[CANCER RESEARCH 64, 5154 –5161, August 1, 2004] Truncated RON Tyrosine Kinase Drives Tumor Cell Progression and Abrogates Cell-Cell Adhesion Through E-Cadherin Transcriptional Repression Chiara Bardella, 1 Barbara Costa, 1 Piera Maggiora, 1 Salvatore Patane’, 1 Martina Olivero, 1 Guglielmina N. Ranzani, 4 Michele De Bortoli, 2 Paolo M. Comoglio, 3 and Maria Flavia Di Renzo 1 1 Laboratory of Cancer Genetics, 2 Functional Onco-Genomics Center, and 3 Division of Molecular Oncology, Institute for Cancer Research and Treatment, University of Torino Medical School, Candiolo, Turin, Italy, and 4 Department of Genetics and Microbiology, University of Pavia, Pavia, Italy ABSTRACT RON is a tyrosine kinase receptor that triggers scattering of normal cells and invasive growth of cancer cells on ligand binding. We identified a short RON mRNA, which is expressed in human lung, ovary, tissues of the gastrointestinal tract, and also in several human cancers, including ovarian carcinomas and cell lines from pancreatic carcinomas and leuke- mias. This transcript encodes a truncated protein (short-form RON; sf-RON), lacking most of the RON receptor extracellular domain but retaining the whole transmembrane and intracellular domains. Sf-RON shows strong intrinsic tyrosine kinase activity and is constitutively phos- phorylated. Epithelial cells transduced with sf-RON display an aggressive phenotype; they shift to a nonepithelial morphology, are unable to form aggregates, grow faster in monolayer cultures, show anchorage-indepen- dent growth, and become motile. We show that in these cells, E-cadherin expression is lost through a dominant transcriptional repression pathway likely mediated by the transcriptional factor SLUG. Altogether, these data show that expression of a naturally occurring, constitutively active trun- cated RON kinase results in loss of epithelial phenotype and aggressive behavior and, thus, it might contribute to tumor progression. INTRODUCTION The RON receptor belongs to the MET family of tyrosine kinase receptors (1). This family includes the human and mouse MET and the human RON and its homologues in mouse (stk; Ref. 2), cat (f-stk; Ref. 3), and chicken (sea; Ref. 4). Members of the family share biochem- ical features and biological properties. MET and RON receptors both trigger complex morphogenetic programs of epithelial cells that lead to “invasive growth” (5). In physiological conditions, cell invasive growth occurs during organ development and regeneration. In cancer cells, inappropriate activation of invasive growth might lead cells to invade and metastasize. The RON receptor is expressed in epithelia and hematopoietic cells. First findings (6) indicated that RON receptor activation regulates tissue macrophage motility. Additional findings demonstrated that the RON receptor induces epithelial cell scattering, which involves not only cell motility but also dissociation and matrix invasion, suggesting that RON might be involved in the pathogenesis of certain epithelial cancers (7). Activation of oncogenes of the MET family occurs in cancer and follows rules that are common to all of the tyrosine kinase oncogenes. MET and RON are overexpressed and amplified in human cancers of specific histotypes (7–10). Both MET and RON receptors can be activated by missense mutations in the tyrosine kinase domain. MET mutations have been found in families suffering from hereditary papillary renal cell carcinoma (11, 12). When the same mutations were introduced in the RON cDNA, mutated receptors converted transfected cells into tumorigenic transformants (13). Rearrangement of both MET and RON genes with the dimerizing TPR sequences led to kinase activation, cell transformation, and tumorigenesis (14, 15). Both the human RON and the homologous mouse stk genes give rise to two transcripts: in humans a 5.0 kb and a 2.0 kb mRNAs (16); and in the mouse a 4.5 kb and a 1.9 kb mRNAs (2). In both species, the longer transcript encodes for the full-size receptor. The shorter mouse transcript (sf-stk) was found to be expressed in animals sus- ceptible to Friend leukemia virus-induced erythroleukemia and absent in resistant mice (17). In the latter, sf-stk-enforced expression restored susceptibility. In Friend-infected erythroblasts, the sf-stk protein di- rectly interacts with viral gp55 but not with the erythropoietin-R (18) and plays a key role in the signal transduction that regulates erythro- poietin-independent erythroblast expansion. Conversely, the sf-stk role in cell physiology is still elusive, although the sf-stk transcript is highly expressed in mouse erythroleukemic cells (2) and is detectable in mouse stomach and intestine (19). The shorter (2.0 kb) human RON mRNA was detected, at a very low level, in colon, skin, and lung tissues but not in bone marrow, bone-marrow-derived cells, human muscle, nor in human brain (16, 20). It has been also found in some human tumor cells lines derived from gastric (16) and lung cancers (21). Although it is known that the shorter transcript includes sequences encoding the RON kinase do- main, its role in normal and cancer tissues is still unknown. In this article, we show that a 5'-truncated RON mRNA, analogous to sf-stk transcript, is expressed in human normal and cancer cells including ovarian, pancreatic, gastrointestinal, and leukemia cells. In well-differentiated breast carcinoma cells, its enforced expression induced loss of epithelial features and progression toward a more aggressive phenotype. MATERIALS AND METHODS Cell Lines and Human Tissues. The GTL16 cell line is a clonal cell line derived from a poorly differentiated gastric carcinoma line (22). All of the other cell lines were purchased from American Type Culture Collection (Manassas, VA). Normal and cancer human tissue samples were obtained and processed as described previously (23). Antibodies. The purified polyclonal antibodies C-20 and N-20, antihuman RON, anti-SLUG, and the monoclonal antibodies against N-cadherin were from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal antibodies anti-E-cadherin were from Transduction Laboratories (BD Biosciences, Can- ada); monoclonal pan anticadherins antibodies were from Sigma (Sigma- Aldrich, Germany); and monoclonal antiphosphotyrosine antibodies were from Upstate (Upstate, MA). Monoclonal anti-myc antibodies (clone 9E10) were obtained from Dr. Luca Tamagnone (I.R.C.C., Candiolo, Torino, Italy). End-Point Reverse Transcription-PCR (RT-PCR). Cytoplasmic mRNA was prepared using the Concert Reagent from Invitrogen (Life Technology, Carlsbad, CA). Total RNA from tissue samples was prepared and analyzed as described previously (22). Retrotranscription was carried out with Moloney murine leukemia virus reverse-transcriptase RNase H minus (Promega, Mad- ison, WI) and oligodeoxythymidine nucleotide. To examine RON transcript Received 2/19/04; revised 4/29/04; accepted 5/19/04. Grant support: Italian Ministry of Research and Education (Ministero per l’Istruzione, l’Universita’ e la Ricerca Scientifica; M. Di Renzo, P. Comoglio, M. De Bortoli, and G. N. Ranzani), the Italian National Research Council (Consiglio Nazionale delle Ricerche-Ministero per l’Istruzione, l’Universita’ e la Ricerca Scientifica Progetto Oncologia), and the Italian Association for Cancer Research (Associazione Italiana Ricerca sul Cancro; M. Di Renzo and P. Comoglio). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Maria Flavia Di Renzo, Laboratory of Cancer Genetics, Istituto per la Ricerca e la Cura del Cancro, SP 142, Km 3.95, 10060 Candiolo (TO), Italy. Phone: 39-11-9933343; Fax: 39-11-9933524; E-mail: mariaflavia.direnzo@ircc.it. 5154