Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells

Oisun Jung 2 Suyong Choi 1 Sun-Bok Jang 0 Sin-Ae Lee 1 Ssang-Taek Lim 6 Yoon-Ju Choi 1 Hye-Jin Kim 1 Do-Hee Kim 0 Tae Kyoung Kwak 1 Hyeonjung Kim 1 Minkyung Kang 5 Mi-Sook Lee 1 Sook Young Park 4 Jihye Ryu 1 Doyoung Jeong 1 Hae-Kap Cheong 3 Hyun Jeong Kim 4 Ki Hun Park 7 Bong-Jin Lee 0 David D. Schlaepfer 6 Jung Weon Lee 1 2 0 Department of Pharmaceutics, Research Institute of Pharmaceutical Sciences, Tumor Microenvironment Global Core Research Center, Medicinal Bioconvergence Research Center, College of Pharmacy, Seoul National University , Seoul 151-742 , Korea (Republic of) 1 Department of Pharmacy 2 Interdisciplinary Program in Genetic Engineering 3 Division of Magnetic Resonance, Korea Basic Science Institute , 804-1 Yangcheong-Ri, Ochang, Chungbuk 306-883 , Korea (Republic of) 4 Department of Dental Anesthesiology and Dental Research Institute, School of Dentistry, Seoul National University , Seoul 110-768 , Korea (Republic of) 5 Department of Biomedical Sciences, College of Medicine, Seoul National University , Seoul 110-799 , Korea (Republic of) 6 Department of Reproductive Medicine, Moores Cancer Center, University of California San Diego , La Jolla, CA 92093 , USA 7 Division of Applied Life Science, Gyeongsang National University , Jinju 660-701 , Korea (Republic of) - Summary Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesiondependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cells leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesiondependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer. Introduction Cell migration is critical for development and maintenance of multicellular organisms in addition to the development of pathological conditions, such as cancer metastasis (Yamaguchi and Condeelis, 2007), which involves highly complex processes regulated by coordinated signaling pathways that respond to extracellular matrix (ECM) or soluble factors (Friedl and Wolf, 2009). Cell migration is mediated by the following biological events: (1) leading edges of migrating cells occupying spaces by forming focal adhesions or contacts, (2) rear edges disassembling old focal adhesions, and (3) adhesion-dependent activation of RhoA GTPase regulating cellular actin polymerization and contractility for forward movement (Friedl and Wolf, 2009). As one of the most important signaling molecules activated as a result of cell adhesion and spreading, focal adhesion kinase (FAK) plays a critical role in migration and invasion (Luo and Guan, 2010). FAK overexpression has been detected in diverse primary and metastatic tumor tissues, supporting its protumorigenic and pro-metastatic roles (McLean et al., 2005; Zhao and Guan, 2009). Intensive actin branching and polymerization occur in leading edges of migratory cells, thus forming new focal contacts and adhesions via FAK and RhoA GTPase activation that in turn leads to the activation of downstream actin-organizing effectors, including actin-related protein (Arp2/3), neural WiskottAldrich syndrome protein (NWASP), and cortactin (Sanz-Moreno and Marshall, 2010). Cell adhesion causes phosphorylation and activation of focal adhesion molecules that are critically involved in the regulation of morphological changes, migration, and invasion (Danen, 2009). Among these molecules, FAK is a non-receptor Tyr kinase that is autophosphorylated at Tyr397 upon cell adhesion to the ECM. Phosphorylated Tyr397 is a binding site for Src-homology 2-domain containing molecules such as c-Src or phosphoinositide 3-kinase, which phosphorylate other Tyr residues in FAK or phosphatidylinositol, respectively (Schaller, 2010). Although FAK downstream signaling for most adherent cell functions has been intensively investigated, how FAK is activated during cell adhesion remains unknown (Frame et al., 2010). Membrane-tetraspanning tetraspanins collaborate with integrins for cell adhesion and migration (Berditchevski, 2001). Transmembrane 4 L six family member 5 (TM4SF5) is a membrane glycoprotein with four transmembrane domains whose intracellular loop (ICL; 69R to 91V) and NH2- and COOH-terminal tails are located intracellularly, similar to those of genuine tetraspanins or transmembrane four super-family members (TM4SFs) (Wright et al., 2000). TM4SF5 is highly expressed in diverse clinical cancer tissues, and its overexpression in hepatocytes enhances aberrant proliferation, migration, and invasion (Lee et al., 2011), although it is unknown how cell migration is mechanistically regulated by TM4SF5. TM4SF5 as a membrane glycoprotein with four transmembrane domains is involved in cell adhesion-related signaling (Lee et al., 2011). TM4SF5 in fibroblasts enhances cell adhesion signaling and FA formation via an increased Tyr925 phosphorylation of FAK on ECM, which are inhibited by serum treatment, suggesting a role of TM4SF5 in regulation of cell-ECM adhesion via coordinative cross-talks between different membrane receptors including integrin a2, TM4SF5 and growth factor receptors (Lee et al., 2006). TM4SF5 in liver epithelial cells results in RhoA inactivation via increased interactions between FAK and RhoGAPs, leading to morphological elongation, EMT, and multilayer growth (Lee et al., 2008). Further interestingly, e TM4SF5 in liver epithelial cells in collagen I environment n restricts cell spreading and migration via an interaction between c ie TM4SF5 and integrin a2, but an interruption of the interaction c S recovers spreading and migration (Lee et al., 2009a). TM4SF5 in ll hepatocarcinoma cells also causes secretion of VEGF depending eC on activation of FAK/c-Src complex downstream of integrin a5, f which can enhance angiogenic activity of neighboring endothelial lo cells (Choi et al., 2009). Although these previous reports reveal na that TM4SF5 collaborates with integrins for different cell ru functions, they did not reveal how TM4SF5 directly or o indirectly activates FAK, a major i (...truncated)