of Proton Dynamics in Cancer, Garching, Germany, 10 Oct - 12 Oct, 2013. Presentation Type: Abstract Topic: 7. pH and stroma-tumor interactions, metastasis Citation: Brisson L, Driffort V, Benoist L, Poet M, Counillon L, Antelmi E, Rubino R, Besson P, Labbal F, Chevalier S, Reshkin SJ, Goré J and Roger S (2014). NaV1.5 sodium channels increase breast cancer cell invadopodial activity by both controlling Src kinase-dependent F-actin polymerization and promoting NHE-1-dependent proton efflux and extracellular matrix digestion. Front. Pharmacol. Conference Abstract: 4th Annual Meeting of the International Society of Proton Dynamics in Cancer. doi: 10.3389/conf.fphar.2014.61.00011 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Dec 2013; Published Online: 07 Feb 2014. * Correspondence: Dr. Sébastien Roger, UMR inserm 1069 Université François-Rabelais de Tours, Tours, France, sebastien.roger@univ-tours.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lucie Brisson Virginie Driffort Lauriane Benoist Mallorie Poet Laurent Counillon Ester Antelmi Rosa Rubino Pierre Besson Fabien Labbal Stépan Chevalier Stephan J Reshkin Jacques Goré Sébastien Roger Google Lucie Brisson Virginie Driffort Lauriane Benoist Mallorie Poet Laurent Counillon Ester Antelmi Rosa Rubino Pierre Besson Fabien Labbal Stépan Chevalier Stephan J Reshkin Jacques Goré Sébastien Roger Google Scholar Lucie Brisson Virginie Driffort Lauriane Benoist Mallorie Poet Laurent Counillon Ester Antelmi Rosa Rubino Pierre Besson Fabien Labbal Stépan Chevalier Stephan J Reshkin Jacques Goré Sébastien Roger PubMed Lucie Brisson Virginie Driffort Lauriane Benoist Mallorie Poet Laurent Counillon Ester Antelmi Rosa Rubino Pierre Besson Fabien Labbal Stépan Chevalier Stephan J Reshkin Jacques Goré Sébastien Roger Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na(+) channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na(+)/H(+) exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4-7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.
ABSTRACT Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na + /H + exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3′-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.
Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na+/H+ exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in invasion and metastasis. However, the mechanistic basis for its role remains unknown. Here, we demonstrate that ezrin phosphorylated at T567 is highly overexpressed in the membrane of human breast tumors and positively associated with invasive growth and HER2 overexpression. Further, in the metastatic cell line, MDA-MB-231, p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-localized in a functional complex with NHE1, EGFR, ß1-integrin and phosphorylated-NHERF1. Manipulation by mutation of ezrins T567 phosphorylation state and/or PIP2 binding capacity or of NHE1s binding to ezrin or PIP2 demonstrated that p-ezrin expression and binding to PIP2 are required for invadopodia-mediated ECM degradation and invasion and identified NHE1 as the membrane protein that p-ezrin regulates to induce invadopodia formation and activity.
Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB‑231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1‑EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.
