ORIGINAL ARTICLE Specific interactions between Smad proteins and AP-1 components determine TGFb-induced breast cancer cell invasion A Sundqvist 1 , A Zieba 2,5 , E Vasilaki 1,5 , C Herrera Hidalgo 1 , O So ¨ derberg 2 , D Koinuma 3 , K Miyazono 1,3 , C-H Heldin 1 , U Landegren 2 , P ten Dijke 1,4 and H van Dam 1,4 Deregulation of the transforming growth factor b (TGFb) signal transduction cascade is functionally linked to cancer. In early phases, TGFb acts as a tumor suppressor by inhibiting tumor cell proliferation, whereas in late phases, it can act as a tumor promoter by stimulating tumor cell invasion and metastasis. Smad transcriptional effectors mediate TGFb responses, but relatively little is known about the Smad-containing complexes that are important for epithelial–mesenchymal transition and invasion. In this study, we have tested the hypothesis that specific members of the AP-1 transcription factor family determine TGFb signaling specificity in breast cancer cell invasion. Using a 3D model of collagen-embedded spheroids of MCF10A-MII premalignant human breast cancer cells, we identified the AP-1 transcription factor components c-Jun, JunB, c-Fos and Fra1 as essential factors for TGFb- induced invasion and found that various mesenchymal and invasion-associated TGFb-induced genes are co-regulated by these proteins. In situ proximity ligation assays showed that TGFb signaling not only induces complexes between Smad3 and Smad4 in the nucleus but also complexes between Smad2/3 and Fra1, whereas complexes between Smad3, c-Jun and JunB could already be detected before TGFb stimulation. Finally, chromatin immunoprecipitations showed that c-Jun, JunB and Fra1, but not c-Fos, are required for TGFb-induced binding of Smad2/3 to the mmp-10 and pai-1 promoters. Together these results suggest that in particular formation of Smad2/3-Fra1 complexes may reflect activation of the Smad/AP-1-dependent TGFb-induced invasion program. Oncogene advance online publication, 27 August 2012; doi:10.1038/onc.2012.370 Keywords: invasion; spheroids; TGFb; AP-1; Smad; PLA INTRODUCTION Transforming growth factor b (TGFb) family members are secreted homodimeric proteins that exert a wide range of biological effects on a large variety of cell types, including regulation of proliferation, differentiation, migration and apoptosis. Signaling by TGFb cytokines occurs via ligand-induced heteromeric complex formation of distinct type I and type II serine/threonine kinase receptors, causing the type I receptor to become phosphorylated by the constitutively active type II receptor. The activated TGFb type I receptor kinase propagates the signal within the cell through phosphorylation of the receptor-regulated (R-)Smad proteins Smad2 and Smad3 at their extreme carboxyl-terminal serine residues. 1,2 The activated R-Smads then form heteromeric complexes with the common-partner (Co-)Smad, Smad4, and accumulate in the nucleus, where they can bind DNA and regulate gene expression. The Smads control gene expression in a cell type-specific manner by interacting with other proteins, such as AP-1, AP-2 and Ets transcription factors and specific co-activators and co-repressors. 3–7 These interactions can alter the intensity, duration and specificity of the TGFb-signaling response. Aberrant regulation of TGFb signaling has been implicated in several pathological situations such as carcinogenesis, vascular disorders and fibrosis. 6,8 TGFb has a biphasic role in tumor progression. In the early stages of tumor development, TGFb inhibits cell growth and thus acts as a tumor suppressor. Escape from TGFb/Smad-induced growth inhibition and apoptosis is commonly observed in tumors. In late-stage cancer, TGFb has been shown to function as a tumor promoter, by stimulating dedifferentiation of epithelial cells to malignant invasive and metastatic fibroblastic cells. 6,9 This epithelial–mesenchymal transi- tion (EMT) is a complex process. It involves disruption of polarization of epithelial cells and gain of spindle-shaped morphology with formation of actin stress fibers, reduced cell– cell junctions through delocalization and downregulation of E-cadherin, and increased cellular motility. Transcriptional repres- sors of E-cadherin, such as SIP1, Snail and Slug, are induced by TGFb. 9–11 Induction of EMT by TGFb can be observed in many different epithelial cell types and is promoted by activated Ras, activated Raf or by serum treatment. 12 It can be mediated both by perturbed Smad-dependent pathways and non-Smad signaling pathways, including MAP kinase pathways. 13,14 Knockdown of Smad3 and Smad4, but not of Smad2, inhibits TGFb-induced EMT of NMuMG cells. 15,16 The dimeric Jun/Fos and Jun/ATF AP-1 transcription factor complexes are composed of c-Jun, JunB, JunD, c-Fos, FosB, Fra1, Fra2 and certain ATF members, such as ATF2. These proteins control cell proliferation and differentiation by regulating gene expression in response to a wide range of stimuli, and they can be 1 Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; 2 Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden; 3 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan and 4 Department of Molecular Cell Biology, Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands. Correspondence: Dr H van Dam, Department of Molecular Cell Biology, Centre for Biomedical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands. E-mail: vdam@lumc.nl 5 These authors contributed equally to this work. Received 3 April 2012; revised 11 June 2012; accepted 9 July 2012 Oncogene (2012), 1–10 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc