Tetraspanin 8 is a novel regulator of ILK-driven β1 integrin adhesion and signaling in invasive melanoma cells

// Manale El Kharbili 1, 2, 3, 4 , Clément Robert 1, 2, 3 , Tiffany Witkowski 5, 6 , Emmanuelle Danty-Berger 7 , Laetitia Barbollat-Boutrand 1, 2, 3 , Ingrid Masse 1, 2, 3 , Nicolas Gadot 8 , Arnaud de la Fouchardière 9 , Paul C. McDonald 10 , Shoukat Dedhar 10 , François Le Naour 11, 12 , Françoise Degoul 5, 6 , Odile Berthier-Vergnes 1, 2, 3 1 Université de Lyon, Lyon, France 2 Université Lyon 1, Lyon, France 3 CNRS, UMR5534, Centre de Génétique et de Physiologie Moléculaire et Cellulaire, Villeurbanne, France 4 Current address: Department of Dermatology, University of Colorado, Aurora, Colorado, USA 5 Clermont Université, Université d’Auvergne, Imagerie Moléculaire et Thérapie Vectorisée, Clermont-Ferrand, France 6 Inserm, U990, Clermont-Ferrand, France 7 Laboratoire CarMeN (INSERM 1060, INRA1397, INSA), Université Lyon 1, Lyon, France 8 Université Lyon 1, Fédération de Recherche Santé Lyon-Est, ANIPATH, Faculté Laennec, Lyon, France 9 Département de Biopathologie, Centre Léon Bérard, Lyon, France 10 Department of Integrative Oncology, British Columbia Cancer Research Center, Vancouver, Canada 11 INSERM U602, Villejuif, France 12 Current address: INSERM U1193, Hôpital Paul Brousse, Villejuif, France Correspondence to: Odile Berthier-Vergnes, email: odile.berthier-vergnes@univ-lyon1.fr Keywords: melanoma, matrix, integrin, tetraspanin 8, ILK Received: November 09, 2016      Accepted: January 09, 2017      Published: February 04, 2017 ABSTRACT Melanoma is well known for its propensity for lethal metastasis and resistance to most current therapies. Tumor progression and drug resistance depend to a large extent on the interplay between tumor cells and the surrounding matrix. We previously identified Tetraspanin 8 (Tspan8) as a critical mediator of melanoma invasion, whose expression is absent in healthy skin. The present study investigated whether Tspan8 may influence cell-matrix anchorage and regulate downstream molecular pathways leading to an aggressive behavior. Using silencing and ectopic expression strategies, we showed that Tspan8-mediated invasion of melanoma cells resulted from defects in cell-matrix anchorage by interacting with β1 integrins and by interfering with their clustering, without affecting their surface or global expression levels. These effects were associated with impaired phosphorylation of integrin-linked kinase (ILK) and its downstream target Akt-S473, but not FAK. Specific blockade of Akt or ILK activity strongly affected cell-matrix adhesion. Moreover, expression of a dominant-negative form of ILK reduced β 1 integrin clustering and cell-matrix adhesion. Finally, we observed a tumor-promoting effect of Tspan8 in vivo and a mutually exclusive expression pattern between Tspan8 and phosphorylated ILK in melanoma xenografts and human melanocytic lesions. Altogether, the in vitro, in vivo and in situ data highlight a novel regulatory role for Tspan8 in melanoma progression by modulating cell-matrix interactions through β1 integrin-ILK axis and establish Tspan8 as a negative regulator of ILK activity. These findings emphasize the importance of targeting Tspan8 as a means of switching from low- to firm-adhesive states, mandatory to prevent tumor dissemination.