Gelsolin Induces Colorectal Tumor Cell Invasion via Modulation of the Urokinase-Type Plasminogen Activator Cascade Jingli Zhuo1, Ee Hong Tan2, Benedict Yan3, Lalchhandami Tochhawng 1 , Manikandan Jayapal4, Shiuan Koh 1 , Hwee Kee Tay5, Sutherland K. Maciver6, Shing Chuan Hooi1, Manuel Salto-Tellez7,8, Alan Prem Kumar7,9,10,11, Yaw Chong Goh12, Yaw Chyn Lim1,3*, Celestial T. Yap 1 * 1 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 2 Beatson Institute for Cancer Research, Glasgow, United Kingdom, 3 Department of Pathology, National University Hospital, Singapore, Singapore, 4 Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia, 5 Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom, 6 Department of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom, 7 Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore, 8 Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, United Kingdom, 9 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 10 School of Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, Western Australia, Australia, 11 Department of Biological Sciences, University of North Texas, Denton, Texas, United States of America, 12 Department of Surgery, Singapore General Hospital, Singapore, Singapore Abstract Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin’s influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix- degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/ uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites. Citation: Zhuo J, Tan EH, Yan B, Tochhawng L, Jayapal M, et al. (2012) Gelsolin Induces Colorectal Tumor Cell Invasion via Modulation of the Urokinase-Type Plasminogen Activator Cascade. PLoS ONE 7(8): e43594. doi:10.1371/journal.pone.0043594 Editor: Antonio Moschetta, University of Bari & Consorzio Mario Negri Sud, Italy Received April 4, 2012; Accepted July 23, 2012; Published August 21, 2012 Copyright: 2012 Zhuo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the National Medical Research Council (NMRC) (Grant nos: NMRC/1235/2009, NMRC/1058/2006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: phsyapc@nus.edu.sg (CTY); yawchyn_lim@nuhs.edu.sg (YCL) Introduction Colorectal cancer (CRC) accounts for one of the highest mortality rates from cancer worldwide. The survival rate is highest at about 90% when diagnosed at early stages where tumor growth is localized to primary sites and about 35%–70% in invasive but regional disease. However the occurrence of distant metastasis to the liver or lungs in CRC is a major contributing factor to death, with five-year survival rate at less than 15% [1]. The pathogenesis of CRC from normal colonic epithelium to adenoma is fairly well- characterized and often involves a number of genetic alterations, including mutational activation of oncogenes such as K-ras as well as mutational inactivation of tumor suppressors such as p53 [2] and adenomatous polyposis coli (APC) gene [3]. In contrast, less is known about the molecular mechanisms which convert a non- invasive colorectal neoplasm to one with an invasive phenotype. In most solid tumors, the spread of tumor cells is facilitated by events which result in the detachment of malignant cells from the primary site and subsequent dissemination through tissues and vasculature [4]. This metastatic cascade is critically dependent on the integration of migratory and invasive signals involving cytoskele- ton and extracellular matrix (ECM) remodeling [5]. Gelsolin is an actin-binding protein which severs and caps actin filaments [6], and regulates cytoskeletal turnover. Gelsolin appears to have complex roles in tumor biology, with evidence supporting its contradictory involvement in both tumor suppression as well as malignant progression. Gelsolin is reported to be down-regulated in tumors including breast [7] and lung [8] carcinomas, suggesting that loss of gelsolin promotes oncogenesis. Consistent with this, knockdown of gelsolin by small-interfering RNA (siRNA) in the PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43594