The EDA-containing cellular fibronectin induces epithelial–mesenchymal transition in lung cancer cells through integrin α9β1-mediated activation of PI3-K/AKT and Erk1/2

Advance Access publication August XiaojuanSun 1 2 3 PingpingFa 0 2 ZhiwenCui 0 2 4 YeXia 0 2 4 LiangSun 2 3 ZesongLi 2 3 AifaTang 2 3 YaotingGui 0 2 ZhimingCai 2 3 0 Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Centre , Shenzhen 518036 , China 1 Department of Central Lab, Affiliated Hospital of Guangdong Medical College , Zhanjiang 524023 , China 2 Shenzhen Tumor Clinical Immune Gene Therapy Engineering Lab, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University , 3002 West Sungang Road, Shenzhen 518035 , China. Tel: 3 Department of Biobank, Shenzhen Tumor Clinical Immune Gene Therapy Engineering Lab, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University , Shenzhen 518035 , China 4 Department of Urology, Peking University Shenzhen Hospital , Shenzhen 518036 , China - Cellular fibronectin (cFN) is one of the main components of tissue extracellular matrices and is involved in multiple physiologic and pathologic processes such as embryogenesis, wound healing, inflammation and tumor progression. The function of fibronectin in regulating normal cell adhesion and migration is well documented, but its function in cancer progression is only partially unraveled. We have reported previously that fibronectin stimulates the proliferation and survival of non-small lung carcinoma cells through upregulation of pro-oncogenic signals related to cyclooxygenase-2/ phosphatidylinositol-3-kinase/protein kinase B (COX-2/PI3-K/ AKT)/mammalian target of rapamycin triggered by activation of the integrin 51. Here, we extend these studies by showing that fibronectin promotes epithelialmesenchymal transition (EMT) in lung cancer cells. We found that cFN, but not plasma fibronectin or type 1 collagen, induces lung carcinoma cell scattering in vitro, promotes cell migration and invasion of Matrigel and stimulates the expression of the mesenchymal marker -smooth muscle actin while decreasing the expression of the epithelial marker E-cadherin through PI3-K and Erk pathways. Interestingly, the extra domain A(EDA) within cFN was found to be crucial for this process, as confirmed by testing cells overexpressing EDA or cells exposed to EDA-containing matrices. We found that the integrin 9, but not 5, mediated cFN-induced EMT as silencing integrin 9 neutralized cFN-induced EMT. Overall, our findings show that the EDA domain within cFN induces EMT in lung carcinoma cells through integrin 9-mediated activation of PI3-K and Erk. Introduction Lung cancer is the leading cause of cancer death in the USA and worldwide (1). In most cases, the main cause of death from lung cancer relates to tissue invasion and metastasis by carcinoma cells (2,3). One of the first steps in tumor metastasis is the acquisition of cellular motility and invasiveness. During this process, tumor cells partially lose their epithelial markers and gain mesenchymal Abbreviations: -SMA, -smooth muscle actin; cFN, cellular fibronectin; ECM, extracellular matrix; EDA, extra domain A; EDB, extra domain B; EMT, epithelialmesenchymal transition; FBS, fetal bovine serum; NSCLC, non-small lung carcinoma; PBS, phosphate-buffered saline; pFN, plasma fibronectin; siRNA, short interfering RNA; TGF, transforming growth factor. These authors contributed equally to this work. characteristics, a process termed epithelialmesenchymal transition (EMT). Although EMT has been recognized as a central feature of normal embryonic development, governing the formation of gastrula, neural crest and the heart, recent studies have revealed that a similar transition occurs during the progression of tumors, and much evidence has accumulated in favor of a role for EMT in tumor metastasis (4,5). Thus, defining the factors that promote EMT in the setting of lung carcinoma is likely to lead to the identification of new targets for therapy. Tumor cellstroma interactions are becoming increasingly recognized as important determinants of tumor cell fate (2,6,7). Studies in human lung, breast, colon and prostate cancer showed that carcinoma cells are submerged in a microenvironment with fibroblasts and extracellular matrix (ECM) proteins such as fibronectin, collagens, tenascins, proteoglycans, glycosaminoglycans and laminin (2,68). As the result of complex interactions between cells and their surrounding stroma, ECMs may affect tumor cell behavior including metastasis (9,10). Fibronectin is among the ECM proteins present in tumor tissue, and the fragmentation of pericellular fibronectin with the exposure of cryptic molecular binding sites is considered an early sign of malignancy (11). Interestingly, the amount of fibronectin messenger RNA in stroma has been found to be 713 times higher in carcinoma, which is abnormally high compared with normal tissue (12). Furthermore, we and others have reported that fibronectin promotes cancer cells proliferation through effects on pro-oncogenic pathways (1315). These and other observations have promoted investigations into fibronectin as a potential target for tumor therapy (16). Fibronectin is a high-molecular-weight adhesive glycoprotein that exists in two main forms, as an insoluble glycoprotein dimer present within the ECM or as a soluble disulphide-linked dimer in plasma. Each dimer consists of two nearly identical polypeptide chains and three types of homologous repeating modules termed types I, II and III (17). Cells may recognize fibronectin through one or more receptors of the integrin family. Eleven different integrin heterodimers have been found to bind to fibronectin, and four of them, 51, v3, 41 and IIb3, trigger fibronectin fibril formation in vitro (18). The integrin 51 is the best-studied fibronectin-binding integrin, it recognizes the minimal integrin recognition peptide sequence ArgGly-Asp within the fibronectin monomer (19). Different isoforms of fibronectin are generated by alternative splicing of combinations of three exons: extra domain A (EDA/ EIIIA), extra domain B (EDB/EIIIB) and connecting segment III (V). The so-called plasma fibronectin (pFN), produced by hepatocytes and abundant in plasma, lacks both the EDA and EDB domains (17,19), whereas cellular fibronectin (cFN), produced by fibroblasts, epithelial cells and other cell types, contains the EDA and/or EDB segments. Although the functions of EDA and EDB domains have not been fully elucidated, their ablation leads to embryonic lethality within EDA/EDB double-null animals (20). Fibronectin containing EDA has been implicated in the regulation of wound healing (21) and is more potent than fibronectin lacking EDA in promoting cell spreading and cell migration irrespective of the presence or absence of EDB (22). The EDGIHEL sequence within the EDA variant facilitates binding to 41 and 91 integrins (23,24). In this study, we show that cFN prevents the clustering of lung a (...truncated)