ORIGINAL ARTICLE Targeting Lyn regulates Snail family shuttling and inhibits metastasis D Thaper 1,2 , S Vahid 1,2 , KM Nip 1,2 , I Moskalev 1 , X Shan 3 , S Frees 1 , ME Roberts 4 , K Ketola 1 , KW Harder 4 , C Gregory-Evans 3 , JL Bishop 1 and A Zoubeidi 1,2 The acquisition of an invasive phenotype by epithelial cells occurs through a loss of cellular adhesion and polarity, heralding a multistep process that leads to metastatic dissemination. Since its characterization in 1995, epithelial–mesenchymal transition (EMT) has been closely linked to the metastatic process. As a defining aspect of EMT, loss of cell adhesion through downregulation of E-cadherin is carried out by several transcriptional repressors; key among them the SNAI family of transcription factors. Here we identify for the first time that Lyn kinase functions as a key modulator of SNAI family protein localization and stability through control of the Vav-Rac1-PAK1 (Vav-Rac1-p21-activated kinase) pathway. Accordingly, targeting Lyn in vitro reduces EMT and in vivo reduces metastasis of primary tumors. We also demonstrate the clinical relevance of targeting Lyn as a key player controlling EMT; patient samples across many cancers revealed a strong negative correlation between Lyn and E-cadherin, and high Lyn expression in metastatic tumors as well as metastasis-prone primary tumors. This work reveals a novel pancancer mechanism of Lyn- dependent control of EMT and further underscores the role of this kinase in tumor progression. Oncogene advance online publication, 13 March 2017; doi:10.1038/onc.2017.5 INTRODUCTION Since its discovery in 1979, the 'proto-oncogene' Src has led research into the field of non-receptor tyrosine kinases. 1 The Src family of kinases (SFKs) has 11 members that contain a kinase domain, an SH2 domain and a SH3 domain. 2 Combined, these SFKs have a major role in the cellular events of different biological systems, ranging from the immune system 3 to the nervous system. 4 Owing to their pleiotropic nature, it is no surprise that aberrant activation of SFKs is associated with carcinogenesis and tumor progression. Aside from their fundamental roles in cell proliferation, survival and angiogenesis, SFKs enhance cell migration and invasion across different cancers. Increased migratory and invasive attributes of cells in cancer occurs through a process called epithelial–mesenchymal transition (EMT), which is essential for metastasis. 5 Metastasis is the sequential process whereby mesenchymal cells penetrate the basement membrane, intravasate into blood/lymphatic vessels, survive in the vascula- ture, extravasate to secondary sites and adapt to new host environments. 6 The colonization and tumor growth at the distant site is dictated by conflicting pressures such as proliferation, dormancy, angiogenesis and apoptosis; all pathways that, like EMT, are regularly modulated by SFKs. 7 In particular, there is increasing evidence that the Lyn tyrosine kinase is an SFK that is associated with metastasis and thus may promote EMT. 8 For example, we have recently reported increased expression of Lyn in metastatic castration-resistant prostate cancer (PCa). 9 In addition to prostate, Lyn has been implicated in other cancers, including breast, 10 colon 11 and glioblastoma. 12 Lyn regulates cellular events, like proliferation and survival, in multiple cancer types 13 and targeting Lyn in Ewing’s sarcoma reduces metastatic potential. 14 Higher Lyn protein expression has been implicitly linked to highly aggressive triple-negative breast cancer (TNBC), whereby 46% of TNBC cases were Lyn(+). In addition, 79% of Lyn(+) patients were TNBC and Lyn expression was linked to reduced patient survival. 15 In this study, Lyn knockdown using small interfering RNA (siRNA) in TNBC cell lines reduced cell migration and invasion. Additionally, Lyn kinase was strongly implicated in zebrafish wound healing as its expression was induced in the leading edge of the closing wound. 16 With the availability of a well-tolerated and clinically active inhibitor, Bafetinib, 17 targeting Lyn presents as a viable strategy. Therefore, the underlying mechanism by which Lyn modulates metastasis warrants exploration. The gain of mesenchymal markers vimentin and fibronectin with a concomitant loss of E-cadherin are hallmarks of EMT. Central to this process is an increase in transcription factors of the SNAI family, which lead to transcriptional repression of E-cadherin and start the transformation to a mesenchymal phenotype. 18 In this study, we provide mechanistic insight into how Lyn regulates EMT in PCa , breast cancer (BrCa) and bladder cancer (BlCa). In vitro and in vivo studies showed Lyn promotes EMT via initiating the Vav-Rac1-PAK1 (Vav-Rac1-p21-activated kinase) signal cas- cade; this leads to protein localization and stability of the SNAI transcription factors Slug and Snail, upregulation of EMT-related genes, invasion and metastasis. Furthermore, we established the importance of Lyn in human metastatic cancer, demonstrating that Lyn expression across multiple tumor types is, (i) increased in metastatic tumors and primary tumors that eventually metastasize, (ii) negatively correlates with E-cadherin expression, (iii) positively correlates with the expression of EMT markers 1 Department of Urology, Vancouver Prostate Centre, University of British Columbia, Vancouver, BC, Canada; 2 Faculty of Medicine, Department of Urologic Science, University of British Columbia, Vancouver, BC, Canada; 3 Faculty of Medicine, Department Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, BC, Canada and 4 Faculty of Science, Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada. Correspondence: Dr A Zoubeidi, Department of Urology, Vancouver Prostate Centre, University of British Columbia, 2660 Oak Street, Vancouver, BC, Canada V6H3Z6. E-mail: azoubeidi@prostatecentre.com Received 18 July 2016; revised 27 November 2016; accepted 27 December 2016 Oncogene (2017), 1 – 12 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0950-9232/17 www.nature.com/onc