Expression profiling of epithelial plasticity in tumor progression Martin Jechlinger 1,5 , Stefan Grunert 1 , Ido H. Tamir 1 , Elzbieta Janda 1,6 , Susanna Lu¨demann 1,7 , Thomas Waerner 1 , Peter Seither 2 , Andreas Weith 2 , Hartmut Beug* ,1,3 and Norbert Kraut 2,3,4 1 IMP, Research Institute for Molecular Pathology, Dr Bohrgasse 7, Vienna A-1030, Austria; 2 Boehringer Ingelheim Pharma KG, Genomics Group, Birkendorfer Str. 65, Biberach D-88397, Germany Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlat- ing gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and trans- differentiation to mesenchymal-like cells with invasive behavior. Oncogene (2003) 22, 7155–7169.doi:10.1038/sj.onc.1206887 Keywords: epithelial–mesenchymal transition; microar- ray; ras; TGFb Introduction Epithelial tumors (carcinomas) comprise more than 80% of all tumors in man. They arise through a multistep process, best studied in human colon cancer and mouse skin tumor progression (Hanahan and Weinberg, 2000). While many molecular players are known (Christofori and Semb, 1999; Hanahan and Weinberg, 2000), it remains ill understood as to how oncogenes/tumor suppressors contribute to local inva- sion and metastasis. However, evidence accumulates that epithelial-to-mesenchymal transition (EMT) repre- sents an important in vitro correlate of late-stage tumor progression (Boyer et al., 2000; Thiery, 2002; Petersen et al., 2003). During EMT, cells lose epithelial polarity, and acquire a spindle-shaped, highly motile fibroblas- toid phenotype. This includes loss or redistribution of tight- and adherens-junction proteins, a switch to a mesenchymal gene-expression program and the ability to pass through the basement membrane (Reichmann et al., 1992; Hay, 1995; Oft et al., 1996). EMT also occurs during embryonic development, and is regulated by an increasingly complex pattern of signaling path- ways (Hay, 1995; Thiery, 2002; Gru¨nert et al., 2003). Ras family proteins regulate cell fate and prolifera- tion, cause oncogenic transformation and are mutated/ amplified in a high proportion of human cancers. In multiple epithelial cell models, oncogenic Ras protects cells from transforming growth factor (TGF) b-induced cell cycle arrest and apoptosis. More importantly, Ras cooperates with TGFb to induce EMT, which is stabilized by autocrine production of TGFb (Oft et al., 1996; Lehmann et al., 2000; Gotzmann et al., 2002). Oncogenic Ras acts through numerous effectors, such as Raf kinase and phosphoinositide 3-OH kinase (PI3- kinase), which trigger the ERK/MAP- and the PKB/ Akt-kinase pathways, respectively (Downward, 1998). To dissect Ras-downstream signaling with respect to EMT, two Ras effector-specific mutants which predo- minantly signal either through the ERK/MAPK path- way (S35-V12Ras) or through the PI3K-PKB/AKT pathway (C40-V12Ras; Rodriguez-Viciana et al., 1997) were expressed in polarized mammary epithelial cells (EpH4). Exposure of these cells to TGFb demonstrated that EMT requires hyperactivation of the ERK/MAPK pathway by Ras-S35 while hyperactive PI3K signaling induced by Ras-C40 enabled TGFb-induced ‘scattering’ Received 1 May 2003; accepted 18 June 2003 *Correspondence: H Beug; E-mail: beug@nt.imp.univie.ac.at 3 These two authors contributed equally to this work 4 Current address: Boehringer Ingelheim Austria GmbH, Department of NCE Lead Discovery, Dr Boehringer Gasse 5-11, Vienna A 1121, Austria 5 Current address: Memorial Sloan Kettering Cancer Center, 1275 York Ave, Box 62, New York, NY 10021, USA 6 Current address: European Patent Office Munich, Erhardtstrsse 27, 80331 Munich, Germany 7 Current address: Dipartimento di Medicina Sperimentale e Clinica Universita degli studi Magna Graecia, Policlinico Mater Domini Piano 5, Via Campanella 115, 88100 Catanzaro, Italy Oncogene (2003) 22, 7155–7169 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc