Oncogene (2011) 30, 2230–2241 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE TGF-b1 signaling targets metastasis-associated protein 1, a new effector in epithelial cells

SB Pakala1, K Singh1,3, SDN Reddy1, K Ohshiro1, D-Q Li1, L Mishra2 and R Kumar1

1Department of Biochemistry and Molecular Biology and Institute of Coregulator Biology, The George Washington University Medical Center, Washington, DC, USA and 2Department of Gastroenterology, Hepatology and Nutrition, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

In spite of a large number of transforming growth factor b1 gene chromatin in response to upstream signals. The (TGF-b1)-regulated genes, the nature of its targets with TGF-b1-signaling is largely mediated by Smad proteins roles in transformation continues to be poorly understood. (Massague et al., 2005) where Smad2 and Smad3 are Here, we discovered that TGF-b1 stimulates transcription phosphorylated by TGF-b1-receptors and associate with of metastasis-associated protein 1 (MTA1), a dual master the common mediator Smad4, which translocates to the coregulator, in epithelial cells, and that MTA1 status is a nucleus to participate in the expression of TGF-b1-target determinant of TGF-b1-induced epithelial-to-mesenchymal genes (Deckers et al., 2006). Previous studies have shown transition (EMT) phenotypes. In addition, we found that that CUTL1, also known as CDP (CCAAT displacement MTA1/polymerase II/activator protein-1 (AP-1) co-activator protein), a target of TGF-b1, is needed for its short-term complex interacts with the FosB-gene chromatin and stimu- effects of TGF-b1 on cell motility involving Smad4- lates its transcription, and FosB in turn, utilizes FosB/histone dependent pathway (Michl et al.,2005). deacetylase 2 complex to repress E-cadherin expression in In addition to TGF-b1-signaling, the process of breast TGF-b1-stimulated mammary epithelial cells. These find- cancer progression is profoundly influenced by the levels ings suggest that TGF-b1 regulates the components of EMT of metastasis-associated protein 1 (MTA1), an integral via stimulating the expression of MTA1, which in turn, role in nucleosome remodeling and histone deacetylation induces FosB to repress E-cadherin expression and thus, complex (Manavathi and Kumar, 2007). The MTA1 revealed an inherent function of MTA1 as a target and regulates the transcriptional outcome of its targets effector of TGF-b1 signaling in epithelial cells. because of its ability to modify the acetylation status on Oncogene (2011) 30, 2230–2241; doi:10.1038/onc.2010.608; the target gene chromatin and thus, controlling accessi- published online 24 January 2011 bility of various coregulators to DNA (Gururaj et al., 2006). In addition of its interaction with histone deacety- Keywords: TGF-b1 signaling; MTA1; FosB; target gene lases (HDACs) and nucleosome remodeling and histone transcription; epithelial-to-mesenchymal transition deacetylation complex, it functions as a corepressor on its targets (Mazumdar et al., 2001; Molli et al., 2008), where as MTA1 also interacts with RNA polymerase II (Pol II) Introduction and acts as a co-activator on some targets (Gururaj et al., 2006; Balasenthil et al., 2007). Earlier studies from this and Inappropriate transforming growth factor b1 (TGF-b1)- other laboratories have shown that increased MTA1 signaling has been linked with dedifferentiation, epithe- expression in murine mammary epithelial or tumor cells lial–mesenchymal transition (EMT) and tumor progres- is associated with not only increased invasiveness but also sion (Condeelis and Segall, 2003; Peinado et al., 2003). a reduced expression of E-cadherin, a hallmark of EMT During EMT, epithelial cells lose their polarity and repress (Zhang et al., 2006; Tong et al., 2007; Dannenmann et al., E-cadherin but express mesenchymal markers such as 2008). However, in spite of an inverse correlation in the vimentin and a-smooth muscle actin (a-SMA) (Thiery, levels of MTA1 and E-cadherin in tumor cells, it remains 2002; Radisky, 2005; Zavadil and Bottinger, 2005; Hugo unknown whether TGF-b1-signaling, a known modifier of et al., 2007). The process of cancer progression involves E-cadherin expression and EMT, require MTA1. Here, we a rapid modulation of TGF-b1-responsive genes with examined the role of MTA1 in TGF-b1-signaling and roles in EMT and metastasis via remodeling of target discovered that MTA1 is a target and an essential modifier of TGF-b1-signaling in epithelial cells. Correspondence: Professor R Kumar, Department of Biochemistry and Molecular Biology and Institute of Coregulator Biology, The George Washington University Medical Center, Washington, DC Results 20037, USA. E-mail: [email protected] MTA1 is required for TGF-b1-signaling in epithelial cells 3Current address: Department of Cancer Biology, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195, USA. In spite of a widespread upregulation of MTA1 in breast Received 3 October 2010; revised 17 November 2010; accepted 4 cancer and metastasis, it remains unknown whether December 2010; published online 24 January 2011 MTA1 has any role in EMT, a prerequisite for tumor MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2231 metastasis. Hence, we wished to investigate whether and CEBPa (CAAT enhancer-binding protein) inhibits MTA1 has a role in TGF-b1-induced EMT using HC11 TGFb1 signaling (Lee et al., 1994; MacLellan et al., cells as a model system. HC11 cells are mammary 1994; Kurisaki et al., 2003; Gery et al., 2005). As TGF- epithelial cells with polygonal morphology while TGF- b1 signaling stimulates (and not inhibits) MTA1 b1 stimulates its trans-differentiation into mesenchymal transcription, we choose to concentrate on CDP in the phenotype with an elongated morphology (Figure 1a) subsequent studies. (Radisky, 2005). We found that MTA1-depletion by The presence of seven consensus motifs for CDP on selective small interfering RNA (siRNA) compromises the MTA1 promoter provided clues that MTA1 might the ability of TGF-b1 to induce elongated morphology be regulated by TGF-b1-responsive transcription fac- in HC11 cells (Figure 1b). In parallel, MTA1-knock- tors. We found that transient expression of CDP alone is down also suppressed the effect of TGF-b1 to upregu- sufficient to induce MTA1 promoter activity (Figure 2e), late a-SMA (Figure 1c) and to downregulate epithelial suggesting that TGF-b1 may indeed, utilize CDP to marker E-cadherin (Figure 1c). Furthermore, HC11 stimulate MTA1 transcription (Supplementary Figure cells stably overexpressing MTA1 exhibited a distinct S2a). To validate this finding, we next examined the mesenchymal morphology (Figure 1d) as well as impact of CDP-knockdown on the ability of TGF-b1to downregulation of E-cadherin (Figure 1e). MTA1 over- stimulate MTA1 transcription. We found that CDP- expression or knockdown in the NMuMG primary depletion prevents TGF-b1 stimulation of MTA1 mammary cells also modulated the levels of E-cadherin promoter activity (Figure 2f). As expected, TGF-b1 and a-SMA (Figures 1f and g). These findings suggested stimulates the expression of CDP and a-SMA. Interest- that MTA1 is a modifier of TGF-b1-signaling and its net ingly, CDP-knockdown also abolished the stimulation levels affect EMT. of the endogenous MTA1 and a-SMA by TGF-b1 (Figure 2g). As Smad4-dependent signaling is involved TGF-b1-signaling induces MTA1 expression through CDP in the transcriptional regulation of CDP (Michl et al., During the course of preceding studies, we consistently 2005), we next examined the effect of Smad4-knock- noticed that TGF-b1-stimulation of cells accompanies down on MTA1 transcription. We found that Smad4- with an increased expression of MTA1 protein (Figures 1c depletion prevents the ability of TGF-b1 stimulation of and f), raising the hypothesis that MTA1 might be MTA1 transcription (Figure 2h). These results suggest a regulated by TGF-b1-signaling. To test this notion, we mechanistic role of CDP in TGF-b1 stimulation of next treated HC11 cells with TGF-b1 and showed that MTA1 transcription. indeed, TGF-b1 stimulates the level of MTA1 protein To gain a deeper insight into the molecular mechan- and mRNA (Figures 2a and b) and represses the ism involved in CDP regulation of MTA1 stimulation, expression of E-cadherin protein (Figure 2a) and that we next mapped the recruitment of CDP to MTA1 transcriptional inhibitor actinomycin D substantially promoter by chromatin immunoprecipitation (ChIP). reduces the levels of TGF-b1-inducible MTA1 mRNA CDP was recruited to regions II, IV, V and VI of the and protein (Figures 2b and c). These findings suggested MTA1 promoter (Figure 3a). Interestingly, CDP inter- that MTA1 may be a target of TGF-b1. action at these regions was further increased on TGF-b1 MTA1 gene resides on chromosome 14q32.3 locus in treatment. To further characterize the responsive active human and chromosome 12f1 locus in mouse. To study sites on MTA1 promoter, we determined the status of the TGF-b1 regulation of MTA1 transcription, we acetylated histone H4 at all these regions. We found that cloned the 5 kb promoter region of mouse MTA1 and TGF-b1 stimulation leads to recruitment of acetylated generated a series of promoter-luc reporter systems histone H4 at region IV and VI, suggesting that these (Supplementary Figure S1a). The MTA1 region of could be the functional regions in vivo (Figure 3b). On À5200 to À2872-bp upstream to the first codon ATG the other hand, there was no recruitment of acetylated showed the maximum potential for promoter-like histone H3 onto MTA1 promoter in TGF-b1-stimulated sequence. Both the minimal and full-length MTA1 cells (Supplementary Figure S2b). Sequential double- promoter was equally responded to TGF-b1 stimulation ChIP analysis involving CDP followed by acetylated H4 (Figure 2d and Supplementary Figure S1a). antibodies indicated that CDP and acetyl histone H4 As MTA1 is involved in promoting tumor invasive- are co-recruited only to region VI of MTA1 promoter ness and motility (Mahoney et al., 2002; Hofer et al., (Figure 3c). 2004) and is now induced by TGF-b1 (this study), we Next, we examined whether CDP directly binds to the next performed Transfac analysis of the MTA1 promo- region VI of MTA1 promoter, which harbors two ter to identify the bindings sites for transcription factors putative sites for CDP. Probes for both sites, designated that might be responsive to TGF-b1 signaling and thus, BS1 and BS2, were used to examine binding to involved in the noted TGF-b1 stimulation of MTA1 glutathione S-transferase–CDP by electrophoretic transcription. We found that MTA1 promoter harbors mobility shift assay (EMSA). Results indicated that binding sites for TGF-b1-related factors CDP, CEBPa CDP does not bind to region BS1 but effectively binds and YY1 (Supplementary Figure S1b and Supplemen- to region BS2, indicating that BS2 may represent the tary Table S1). CDP is a direct target of TGF-b1 actual CDP-binding site in the region VI of MTA1 signaling and in turn, regulates other TGF-b1-respon- promoter (Figure 3d and Supplementary Figure S2c). sive genes and promotes tumor cell invasiveness (Michl Accordingly, results from the nuclear extracts et al., 2005; Michl and Downward, 2006), while YY1 from control and TGF-b1-stimulated HC11 cells

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2232

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2233 revealed the formation of CDP–DNA complex(s) at the expression, which cannot be rescued by TGF-b1 BS2 region and the observed complex could be super- stimulation (Figure 4g). This may be due to the fact shifted by an anti-CDP-Ab but not control immunoglo- that actinomycin D is a broad range transcriptional bulin G (IgG) (Figure 3e). To further confirm the inhibitor and as expected, it also blocked TGF-b1 binding of CDP to the BS2 motif in the region VI in vivo, stimulation of MTA1, a new FosB co-activator (this we performed ChIP for CDP recruitment to these study). Consistent with these results, we found that regions using specific primers and found that, indeed, MTA1 overexpression induces FosB-promoter activity CDP-binding sites are limited to the BS2 motif in region in HC11 cells (Figure 4h). VI of MTA1 promoter in TGF-b1-stimulated cells To determine the underlying mechanism of MTA1 (Figure 3f). These findings suggest that TGF-b1 utilizes regulation of FosB expression, we next carried out a CDP to stimulate MTA1 transcription (Figure 3g). ChIP-based promoter walk in HC11 cells treated with or without TGF-b1. We found the recruitment of MTA1 to TGF-b1-mediated FosB expression requires MTA1 only region III (À644 to À794) of the FosB-promoter To understand the physiologic significance of TGF-b1 and TGF-b1 stimulation further enhances such recruit- regulation of MTA1 expression, we next investigated the ment (Figure 5a). We also found that RNA Pol II and possibility of a role of MTA1 in TGF-b1 signaling. MTA1/Pol II co-activator complex is also recruited onto Although these studies were in-progress, results from region III of FosB-promoter (Figure 5a). Interestingly, ongoing, in-house, genome-wide analyses of MTA1 complexes containing MTA1/acetyl hitsone H4 but not targets in MCF-7 cells using NimbleGen promoter MTA1/HDAC2 is recruited to the same region, platform revealed that MTA1 might be recruited to supporting the functional active nature of the region genes involved in the TGF-b1 pathway (Supplementary (Figure 5a). A schematic representation of recruitment Table S2). Since previous study has implicated Fos of MTA1, RNA Pol II and acetyl histone H4 onto FosB- family genes in TGF-b1-induced EMT (Eferl and promoter is presented in Supplementary Figure S3a. Wagner, 2003) and because FosB is now being identified Transfac analysis of the region III of FosB-promoter as a MTA1 target in our ChIP-on-ChIP analysis (this revealed the presence of one potential AP-1 site (50- study), we decided to focus on FosB as a potential CGAGTCACAC-30, À708 to À717). Members of the MTA1 target and asked whether MTA1 is required Jun/Fos family form hetero- or homo-dimers and for TGF-b1 induction of FosB. We found that both constitute AP-1 transcription factors. Hence, we next TGF-b1 stimulation as well as MTA1 overexpression carried out single or sequential c-Jun-Ab-based ChIP alone induces FosB mRNA or protein in HC11 cells analysis onto FosB-promoter. To our surprise, c-Jun (Figures 4a and b). Further selective siRNA-mediated and MTA1/c-Jun complexes are also recruited to the knockdown of MTA1 in HC11 cells reduced FosB mRNA region III of FosB-promoter (Figure 5a). To determine and protein (Figures 4c and d). Since the outcome of whether c-Jun has a mechanistic role along with MTA1 siRNA-mediated knockdown studies is dependent on the in TGF-b1-mediated stimulation of FosB expression, we extent of target knockdown, we next examined the effect examined the effects of c-Jun alone or in combination of of MTA1-depletion in mouse embryonic fibroblasts MTA1 on FosB-promoter (À1000 to þ 307) and a (MEFs) from wild-type and MTA1À/À mice treated with truncated FosB-promoter lacking AP-1 site (À603 to TGF-b1. We found that MTA1-deficiency substantially þ 307) in TGF-b1-stimulated cells. We found that compromised the ability of TGF-b1 to induce FosB MTA1 and c-Jun stimulates FosB-promoter activity in protein expression and promoter activity in MEFs a cooperative manner and that AP-1 site on FosB- (Figures 4e and f). Further co-transfection of MTA1 promoter is indispensable for stimulation of Fos-B in MTA1À/À MEFs results in increased FosB-promoter activity by MTA1 and/or TGF-b1 (Figure 5b). Collec- activity (Figure 4f), implying an important role of tively, these results suggest that MTA1/Pol II/c-Jun may MTA1 in the noted upregulation of FosB in TGF-b1- exist in the same complex under physiologic condition. stimulated cells. The observed effect of TGF-b1on To support this notion, we showed that indeed, RNA FosB expression was transcriptional in nature as Pol II and c-Jun could be co-immunoprecipitated along inclusion of actinomycin D alone repressed FosB with MTA1 in both basal and TGF-b1-stimulated HC11

Figure 1 MTA1 induces EMT in the mammary epithelial cells. (a) Bright-phase microscopic analysis of HC11/pcDNA and HC11/ MTA1 stable clones treated with and without TGF-b1. (b) F-actin staining of HC11 cells transfected with MTA1 siRNA with or without TGFb1. Upper panel, western blot analysis for MTA1. (c) Western blot analysis for E-cadherin, a-SMA and MTA1 expression from the cell lysates isolated from HC11 cells treated with MTA1–siRNA in presence and absence of TGF-b1. Vinculin was used as the loading control. (d) F-actin staining of HC11 pcDNA and MTA1 stable clones with and without TGF-b1. Upper panel represents the western blot analysis showing the expression of MTA1 in HC11 pcDNA and MTA1 stable clones. (e) Confocal staining (upper panel) and western blot analysis (lower panel) for E-cadherin in HC11 pcDNA and MTA1 stable clones with and without TGF-b1 treatment. Nucleus is stained with DAPI. (f) Western blot analysis for E-cadherin, a- SMA and MTA1 expression in the cell lysates of NMuMG (normal murine mammary gland) cells transfected with pcDNA and MTA1 in presence and absence of TGF-b1. (g) Western blot analysis for E-cadherin and a-SMA expression from the cell lysates of NMuMG cells treated with specific MTA1– siRNA in presence and absence of TGF-b1. NMuMG cells were transfected with control siRNA and MTA1-siRNA. After 48 h of transfection, cells were serum starved and treated with TGF-b1 for 12 h. Cells were harvested and western blot analysis was carried out with specified antibodies. (a–g) cells were treated with 2 ng/ml of TGF-b1 for 12 h.

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2234

Figure 2 TGF-b1 induces MTA1 expression in HC11 cells. (a) Western blot analysis for MTA1 and E-cadherin proteins from the cell lysates isolated from HC11 cells treated with TGF-b1 (2 ng/ml) for 4, 8, 12 and 24 h. (b) Quantitative PCR (qPCR) analysis of MTA1 mRNA expression in HC11 cells treated with TGF-b1 (2 ng/ml for 12 h) in presence and absence of actinomycin D (250 ng/ml). Cells were treated with actinomycin D 1 h before TGF-b1 treatment. Results are presented in-terms of a fold change after normalizing MTA1 mRNA levels with b-actin mRNA. Each value represents the mean of±s.e. of three independent experiments. (c) Western blot analysis for MTA1 from the cell lysates isolated from HC11 cells treated with TGF-b1 in presence and absence of actinomycin D (250 ng/ml) (upper panel) and its quantitation using image quant (lower panel). (d) MTA1 promoter (both minimal and full length) activity in HC11 cells treated with TGF-b1 (2 ng/ml for 12 h). HC11 cells were transfected with murine MTA1-pGL3 luciferase reporter plasmid and pCMVb vector carrying b-galactosidase gene as an internal control for transfection efficiency. After 36 h of transfection, cells were serum starved for 14 h, treated with TGF-b (2 ng/ml for 12 h) and luciferase activities in triplicate samples were measured. Results were presented in terms of fold change and the values represent the mean of±s.e. from three independent transfection experiments. (e) MTA1 promoter activity after co-transfecting with CDP-pMX in presence of TGF-b1 (2 ng/ml for 12 h) in HC11 cells. CDP–pMX plasmid with different concentration (0.25 and 1 mg) was transfected along with MTA1-pGL3 luciferase reporter plasmid and promoter luciferase assay was carried out as described in figure legend 2d.(f) MTA1 promoter activity in TGF-b1 (2 ng/ml for 12 h) activated HC11 cells with and without CDP knockdown. HC11 cells were transfected with CDP–siRNA and after 36 h of transfection MTA1-pGL3 luciferase reporter plasmid and pCMVb vector carrying b-galactosidase gene were transfected and luciferase assay was carried out as described in figure legend 2d.(g) Western blot analysis of MTA1, CDP and a-SMA protein expression from the cell lysates of HC11 treated with TGF-b1 (2 ng/ml for 12 h) after selective knock down of CDP. (h) MTA1 promoter activity in TGF-b1 (2 ng/ml for 12 h) activated HC11 cells with and without Smad4 knockdown. After Smad4 knockdown using Smad4-siRNA, HC11 cells were transfected with MTA1-pGL3 luciferase reporter plasmid following the method described earlier.

cells but the amount of co-IP proteins were significantly tion of cells promotes the formation of distinct protein/ increased in stimulated cells, presumably because of DNA complexes. To gain insights into the components increased levels of MTA1 protein (Figure 5c). To of complexes, we attempted to supershift the protein/ demonstrate a potential direct binding of c-Jun to DNA band by inclusion of anti-MTA1-Ab, anti-c- FosB-promoter, we next performed EMSA using the Jun-Ab or both (Figure 5d). Interestingly, anti-MTA1 PCR product encompassing region III of the murine or anti-c-Jun-antibodies had similar effects on the FosB-promoter, and nuclear extracts from TGF-b1- mobility of the protein/DNA complex. However, the stimulated HC11 cells. We found that TGF-b1 stimula- complex could be further super shifted by co-treatment

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2235

Figure 3 TGF-b1 induces MTA1 expression through CDP. (a) Recruitment of CDP on to the mouse MTA1 promoter in HC11 cells treated with TGF-b1. HC11 cells stimulated with TGF-b1 were treated with 1% formaldehyde to crosslink the histones to DNA and lysed by sonication, immunoprecipitated either by anti-CDP antibody or IgG antibody. The immunoprecipitants were then collected by adding beads, washed, eluted the DNA from the beads and purified DNA was subjected to PCR. (b) Recruitment of acetyl histone H4 (AcH4) on to mouse MTA1 promoter in HC11 cells treated with TGF-b1. ChIP analysis was carried out with AcH4 antibody as described earlier. (c) Recruitment of CDP followed by AcH4 complex onto MTA1 promoter (À3368 to À3604) by sequential double ChIP assay in HC11 cells treated with TGF-b1. The first ChIP was carried out with anti-CDP antibody followed by second ChIP with anti-AcH4 antibody. (d) EMSA analysis of glutathione S-transferase (GST)–CDP binding to the binding site 2 (BS2) of region VI of mouse MTA1 promoter in the nuclear extracts prepared from TGF-b1-stimulated HC11 cells. (e) EMSA analysis showing super shift with CDP antibody on BS2 of region VI of mouse MTA1 promoter in the nuclear extract prepared from TGF-b1-stimulated HC11 cells. (f) Recruitment of CDP on to the BS2 of region VI of mouse MTA1 promoter in the nuclear extracts prepared from the HC11 cells treated with TGF-b1. (g) Model diagram showing the requirement of CDP for the induction of MTA1 expression by TGF-b1. (a–f) Cells were treated with 2 ng/ml of TGF-b1 for 12 h. with both antibodies, suggesting the existence of MTA1- cadherin (Ohnishi et al., 2008). Having shown that cJun complex and its ability to interact FosB–DNA. In MTA1 is a target of TGF-b1 signaling and has an brief, these findings reveal that MTA1 behaves as a co- important role in TGF-b1-mediated stimulation of FosB activator in stimulating the expression of Fos-B in TGF- expression, we next investigated the role of this newly b1-stimulated epithelial cells. identified MTA1-FosB pathway in the regulation of E-cadherin. We next examined the effects of MTA1 or FosB on the regulation of E-cadherin transcription. MTA1/HDAC2/FosB corepressor complex Co-expression of either MTA1 or FosB substantially downregulates E-cadherin downregulates E-cadherin promoter activity in HC11 On the basis of the complementary DNA microarray cells and there is no addition effect of TGF-b1 analysis, an earlier study has suggested but not stimulation (Figure 6a). To demonstrate a role of established that FosB may negatively regulate E- MTA1 in the regulation of E-cadherin transcription,

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2236

Figure 4 TGF-b1-mediated FosB expression requires MTA1. (a) Quantitative PCR (qPCR) analysis of Fos B and MTA1 mRNA expression in HC11 cells after co-transfecting with pcDNA and MTA1 with and without TGF-b1 treatment. Results are presented in- terms of a fold change after normalizing with b-actin mRNA. Each value represents the mean of±s.e. of three independent experiments. (b) Western blot analysis for FosB and MTA1 protein expression from the cell lysates prepared from HC11 cells treated with TGF-b1 (2 ng/ml) for 4, 8, 12 and 24 h. (c) qPCR analysis of FosB mRNA expression in TGF-b1-stimulated HC11 cells after selective knockdown of MTA1 using MTA1–siRNA. Results are presented in-terms of a fold change after normalizing the FosB and MTA1 mRNA expression levels with b-actin mRNA. Each value represents the mean of±s.e. of three independent experiments. (d) Western blot analysis for FosB protein expression in the cell lysates prepared from TGF-b1-stimulated HC11 cells after selective knockdown of MTA1 using MTA1–siRNA. (e) Western blot analysis for FosB protein in MTA1 þ / þ and MTA1À/À MEFs after treating with TGF-b1. (f) FosB-promoter activity in MTA1 þ / þ and MTA1À/À MEFs after co-transfecting with MTA1 in presence and absence of TGF-b1. FosB-pGL3 luciferase reporter plasmid and pCMVb vector carrying b-galactosidase gene were transfected and after 36 h of transfection cells were serum starved and treated with TGF-b1. Luciferase assay was carried out as mentioned in figure legend 2d.(g) qPCR analysis for FosB mRNA expression in HC11 cells treated with TGF-b1 in presence and absence of actinomycin D (250 ng/ml). Actinomycin D was added 1 h before the TGF-b1 treatment. The results were represented in terms of fold change after normalizing the FosB mRNA levels with b-actin mRNA. Each value represents the mean of±s.e. of three independent experiments. (h) FosB-promoter activity in HC11 cells after co-transfecting with MTA1 in presence and absence of TGF-b1. Luciferase assay was carried out in triplicates and results were presented in-terms of fold change and the values represent the mean of±s.e. from the three independent transfection experiments. (a, c–f, h) Cells were treated with 2 ng/ml of TGF-b1 for 12 h.

we showed a significant reduction in the ability of TGF- MTA1–FosB pathway, we next performed a ChIP- b1 to inhibit E-cadherin promoter activity in MTA1À/À based promoter walk onto E-cadherin promoter. We MEFs (Figure 6b). However, overexpression of FosB, a found the recruitment of MTA1, FosB and MTA1/FosB target of MTA1, could inhibit E-cadherin promoter complex to the regions II and III of E-cadherin promoter activity in MTA1À/À MEFs. To independently validate (Figure 7a) but not to region I, IV, V and VI these results, we next showed that FosB knockdown (Supplementary Figure S3b) in HC11 cells. Interest- in HC11 cells (Figure 6c) antagonizes the ability of ingly, TGF-b1 stimulation of HC11 cells results in an TGF-b1 stimulation or MTA1 overexpression to inhibit enhanced recruitment of these proteins onto E-cadherin E-cadherin transcription (Figure 6c). These findings promoter (Figure 7a). As MTA1 is known to interact suggest that FosB may constitute a downstream effect with HDACs (Molli et al., 2008) and the fact that both of MTA1 in repressing E-cadherin transcription, at-least MTA1 and HDAC2 are components of the nucleosome in-part. remodeling and histone deacetylation corepressor (Man- To delineate the molecular mechanism involved in the avathi and Kumar, 2007) we next showed that HDAC2 observed repression of E-cadherin transcription by as well as FosB/HDAC2 complex are also recruited to

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2237

Figure 5 MTA1 regulation of FosB transcription. (a) Recruitment of MTA1, Pol II, MTA14Pol II, MTA14AcH4, MTA14 HDAC2, c-Jun and MTA14 c-Jun on to the FosB-promoter (either on to region I or II or III) in HC11 cells after treatment with TGF- b1. Single ChIP analysis was carried out with MTA1, Pol II and c-Jun antibodies where as double ChIP analysis was carried out either with MTA1 followed by Pol II (first ChIP: MTA1 Ab; second ChIP: Pol II Ab) or MTA1 followed by c-Jun (first ChIP: MTA1 Ab; second ChIP: c-Jun Ab) or MTA1 followed by AcH4 (first ChIP: MTA1 Ab; second ChIP: AcH4 Ab) or MTA1 followed by HDAC2 antibody (first ChIP: MTA1 Ab; second ChIP: HDAC2 Ab) in TGF-b1-stimulated HC11 cells. (b) FosB-promoter activity in HC11 cells after co-transfecting either with MTA1-pcDNA or c-Jun-pcDNA or both in presence of TGF-b1. (c) Western blot analysis for Pol II, c-Jun and HDAC2 after IP with MTA1 in HC11 cells treated with TGF-b1. (d) EMSA analysis of c-Jun and MTA1 binding to the FosB-promoter using the PCR product encompassing the region III (À644 to À794) of FosB-promoter in the nuclear extract of HC11 cells treated with TGF-b1. (a–d) Cells were treated with 2 ng/ml of TGF-b1 for 12 h. the regions II and III of E-cadherin promoter in TGF-b- MTA1 and FosB antibodies resulted in an additional responsive manner (Figure 7a). We also showed that retardation of mobility of the complex (Figure 7d). knockdown of FosB in HC11 cells inhibits MTA1 These findings suggest a potential role of MTA1/FosB/ recruitment onto E-cadherin DNA, while there was no HDAC2 corepressor complex in TGF-b1-mediated sup- effect of MTA1–siRNA on the recruitment of FosB pression of E-cadherin transcription. Consistent with to E-cadherin DNA (Figure 7b). In addition to this, no these results, we showed that FosB downregulation in effect was observed on the recruitment of FosB to HC11 leads to an enhanced E-cadherin expression E-cadherin promoter even in MTA1 þ / þ and MTA1À/À (Figure 7e). In summary, findings presented here suggest MEFs (Supplementary Figure S3c). This is presumably, that TGF-b1 signaling targets MTA1 and that MTA1 due to downstream nature of FosB in relation to MTA1. has an important role in the action of TGF-b1and in These findings suggested the possibility of an enhanced supporting the expression of FosB, which in turn, interaction among MTA1, HDAC2 and FosB in TGF- facilitates the repression of E-cadherin—a target of b1-stimulated cells. Indeed, we found that both MTA1 both TGF-b1 and MTA1 in epithelial cells (Figure 8). and HDAC2 could be efficiently co-immunoprecipated along with FosB in TGF-b1-stimulated HC11 cells (Figure 7c). To demonstrate a potential direct binding of FosB to Discussion the mouse E-cadherin-DNA, we next performed EMSA using the PCR product encompassing region II or Our core findings that MTA1 downregulates the III (Figure 7d) and nuclear extracts from TGF-b1- expression of E-cadherin has introduced a new regula- stimulated HC11 cells. We found the formation of tory player in the event of EMT. E-cadherin down- distinct protein/DNA complexes. Interestingly, the regulation is one of the principal events during EMT in binding of these complexes to the test DNA could be embryonic development and cancer progression. EMT super shifted by the inclusion of anti-FosB or anti- represents a key event in the progression of tumor cells MTA1 but not control IgG; while co-incubation of anti- to dedifferentiated and invasive phenotypes (Thiery,

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2238 2002) and in cancer metastasis (Radisky, 2005; Hugo et al., 2007). It is evident that many transcriptional factors, such as Snail, Slug, SIP1, Twist, LIV1 and dEF were involved in the repression of E-cadherin (Thiery, 2002; Zavadil and Bottinger, 2005) and there-by stimulates the process of epithelial plasticity in breast cancer cells. Although many signaling pathways con- verge with the function and expression of E-cadherin, the transcriptional regulation of E-cadherin expression continue to be poorly understood. In this context, this study establishes a definitive contribution of MTA1 in downregulation of E-cadherin expression, leading to EMT. As MTA1 regulates its target genes by acting either as a transcriptional corepressor or co-activator (Mazumdar et al., 2001; Gururaj et al., 2006; Balasenthil et al., 2007; Molli et al., 2008), our results show that E-cadherin is target of MTA1 and it downregulates E-cadherin expression at the level of transcription by directly interacting with its promoter as a part of FosB/HDAC2 complex. MTA1 overexpression is associated with metastatic growth of cell lines and with invasive growth of tumors (Manavathi and Kumar, 2007). Studies presented here provide a mechanistic basis of earlier correlative studies between MTA1 and E-cadherin (Zhang et al., 2006; Tong et al., 2007; Dannenmann et al., 2008). Recently, von Burstin et al. (2009) reported the role of HDACs in silencing of E-cadherin during the metastatic process of pancreatic cancer cells. Although there is existence of a strong relationship between the MTA family, HDACs and E-cadherin, the study explains the mechanistic basis of the regulation of E-cadherin by these molecules during breast cancer progression. In this context, our study demonstrates the contribution of MTA1 in the regulation of TGF-b1-mediated E-cadherin repression, which may be one of the mechanisms that attributes to the metastatic function of MTA1. Overall, our results strongly suggest that MTA1 induces EMT by regulating the expression of E-cadherin, which has a key role in regulating the motility, invasion and metastatic poten- tial of cancer cell.

Materials and methods

Antibodies and cell culture Antibodies against MTA1 (A300-280A), and RNA Pol II (A300-653A) were purchased from Bethyl labs, Montgomery, TX, USA. HDAC2 (sc-9959), FosB (sc-48), E-cadherin (sc- 1500), c-Jun (sc-1694) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). a-SMA (A2547), normal mouse IgG, rabbit IgG and antibodies against vinculin were purchased from Sigma Chemical Co (St Louis, MO, Figure 6 Effect of MTA1 and FosB expression on E-cadherin transcription. (a) E-cadherin promoter activity in HC11 cells after USA). Acetyl histone H3 (06-599) and H4 (06- 598) antibodies co-transfecting either with pcDNA or MTA1 or FosB in presence were purchased from United Biotech Inc. (Mountain View, of TGF-b1. (b) E-cadherin promoter activity in MTA1À/À MEFs CA, USA). All cells were cultured in Dulbecco’s modified after co-transfecting with either pcDNA or FosB in presence and Eagle’s medium/F12 medium supplemented with 10% fetal absence of TGF-b1. (c) E-cadherin promoter activity in HC11 cells bovine serum. HC11 cells were maintained in RPMI-1640 after co-transfecting with MTA1 followed by selective knockdown medium supplemented with 10% fetal calf serum, 10 ng/ml of FosB in presence of TGF-b1. The upper panel represents the epidermal growth factor and 5 mg/ml insulin. TGF-b1 was western blot analysis showing the knock down of FosB in HC 11 obtained from PeproTech Inc., Rocky Hill, NJ, USA. Cells cells from the luciferase samples. (a–c) Cells were treated with 2 ng/ were treated with TGF-b1 (2 ng/ml of the medium for 12 h) ml of TGF-b1 for 12 h. after 12–14 h of serum starvation in serum-free medium.

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2239

Figure 7 Downregulation of E-cadherin expression by MTA1/FosB/HDAC2 complex. (a) Recruitment of MTA1, FosB, MTA14FosB, HDAC2, FosB 4HDAC2 on to the region II (À521 to À709) and III (À979 to À1151) of E-cadherin promoter in TGF-b1-stimulated HC11 cells. Single ChIP analysis was carried out with either MTA1 or FosB or HDAC2 antibodies whereas double ChIP analysis was carried out either with MTA1 followed by FosB antibody (first ChIP: MTA1 Ab, second ChIP: FosB) or FosB followed by HDAC2 (first ChIP: FosB; second ChIP: HDAC2) in the TGF-b1-stimulated HC11 cells. (b) Recruitment of MTA1 after selective knockdown of FosB (upper panel) and FosB recruitment after MTA1 knockdown (lower panel), on to the region II and III of E-cadherin promoter in HC11 cells stimulated with TGF-b1. (c) Western blot analysis showing the interaction of FosB with MTA1 and HDAC2 in TGF-b1-stimulated HC11 cells. Immunoprecipitation was carried out with anti-FosB antibody from the HC11 cells treated with or with TGF-b1 for 12 h and western blot analysis was carried out with MTA1, HDAC2 and FosB antibodies. (d) EMSA analysis of FosB and MTA1 binding to the E-cadherin promoter using the PCR product encompassing the region II (À521 to À709) and III (À979 to À1151) of E-cadherin promoter in the nuclear extract of HC11 cells treated with TGF-b1. (e) Confocal analysis for E-cadherin expression after selective knock down of FosB using FosB–siRNA in HC11 cells. (a–e) Cells were treated with 2 ng/ml of TGF-b1 for 12 h.

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2240

Figure 8 Schematic representation of MTA1 regulation of FosB and E-cadherin expression: TGF-b1 stimulation of mammary epithelial cells results in the induction of MTA1. Once MTA1 is induced, it forms a co-activator complex along with c-Jun and Pol II and gets recruited to FosB-promoter leading to induced FosB expression, which in turn, represses the transcription of E-cadherin by recruiting on to its chromatin along with MTA1 and HDAC2. Dotted line shows MTA1-independent regulation of E-cadherin by other mechanisms not investigated here.

Quantitative PCR ChIP assay For quantitative PCR total RNA was extracted using TRIzol ChIP assays were performed as described previously (Mazumdar reagent (Invitrogen, Carlsbad, CA, USA), and first-strand et al., 2001). Briefly, cells were crosslinked with formaldehyde complementary DNA synthesis was carried out with Super- (1% final concentration) and sonicated on ice to fragment the Script II reverse transcriptase (Invitrogen) using 2 mg of total chromatin into an average length of 500 bp–1 kb. The lysates RNA and poly (dT) primer. Quantitative reverse transcrip- were diluted using chromatin-dilution buffer. MTA1 or CDP or tase–PCR was performed with the gene-specific primers listed acetyl histone H4 or acetyl Histone H3 or Pol II or HDAC2 or in Supplementary Table 3 using a 7900HT sequence detection FosB or c-Jun or mouse IgG were used to immunoprecipitate system (Applied Biosystems, Foster City, CA, USA). the respective antigens at 4 1C overnight. Protein A sepharose The levels of mRNA of all the genes were normalized to that beads saturated with bovine serum albumin and single-strand of b-actin mRNA. DNA were added to the lysate to isolate the antibody-bound complexes. The beads were washed to remove nonspecific binding, and the antibody-bound chromatin was eluted. The Reporter and immunoprecipitation assays eluate was ‘de-crosslinked’ by heating at 65 1Cfor6h.RNase MTA1-luc, FosB-luc and E-cadherin-luc assays were per- was added during this step to digest the RNA contaminants. formed according to the manufacturer’s instructions (Promega, Samples were treated with proteinase K for 1 h at 45 1C to digest Madison, WI, USA), and the results were standardized against the immunoprecipitated proteins, and finally, the DNA was the b-galactosidase activity, an internal control. Some assays extracted using the phenol chloroform method. For the double were performed in the presence of control siRNA or MTA1 ChIP experiment, an initial ChIP assay was done with either siRNA and FosB–siRNA. Cell lysates were prepared in RIPA MTA1 or FosB antibody to immunoprecipitate MTA1-bound lysis buffer, and western blot analysis was performed as chromatin, which was eluted from the protein A sepharose beads previously described (Kumar et al., 2002). For immunopreci- and subjected to a second ChIP assay with either Pol II or anti- pitation assays, 1 mg of cell lysate was incubated with MTA1 HDAC2 antibody. With the DNA isolated at the end of the or FosB antibody and agarose beads with constant rotation ChIP analysis, PCR was performed using the primers mentioned followed by extensive washing with NP-40 buffer (20 mM in Supplementary Table 4. HEPES [pH 7.6], 150 mM KCl, 1 mM DTT, 0.1% NP-40 and 8% glycerol). The immunoprecipitated proteins were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Electrophoretic mobility shift assay transferred to nitrocellulose membrane and analyzed by Nuclear extracts were prepared using a Nonidet P-40 lysis method western blotting using antibodies for TGF-b1 signaling (Bui-Nguyen et al., 2010) EMSA for CDP or FosB or E-cadherin components. DNA binding was performed using the annealed and [g32P] ATP end-labeled oligonuclotides (Supplementary Table 5) or PCR products in a 20-ml reaction mixture for 15 min at 20 1C. Samples Cloning of murine MTA1 promoter were run on a nondenaturing 5% polyacrylamide gel and imaged Full-length murine MTA1 promoter and its deletion constructs by autoradiography. Specific competitions were performed by were generated by following the procedures described else- adding a 10 folds excess of competitor to the incubation mixture, where (Pakala et al., 2010). and supershift EMSAs were performed by adding 1.5 mlofthe either MTA1 or CDP or FosB or c-Jun antibodies. siRNA transfection siRNA against MTA1 and negative control siRNA were Confocal analysis purchased from Dharmacon (Lafayette, CO, USA). HC11 After transfecting HC11 cells with MTA1-siRNA or FosB-siRNA, cells were seeded at 40% density the day before transfection in E-cadherin and a-SMA expression was determined by indirect six-well plates, and siRNA transfections were performed with immunofluorescence. The cells were grown on sterile glass cover oligofectamine reagent (Invitrogen) according to the man- slips, fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton ufacturer0s instructions. Cells were harvested 48 h after X-100 and blocked in 10% normal goat serum in phosphate- transfection and cell lysates were prepared. buffered saline. Cells were incubated with FosB or E-cadherin

Oncogene MTA1, a new effector of TGF-b1 signaling SB Pakala et al 2241 antibody, washed three times in phosphate-buffered saline, and Conflict of interest then incubated with goat anti-rabbit secondary antibodies conjugated with 488-Alexa from Molecular Probes (Eugene, OR, The authors declare no conflict of interest. USA). The 4,6-diamidino-2-phenylindole (Molecular Probes) was used as a nuclear stain. Microscopic analysis was performed using an Olympus FV300 laser-scanning confocal microscope (Olympus America Inc., Melville, NY, USA) using sequential laser excitation Acknowledgements to minimize fluorescence emission bleed-through. We would like to thank Dr Toshio Matsumoto for FosB- Statistical analysis promoter constructs and Dr Eric R. Fearon for E-cadherin The results are given as the mean±s.e. Statistical analysis of promoter construct. The work was supported by NIH Grant the data was performed by using Student’s t-test. CA98823 (RK).

References

Balasenthil S, Gururaj AE, Talukder AH, Bagheri-Yarmand R, invasion, and anchorage-independent survival of immortalized Arrington T, Haas BJ et al. (2007). Identification of Pax5 as a human keratinocytes. Oncogene 21: 2161–2170. target of MTA1 in B-cell lymphomas. Cancer Res 67: 7132–7138. Manavathi B, Kumar R. (2007). Metastasis tumor antigens, an Bui-Nguyen TM, Pakala SB, Sirigiri RD, Xia W, Hung MC, Sarin SK emerging family of multifaceted master coregulators. J Biol Chem et al. (2010). NF-kappaB signaling mediates the induction of 282: 1529–1533. MTA1 by hepatitis B virus transactivator protein HBx. Oncogene Massague J, Seoane J, Wotton D. (2005). Smad transcription factors. 29: 1179–1189. Genes Dev 19: 2783–2810. Condeelis J, Segall JE. (2003). Intravital imaging of cell movement in Mazumdar A, Wang RA, Mishra SK, Adam L, Bagheri-Yarmand R, tumours. Nat Rev Cancer 3: 921–930. Mandal M et al. (2001). Transcriptional repression of oestrogen Dannenmann C, Shabani N, Friese K, Jeschke U, Mylonas I, Bruning receptor by metastasis-associated protein 1 corepressor. Nat Cell A. (2008). The metastasis-associated gene MTA1 is upregulated in Biol 3: 30–37. advanced ovarian cancer, represses ERbeta, and enhances expres- Michl P, Downward J. (2006). CUTL1: a key mediator of TGFbeta- sion of oncogenic cytokine GRO. Cancer Biol Ther 7: 1460–1467. induced tumor invasion. Cell Cycle 5: 132–134. Deckers M, van Dinther M, Buijs J, Que I, Lowik C, van der Pluijm G Michl P, Ramjaun AR, Pardo OE, Warne PH, Wagner M, Poulsom R et al. (2006). The tumor suppressor Smad4 is required for et al. (2005). CUTL1 is a target of TGF(beta) signaling transforming growth factor beta-induced epithelial to mesenchymal that enhances cancer cell motility and invasiveness. Cancer Cell 7: transition and bone metastasis of breast cancer cells. Cancer Res 66: 521–532. 2202–2209. Molli PR, Singh RR, Lee SW, Kumar R. (2008). MTA1-mediated Eferl R, Wagner EF. (2003). AP-1: a double-edged sword in transcriptional repression of BRCA1 tumor suppressor gene. tumorigenesis. Nat Rev Cancer 3: 859–868. Oncogene 27: 1971–1980. Gery S, Tanosaki S, Bose S, Bose N, Vadgama J, Koeffler HP. (2005). Ohnishi YN, Sakumi K, Yamazaki K, Ohnishi YH, Miura T, Down-regulation and growth inhibitory role of C/EBPalpha in Tominaga Y et al. (2008). Antagonistic regulation of cell-matrix breast cancer. Clin Cancer Res 11: 3184–3190. adhesion by FosB and DeltaFosB/Delta2DeltaFosB encoded by Gururaj AE, Singh RR, Rayala SK, Holm C, den Hollander P, Zhang alternatively spliced forms of fosB transcripts. Mol Biol Cel 19: H et al. (2006). MTA1, a transcriptional activator of breast cancer 4717–4729. amplified sequence 3. Proc Natl Acad Sci USA 103: 6670–6675. Pakala SB, Bui-Nguyen TM, Reddy SD, Li DQ, Peng S, Rayala SK Hofer MD, Menke A, Genze F, Gierschik P, Giehl K. (2004). et al. (2010). Regulation of NF-kappaB circuitry by a component of Expression of MTA1 promotes motility and invasiveness of PANC- the nucleosome remodeling and deacetylase complex controls 1 pancreatic carcinoma cells. Br J Cancer 90: 455–462. inflammatory response homeostasis. J Biol Chem 285: 23590–23597. Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, Williams Peinado H, Quintanilla M, Cano A. (2003). Transforming growth ED et al. (2007). Epithelial—mesenchymal and mesenchymal— factor beta-1 induces snail transcription factor in epithelial cell lines: epithelial transitions in carcinoma progression. J Cell Physiol 213: mechanisms for epithelial mesenchymal transitions. J Biol Chem 374–383. 278: 21113–21123. Kumar R, Wang RA, Mazumdar A, Talukder AH, Mandal M, Yang Radisky DC. (2005). Epithelial-mesenchymal transition. J Cell Sci 118: Z et al. (2002). A naturally occurring MTA1 variant sequesters 4325–4326. oestrogen receptor-alpha in the cytoplasm. Nature 418: 654–657. Thiery JP. (2002). Epithelial-mesenchymal transitions in tumour Kurisaki K, Kurisaki A, Valcourt U, Terentiev AA, Pardali K, Ten progression. Nat Rev Cancer 2: 442–454. Dijke P et al. (2003). Nuclear factor YY1 inhibits transforming Tong D, Heinze G, Schremmer M, Schuster E, Czerwenka K, growth factor beta- and bone morphogenetic protein-induced cell Leodolter S et al. (2007). Expression of the human MTA1 gene in differentiation. Mol Cell Biol 23: 4494–4510. breast cell lines and in breast cancer tissues. Oncol Res 16: 465–470. Lee TC, Zhang Y, Schwartz RJ. (1994). Bifunctional transcriptional von Burstin J, Eser S, Paul MC, Seidler B, Brandl M, Messer M et al. properties of YY1 in regulating muscle actin and c-myc gene (2009). E-cadherin regulates metastasis of pancreatic cancer in vivo expression during myogenesis. Oncogene 9: 1047–1052. and is suppressed by a SNAIL/HDAC1/HDAC2 repressor com- MacLellan WR, Lee TC, Schwartz RJ, Schneider MD. (1994). plex. Gastroenterology 137: 361–371. Transforming growth factor-beta response elements of the skeletal Zavadil J, Bottinger EP. (2005). TGF-beta and epithelial-to-mesench- alpha-actin gene. combinatorial action of serum response factor, ymal transitions. Oncogene 24: 5764–5774. YY1, and the SV40 enhancer-binding protein, TEF-1. J Biol Chem Zhang H, Stephens LC, Kumar R. (2006). Metastasis tumor antigen 269: 16754–16760. family proteins during breast cancer progression and metastasis in a Mahoney MG, Simpson A, Jost M, Noe M, Kari C, Pepe D et al. reliable mouse model for human breast cancer. Clin Cancer Res 12: (2002). Metastasis-associated protein (MTA)1 enhances migration, 1479–1486.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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