ORIGINAL PAPER Journal of Pathology J Pathol 2010; 221: 343–356 Published online 1 April 2010 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/path.2708 SERPINB3 induces epithelial–mesenchymal transition Santina Quarta, 1 Laura Vidalino, 1 Cristian Turato, 2 Mariagrazia Ruvoletto, 1 Fiorella Calabrese, 3 Marialuisa Valente, 3 Stefania Cannito, 4 Giorgio Fassina, 5 Maurizio Parola, 4 Angelo Gatta 1 and Patrizia Pontisso 1 * 1 Department of Clinical and Experimental Medicine, University of Padova, Italy 2 Istituto Oncologico Veneto (IOV, IRCCS), Padova, Italy 3 Department of Diagnostic Medical Sciences and Special Therapies, University of Padova, Italy 4 Department of Experimental Medicine and Oncology, University of Torino, Italy 5 Xeptagen SpA, VEGA Science Park, Marghera, VE, Italy *Correspondence to: Patrizia Pontisso, Clinica Medica 5, Department of Clinical and Experimental Medicine, Via Giustiniani 2, 35128 Padova, Italy e-mail: patrizia@unipd.it Abstract Epithelial –mesenchymal transition is believed to facilitate invasion and metastasis formation of epithelial tumour cells. SERPINB3 is a serine protease inhibitor, physiologically found in normal squamous epithelium but over- expressed in epithelial tumours and known to inhibit apoptosis. We tested the hypothesis that SERPINB3 has a role in invasion by modulating the epithelial –mesenchymal transition programme, using morphological, molecular and cell biology techniques on HepG2 cell clones transfected with the human SERPINB3 gene. The paracrine effect of this serpin was determined by the addition of exogenous recombinant SERPINB3 protein to HepG2 and MDCK cell line. SERPINB3 expression leads to changes in transfected cells morphology, characterized by clusters of loosely connected cells with elongated shape. Ultrastructural analysis confirmed the decrease of desmosomal junctions and widening of intercellular spaces. These alterations were associated with a reduction of E-cadherin and an increase of β-catenin, with a parallel increase of cell proliferation. SERPINB3 clones, untransfected HepG2 and MDCK cells treated with exogenous SERPINB3 expressed vimentin, undetectable in controls. SERPINB3 induced significant cell scattering, migration and invasiveness in untransfected cells. These effects were not dependent on the anti-protease activity of the protein, as documented by the results obtained with an active loop-deleted recombinant SERPINB3 protein. Scatter activity was inhibited by an anti-SERPINB3 antibody in a dose-dependent manner and SERPINB3-transfected cells formed a significantly higher number of colonies on soft agar than controls. In conclusion, the observed results indicate that SERPINB3 induces deregulation of adhesion processes and increases the invasiveness potential supported by features of epithelial–mesenchymal transition, acting at both the autocrine and the paracrine level. Copyright  2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: SERPINB3; invasion; cell adhesion; epithelial–mesenchymal transition; metastasis Received 28 August 2009; Revised 22 February 2010; Accepted 27 February 2010 No conflicts of interest were declared. Introduction Epithelial–mesenchymal transition (EMT) has been identified as a crucial process during embryonic devel- opment, chronic inflammation and fibrosis, as well as cancer progression, favouring invasion and metastasis formation in cancers of epithelial origin [1–3]. Epithe- lial cells undergoing EMT are characterized by pheno- typic changes resulting in altered cell–cell interactions, mainly due to loss of E-cadherin, the ability to invade the extracellular matrix and a gain of cell motility and invasiveness potential [4]. The hallmark of malignancy is the metastatic dissemination of the primary tumour [5], where a small number of cells may bypass the fun- damental rules of the normal behaviour, detach from neighbouring ones, migrate and locally invade the sur- rounding tissue. In vitro and in vivo model systems have allowed the characterization of various signalling pathways leading to EMT and EMT-like phenotypes, with c-Akt, GSK3β, Rho-GTPases and SMAD sig- nalling being the most relevant [6]. In addition, hypoxia promotes EMT by favouring increased invasiveness in different cancer cells of epithelial origin [7]. Extracellular matrix components are central to the metastatic process, where breakdown of tissue bound- aries and rearrangement of tissue architecture allows cancer cell dissemination. Various types of proteolytic enzymes, including matrix metalloproteinases (MMPs) [8] and serine proteases [9], play important roles in the control of degradation and remodelling of extra- cellular matrix. Serine protease inhibitors (serpins), such as plasminogen activator inhibitor (PAI)-1 and -2, due to their ability to reduce proteolysis, are predicted to impair extracellular matrix degradation and conse- quently cancer cell invasion and metastasis. However, Copyright  2010 Pathological Society of Great Britain and Ireland. J Pathol 2010; 221: 343–356 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com