Oncogene (2013) 32, 3606–3615 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Specific interactions between Smad proteins and AP-1 components determine TGFb-induced breast cancer cell invasion A Sundqvist1, A Zieba2,5, E Vasilaki1,5, C Herrera Hidalgo1,OSo¨ derberg2, D Koinuma3, K Miyazono1,3, C-H Heldin1, U Landegren2, P ten Dijke1,4 and H van Dam1,4 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 (2013) 32, 3606–3615; doi:10.1038/onc.2012.370; published online 27 August 2012 Keywords: invasion; spheroids; TGFb; AP-1; Smad; PLA INTRODUCTION inhibits cell growth and thus acts as a tumor suppressor. Escape Transforming growth factor b (TGFb) family members are secreted from TGFb/Smad-induced growth inhibition and apoptosis is homodimeric proteins that exert a wide range of biological effects commonly observed in tumors. In late-stage cancer, TGFb has on a large variety of cell types, including regulation of been shown to function as a tumor promoter, by stimulating proliferation, differentiation, migration and apoptosis. Signaling dedifferentiation of epithelial cells to malignant invasive and by TGFb cytokines occurs via ligand-induced heteromeric complex metastatic fibroblastic cells.6,9 This epithelial–mesenchymal transi- formation of distinct type I and type II serine/threonine kinase tion (EMT) is a complex process. It involves disruption of receptors, causing the type I receptor to become phosphorylated polarization of epithelial cells and gain of spindle-shaped by the constitutively active type II receptor. The activated TGFb morphology with formation of actin stress fibers, reduced cell– type I receptor kinase propagates the signal within the cell cell junctions through delocalization and downregulation of through phosphorylation of the receptor-regulated (R-)Smad E-cadherin, and increased cellular motility. Transcriptional repres- proteins Smad2 and Smad3 at their extreme carboxyl-terminal sors of E-cadherin, such as SIP1, Snail and Slug, are induced by serine residues.1,2 The activated R-Smads then form heteromeric TGFb.9–11 Induction of EMT by TGFb can be observed in many complexes with the common-partner (Co-)Smad, Smad4, and different epithelial cell types and is promoted by activated Ras, accumulate in the nucleus, where they can bind DNA and regulate activated Raf or by serum treatment.12 It can be mediated both by gene expression. The Smads control gene expression in a cell perturbed Smad-dependent pathways and non-Smad signaling type-specific manner by interacting with other proteins, such as pathways, including MAP kinase pathways.13,14 Knockdown of AP-1, AP-2 and Ets transcription factors and specific co-activators Smad3 and Smad4, but not of Smad2, inhibits TGFb-induced EMT and co-repressors.3–7 These interactions can alter the intensity, of NMuMG cells.15,16 duration and specificity of the TGFb-signaling response. The dimeric Jun/Fos and Jun/ATF AP-1 transcription factor Aberrant regulation of TGFb signaling has been implicated in complexes are composed of c-Jun, JunB, JunD, c-Fos, FosB, Fra1, several pathological situations such as carcinogenesis, vascular Fra2 and certain ATF members, such as ATF2. These proteins disorders and fibrosis.6,8 TGFb has a biphasic role in tumor control cell proliferation and differentiation by regulating gene progression. In the early stages of tumor development, TGFb expression in response to a wide range of stimuli, and they can be 1Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; 2Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden; 3Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan and 4Department 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: [email protected] 5These authors contributed equally to this work. Received 3 April 2012; revised 11 June 2012; accepted 9 July 2012; published online 27 August 2012 Smads and AP-1 in breast cancer invasion A Sundqvist et al 3607 induced and/or activated through multiple signaling pathways, for example, MAP kinase pathways.17–20 Certain AP-1 components have been implicated in carcinogenesis and tumor cell invasion.20–23 In the case of TGFb family signaling, both Smad- dependent and Smad-independent pathways can contribute to AP-1 activation.4,14 Previously, we established a 3D model of collagen-embedded spheroids of non-malignant, premalignant and metastatic MCF10A human breast cancer cells. We showed that in this system TGFb-receptor I kinase and Smad4 and Smad3 are essential factors for TGFb-induced invasion, by activating among others the secreted proteases MMP-2 and MMP-9.24 In the present study, we have used this breast cancer invasion model to analyze the role of specific transcription factor AP-1 components in TGFb/ Smad-induced invasion. RESULTS TGFb signaling induces increased levels of various AP-1 components in MCF10A MII cells We previously showed that Smad4 and Smad3 have critical roles in TGFb-induced invasion of both premalignant (MII) and malignant (MIV) MCF10A breast epithelial cells.24 As Smads can regulate gene expression in cooperation with AP-1 transcription factors, and various AP-1 components have been implicated in tumor cell invasion, we first examined whether the expression Figure 1. TGFb-induced AP-1 activity in MCF10A MII cells. MCF10A of AP-1 components is regulated by TGFb in MCF10A MII cells. MII cells were serum-starved for 16 h, treated with TGFb3 and the As MII cells are Ras-transformed cells that can secrete various TGFbRI inhibitor SB-505124 as indicated, and analyzed by immuno- growth factors and cytokines themselves, including TGFb and/or blotting. SB-505124 was added 15 min before TGFb3. TGFb-related cytokines,24 we examined non-stimulated and TGFb- stimulated cells both in the absence and presence of the TGFbRI kinase inhibitor SB-505124. Activation (phosphorylation) of Smad2 and Smad3 was analyzed for comparison. Stockholm, Sweden) smart-pool siRNAs (Figure 2a). Knockdown We used TGFb3 in this study because little is known about the of individual AP-1 components had only minor effects on the response of breast cancer cells to TGFb3, in contrast to TGFb1. All other members, with the exception of c-Fos and Fra1 that were three TGFb isoforms are expressed during breast cancer progres- significantly decreased by knockdown of c-Jun or JunB. These sion, but some contradictory results have been obtained with effects of AP-1 proteins on other family members are most likely respect to the correlation between expression levels and due to the fact that various AP-1 components are (auto-)regulated prognosis (reviewed in Laverty et al.25). Importantly, both TGFb1 via AP-1-binding sites in their promoters, and the fact that Fos family members can be stabilized by dimerization to Jun family and TGFb3 can bind directly to the type II receptor (TGFbRII), 18,20,27 whereas TGF-b2 requires the presence of the co-receptor b-glycan. members. The reduced c-Jun levels upon Smad4 knock- down in Figure 2a indicate that the late TGFb induction of c-jun is Moreover, we found the binding pattern of TGFb3 in M2 cells to 4,14 closely match the pattern reported for TGFb1, binding TGFbRII, Smad dependent, which is in line with previous studies. ALK5/TGFbRI and b-glycan.24–26 However, in certain assays TGFb3 The effect of AP-1 knockdown on invasion was subsequently appears to be a slightly more potent stimulator than TGFb1.25 compared with the effect of the TGFbRI-kinase inhibitor SB-505124 As shown in Figure 1 and Supplementary Figure 1, we found and Smad4 siRNA, as our previous studies had shown that TGFb to strongly increase the protein levels of the AP-1 TGFbRI-kinase inhibition nearly completely blocks TGFb-induced collagen invasion of MII spheroids, whereas knockdown of components c-Jun, JunB, c-Fos and FosB. The TGFbRI kinase 24 inhibitor SB-505124 inhibited the induction of c-Jun, JunB, c-Fos Smad4 only has a partial effect.