The Anti-proliferative Function of the TGF-β1 Signaling Pathway Involves the Repression of the Oncogenic TBX2 by Its Homologue TBX3*

Background: The transcription factor TBX3 regulates the anti-proliferative function of the TGF-β1 pathway but its downstream target(s) responsible for this is unknown. Results: TBX3 downstream of TGF-β1 signaling represses TBX2 to inhibit proliferation. Conclusion: The down-regulation of TBX2 is required for the anti-proliferative function of the TGF-β1-TBX3 axis. Significance: This study sheds light on the mechanisms involved in the anti-proliferative role of TGF-β1. A growing body of work has shown that the highly homologous T-box transcription factors TBX2 and TBX3 play critical but distinct roles in embryonic development and cancer progression. For example, TBX2 and TBX3 are up-regulated in several cancers and recent evidence suggests that whereas TBX2 functions as a pro-proliferative factor, TBX3 inhibits cell proliferation but promotes cancer cell migration and invasion. While the molecular mechanisms regulating these functions of TBX2 and TBX3 are poorly understood we recently reported that the TGF-β1 signaling pathway up-regulates TBX3 expression to mediate, in part, its well described anti-proliferative and pro-migratory roles. The TBX3 targets responsible for these functions were however not identified. Here we reveal for the first time that the TGF-β1 signaling pathway represses TBX2 transcriptionally and we provide a detailed mechanism to show that this is mediated by TBX3. Furthermore, we implicate the down-regulation of TBX2 in the anti-proliferative function of the TGF-β1-TBX3 axis. These findings have important implications for our understanding of the regulation of TBX2 and TBX3 and shed light on the mechanisms involved in the anti-proliferative and pro-migratory roles of TGF-β1.

A growing body of work has shown that the highly homologous T-box transcription factors TBX2 and TBX3 play critical but distinct roles in embryonic development and cancer progression. For example, TBX2 and TBX3 are up-regulated in several cancers and recent evidence suggests that whereas TBX2 functions as a pro-proliferative factor, TBX3 inhibits cell proliferation but promotes cancer cell migration and invasion. While the molecular mechanisms regulating these functions of TBX2 and TBX3 are poorly understood we recently reported that the TGF-␤1 signaling pathway up-regulates TBX3 expression to mediate, in part, its well described anti-proliferative and promigratory roles. The TBX3 targets responsible for these functions were however not identified. Here we reveal for the first time that the TGF-␤1 signaling pathway represses TBX2 transcriptionally and we provide a detailed mechanism to show that this is mediated by TBX3. Furthermore, we implicate the downregulation of TBX2 in the anti-proliferative function of the TGF-␤1-TBX3 axis. These findings have important implications for our understanding of the regulation of TBX2 and TBX3 and shed light on the mechanisms involved in the anti-proliferative and pro-migratory roles of TGF-␤1.
The transforming growth factor ␤1 (TGF-␤1) 2 pathway plays critical roles in a wide range of cellular processes including cell proliferation, apoptosis, differentiation, and the immune response. This multi-functional cytokine generally exerts its effects by binding and activating the TGF␤ receptors I and II, which initiate a cascade of signaling events frequently involving Smad proteins and co-factors but which can also be Smad-independent (1). One of the well-known roles of TGF-␤1 is its ability to inhibit the proliferation of epithelial cells but promote their migration (2). TGF-␤1 has been shown to inhibit proliferation primarily by inducing a G1 cell cycle arrest through its ability to transcriptionally up-regulate expression of the cyclindependent kinase inhibitors p15 Ink4b and p21 Cip1 (3). Its promigratory effects, however, appear to be mainly due to its ability to regulate key players of epithelial-mesenchymal transition (EMT) (4). These anti-proliferative and pro-migratory roles of TGF-␤1 are important in embryonic development, for example during branching morphogenesis of the mammary gland, as well as during oncogenesis (5,6). We recently reported that TGF-␤1 up-regulates expression of the T-box transcription factor, TBX3, through a mechanism involving the co-operation of Smad proteins and JunB. This was shown to be a critical downstream event for mediating TGF-␤1-induced anti-proliferation and pro-migration of breast epithelial cells and keratinocytes but the TBX3 target genes responsible for this were not described (7).
The T-box family of transcription factors are highly conserved in evolution and play critical roles in embryonic development. TBX3 is a member of the TBX2 subfamily and along with its closely related family member, TBX2, it plays an important role in the development of the limbs, heart, and mammary glands (8). TBX2 and TBX3 are overexpressed in a number of cancers, including breast, pancreatic, ovarian, liver, cervical, and melanoma (9 -13). While there is evidence to suggest that they both repress the cell cycle regulators p19 ARF and p21 WAF1/ CIP1/SDII, they have distinct functions in embryonic development and cancer (8 -10, 14 -19). For example, mouse studies examining the effects of loss of function mutations have shown that Tbx3 plays an important role during mammary placode induction while Tbx2 has no significant effects on this process (19 -21). Furthermore, we and others have also shown that TBX2 and TBX3 play distinct roles in cancer progression, with TBX2 functioning as a powerful pro-proliferative factor and TBX3 playing a role in migration and invasion (22,23).
Together with our previously published data showing that TBX2 and TBX3 exhibit mutually exclusive expression in some melanoma cell lines (24), we therefore speculated that the antiproliferative function of TGF-␤1 may be due to its ability to down-regulate TBX2 expression. Indeed, here we show that in human breast epithelial and mouse melanoma cells, TGF-␤1 negatively regulates TBX2 protein and mRNA levels at a transcriptional level and importantly, we demonstrate that this regulation is dependent on increased TBX3 levels. Using both in vitro and in vivo assays we reveal that TBX3 binds and regulates the TBX2 promoter in response to TGF-␤1 through a T-element at Ϫ186 bp. Furthermore, we show that the down-regulation of TBX2 expression is responsible for the decrease in cell proliferation induced by TGF-␤1 and that this is possibly mediated by increased p21 levels.
Plasmid Constructs-The human TBX2 promoter luciferase reporter constructs have been described previously (25). The pcDNA 3.1(ϩ) containing the full-length human TBX2 cDNA was supplied by Prof. van Lohuizen (Antoni van Leeuwenhoek hospital, Amsterdam, The Netherlands). The TBX2 cDNA was then excised and the vector re-ligated to generate the pcDNA3.1 (ϩ) empty vector, into which the human TBX3 cDNA was cloned using the NheI and BamHI sites. The p21-pRc/CMV plasmid, which was prepared in the laboratory of Prof William Kaelin, was obtained from Addgene (Addgene plasmid 20814) (26). The TBX3 N-terminal construct was constructed by excising the C-terminal portion of the pCMV-TBX3 construct with BglII and SpeI and re-ligated.
Generation of Stable Cell Lines Expressing TBX2-To generate stably transfected cell lines, MCF-12A cells were transfected with either the empty expression vector pcDNA3.1 (ϩ) or with this vector containing the full-length human TBX2 cDNA using X-tremeGENE HD DNA transfection reagent (Roche Diagnostics, Basel, Switzerland). Stable transfected cells were selected by 400 g/ml G-418.
Generation of MCF-12A Cell Line Overexpressing TBX3-The MCF-12A cells were transiently transduced using adenovirus with human TBX3 cDNA cloned into the vector component of an Adeno-X Tet-off expression system 1 (Clontech). The virus was purified using the Adeno-X Maxi purification kit (Clontech). All steps were performed according to the manufacturer's instructions.
Transfection and Luciferase Assays-Transient transfections were performed using X-tremeGENE HD DNA transfection reagent (Roche Diagnostics) according to the manufacturer's instructions. MCF-12A cells were plated at 1 ϫ 10 5 cells per well of a 12-well plate, 24 h before transfection. Cells were cotransfected with 400 ng of a TBX2-luciferase reporter plasmid plus 100 ng of the TBX3 expression plasmids or corresponding amounts of an empty-vector plasmid. Luciferase activities were measured using the Luminoskan Ascent luminometer (Thermo Labsystems, Franklin, MA). All transfections were performed in duplicate and at least three independent experiments were done to confirm reproducibility. Firefly luciferase values were expressed relative to empty-vector control.
Chromatin Immunoprecipitation Assays (ChIP)-ChIP assays were carried out as previously described (10). Briefly, MCF-12A cells treated with TGF-␤1 for 24 h were fixed in 1% formaldehyde and the chromatin extracted, sonicated, and immunoprecipitated using antibodies against TBX3 (sc-17871) or IgG (negative control, Santa Cruz Biotechnology). DNA precipitated was analyzed by qRT-PCR using human TBX2-specific primer pairs or a nonspecific promoter region (GUSB, Qiagen). Crossing values (Ct) of TBX3 precipitated DNA were normalized against the Ct values of IgG. Fold enrichment was determined using the ⌬⌬Ct method: Fold enrichment ϭ 2 Ϫ(⌬Ct1-⌬Ct2) , where ⌬Ct1 is the ChIP of interest, and ⌬Ct2 is the IgG.
Growth Curves-Short-term growth of cells was compared over a 3-day period, as described previously (29). 0.5 ϫ 10 4 cells were seeded in triplicate in 24-well plates on day 0 and treated with vehicle or 5 ng/ml TGF-␤1 for 2-3 days.
Statistical Analysis-Statistical analysis was performed by using the 2-sample t test (Excel).

TGF-␤1 Transcriptionally Represses TBX2 Expression in MCF-12A Breast Epithelial and B16 Melanoma Cells-TBX2
and TBX3 are highly homologous T-box factors with distinct oncogenic roles. We recently reported that TBX3 is transcriptionally activated by the TGF-␤1 signaling pathway (7), and we therefore examined whether the expression of TBX2 is also responsive to this pathway. To this end we exposed MCF-12A breast epithelial and B16 mouse melanoma cells to TGF-␤1 or vehicle over a time course spanning 1-36 h. Western blot analyses show that TBX2 levels decrease substantially in response to TGF-␤1 treatment in both cell lines from 8 h in MCF-12A cells and 4 h in B16 cells (Fig. 1, A and B). This robust repression was also observed in WI-38 normal human fibroblasts (data not shown). Quantitative real-time PCR (qRT-PCR) experiments performed on cells treated as above demonstrate a corresponding decrease in TBX2 mRNA levels that precedes the decrease in protein levels ( Fig. 1, C and D). To investigate if this regulation is transcriptional, we tested whether the Ϫ1604 bp TBX2 promoter is responsive to TGF-␤1 treatment using a luciferase assay and the results showed a time-dependent decrease in reporter activity in response to TGF-␤1 ( Fig. 2A). To distinguish the need for de novo protein synthesis, cells were pretreated with cycloheximide (CHX), a protein synthesis inhibitor, which abolished the repression of TBX2 mRNA and protein levels by TGF-␤1 (Fig. 2, B and C). Together these results suggest that TGF-␤1-mediated repression of TBX2 is transcriptional and that it requires the production of nascent protein.
The Down-regulation of TBX2 by TGF-␤1 Is Mediated by TBX3 in Breast Epithelial Cells-To narrow down the region of the TBX2 promoter mediating its repression by TGF-␤1, 5Ј deletion constructs of the human TBX2 promoter driving a firefly luciferase reporter were tested in luciferase assays. The results show that the repression of TBX2 by TGF-␤1 treatment is sustained even in the Ϫ218 bp TBX2 promoter construct (Fig. 3A). To investigate whether Smad3 and Smad4 (Smad3/4) are involved in this regulation, MCF-12A cells were co-transfected with increasing amounts of Smad3/4 expression vectors and the Ϫ218 bp TBX2 promoter luciferase construct. Fig. 3B shows that Smad3/4 had very little effect on TBX2 promoter activity suggesting that the regulation of TBX2 by TGF-␤1 either requires a Smad co-factor(s) or involves a mechanism that does not directly require the Smads. We therefore repeated these experiments in the presence or absence of the well-known Smad co-factors, JunB or Sp1, but no repression was observed (data not shown). Based on our previous findings we speculated that TBX2 may be downstream of TBX3 in the TGF-␤1 pathway. To address this we screened the TBX2 promoter for T-elements and a T-element which is highly conserved between Tbx2 promoters from several species was identified at Ϫ186 bp (Fig. 3C). Indeed, depleting TBX3 levels by siRNA in MCF-12A cells diminished the ability of TGF-␤1 to repress TBX2 protein and mRNA levels (Fig. 3, D and E). It is worth noting that while the fold repression of TBX2 by TGF-␤1 in the siTBX3 cells was still significant, it was lower than that of the control cells and may be due to incomplete knockdown of TBX3. Furthermore, knocking down TBX3 in untreated cells resulted in an increase in TBX2 mRNA and protein levels, suggesting that TBX3 represses basal and TGF-␤1 regulated TBX2 levels.
TBX3 Binds to the TBX2 Promoter at Ϫ186 bp in Response to TGF-␤1 Treatment-We next wanted to confirm that TBX3 can directly repress TBX2 in vivo and to this end we performed a chromatin immunoprecipitation (ChIP) assay. Cross-linked chromatin was prepared from TGF-␤1-treated and control MCF-12A cells and DNA immunoprecipitated with an antibody to TBX3 was subjected to qRT-PCR with primers spanning the Ϫ186 bp putative T-element in the TBX2 promoter. The results obtained show that compared with the untreated control, the binding of TBX3 to the TBX2 promoter was enhanced by 8.5-fold in the presence of TGF-␤1 (Fig. 4A). To determine whether TBX3 binds specifically to the Ϫ186 bp T-element in the TBX2 promoter, we performed a DNA affinity immunoblot (DAI) using nuclear extract isolated from TGF-␤1-treated MCF-12A cells and a biotin-labeled probe containing the wild-type or mutated T-element (Fig. 4B). As an additional control a probe containing a full T-element consensus site was included in these experiments and protein-bound biotinylated DNA was isolated and analyzed by Western blotting using an anti-TBX3 antibody. The results showed that while TBX3 bound strongly to both the consensus and wildtype probes, it displayed a decreased affinity for the probe with the mutated T-element (Fig. 4B). Together, these results suggest that TBX3 binds the TBX2 promoter specifically at the T-element at Ϫ186 bp.
To test whether TBX3 can repress the TBX2 promoter and whether the T-element at Ϫ186 bp mediates this repression, MCF-12A cells were co-transfected with a TBX3 expression vector and either a wild type Ϫ218 bp TBX2 promoter luciferase construct or a construct in which the T-element was mutated by site-directed mutagenesis. The results show that TBX3 repressed the TBX2 promoter by ϳ18-fold and that the ability of TBX3 to repress TBX2 was significantly reduced when DECEMBER 19, 2014 • VOLUME 289 • NUMBER 51

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the Ϫ186 bp T-element was mutated (Fig. 4C). To identify the functional domains of the TBX3 protein that are responsible for this regulation, the wild type Ϫ218 bp TBX2 promoter lucifer-ase construct was co-transfected with vectors expressing either a WT TBX3 protein or a TBX3 protein in which the DNA binding site was mutated or only a N-terminal TBX3 protein

TBX2 Is Repressed by TBX3 in the TGF-␤1 Signaling Pathway
lacking the dominant repression domain. The data obtained indicate that the TBX3 DNA binding domain and C terminus containing a repression domain are important for its ability to repress the TBX2 promoter (Fig. 4D). All TBX3 constructs were expressed at similar levels (Fig. 4, C and D, right).
Ectopic Expression of TBX2 Is Able to Rescue TGF-␤1-inhibited Cell Proliferation-We next investigated whether overexpression of TBX2 in MCF-12A cells could rescue the inhibition of cell proliferation by TGF-␤1. Briefly, a pcDNA-TBX2 expression vector or the empty control vector was transfected into MCF-12A cells and G418-resistant clones were pooled for subsequent analysis. Overexpression of TBX2 was confirmed by Western blotting (Fig. 5A), and growth curves show that while cell proliferation was significantly reduced by TGF-␤1 in the control cells, overexpressing TBX2 was sufficient to rescue this phenotype (Fig. 5B). This was confirmed by flow cytometry, which showed that TGF-␤1 treatment induced a significant G1 cell cycle arrest in the control cells but had no effect on the cell cycle profile of the TBX2-overexpressing cells (Fig. 5C). These results correlated with changes in the protein and mRNA levels of the cyclin-dependent kinase inhibitor p21 (Fig. 5, A and E). Importantly, the increase in p21 mRNA and protein levels in TGF-␤1-treated cells are abrogated when TBX2 is overexpressed suggesting that TBX2 may be the upstream regulator of p21 in the TGF-␤1 pathway. Indeed, when TBX2-overexpressing cells were transfected with a p21 expression vector they had a decreased proliferative ability, which could not be further reduced by TGF-␤1 treatment (Fig. 5D). Interestingly, TBX2 overexpression failed to have any significant effect on the TGF-␤1-induced activation of p15 mRNA levels (Fig. 5F), another cell cycle inhibitor which has been shown to mediate a G1 arrest in response to TGF-␤1 (30). Together, these data imply that the anti-proliferative effect of TGF-␤1 on MCF-12A cells occurs primarily through up-regulation of p21, since the continued repression of p21 in TBX2 overexpressing cells allowed the cells to proliferate even in the presence of TGF-␤1 and despite the up-regulation of p15. To confirm this, p21 expression was knocked down using a siRNA approach and growth curve analyses performed in the presence and absence of TGF-␤1. As expected, cells in which p21 were effectively knocked down proliferated faster than the control cells in the absence of TGF-␤1 (Fig. 6A). Importantly, the p21-depleted cells also failed to show a significant decrease in cell proliferation in response to TGF-␤1. In addition, when the MCF-12A cells were depleted of TBX2 the untreated cells had a significant decrease in cell proliferation which could not be significantly further decreased in response to TGF-␤1 treatment depleted TBX2 in (Fig. 6B). These data together with Western blot results from TBX3 overexpressing cells (Fig. 6C) suggest that the down-regulation of TBX2 by TBX3 mediates the anti-proliferative effect of TGF-␤1 through allowing up-regulation of p21.

DISCUSSION
The TGF-␤1 signaling pathway controls a wide spectrum of cellular functions and deregulated TGF-␤ signaling has been linked to severe human diseases, including cardiovascular disease, skeletal, and muscular disorders and cancer (31). TGF-␤1 is a potent inhibitor of cell proliferation, which is thought to   DECEMBER 19, 2014 • VOLUME 289 • NUMBER 51

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result from its ability to induce a G1 cell cycle arrest through the up-regulation of the cyclin-dependent kinase inhibitors (cdki), p15, and p21 (30). However, it can also stimulate invasiveness by promoting an epithelial to mesenchyme transition. Understanding how TGF-␤1 coordinates its effects on the cell cycle and invasion in different cell types is a key issue, both for development and disease.
Here we have dissected the interrelated roles of the TBX2 and TBX3 transcription factors, which are widely implicated in cancer progression, in the response to TGF-␤1. Previously we demonstrated that the TGF-␤1-mediated growth arrest observed in melanoma could be bypassed by ectopic TBX2 expression (36), and that TBX3, which can promote invasiveness by repressing E-cadherin expression (24), is a downstream target of the TGF-␤1 signaling pathway and plays an important role in exerting its anti-proliferative and pro-invasive effects (7). However, how TBX3 might exert its anti-proliferative function, and what suppresses TBX2 expression was not known. In this study we show that when the TGF-␤1 pathway is stimulated in breast epithelial and melanoma cells, TBX2 expression is repressed through the direct binding of TBX3 to a half T-element in the TBX2 promoter. Our results are also consistent with TBX2 being a powerful pro-proliferative factor (22,23), and indicate that the down-regulation of TBX2 blocks proliferation through the de-repression of the TBX2 target gene, p21, a cdki that initiates the TGF-␤1-induced G1 arrest (32). Together these data provide an explanation for how the TGF-␤1 pathway exerts its anti-proliferative effect and reveal an upstream mech-anism involved in regulating TBX2 and TBX3 in contexts where they are differentially expressed. Moreover, since overexpression of TBX2 can override the anti-proliferative effects of TGF-␤1 without affecting p15 levels, up-regulation of p15 is unlikely to be required for the effect of TGF-␤1 on the proliferative ability of these cells, though it may be involved in other TGF-␤1-mediated functions.
TBX2 and TBX3 are highly homologous T-box transcription factors which have distinct roles during embryonic development and in breast cancer and melanoma cell lines where they are both overexpressed (7, 19 -22). Importantly, whereas TBX2 is essential for promoting cell proliferation, TBX3 inhibits cell proliferation but is required for cell migration. These results together with data from the current study suggest an interesting FIGURE 6. The down-regulation of TBX2 by TBX3 mediates the TGF-␤1 anti-proliferative effect through up-regulation of p21. A, MCF-12A cells were transiently transfected with either 25 nM sip21 or siControl for 12 h followed by 3 days of 5 ng/ml TGF-␤1 treatment. Replicate wells from growth curve analyses (right) were pooled and protein harvested for Western blot analyses (left). Upper panel: sip21#1, lower panel sip21#2. B, growth curve analyses of MCF-12A cells transiently transfected with either 10 nM siTBX2 or siControl for 12 h followed by 3 days of 5 ng/ml TGF-␤1 treatment. Left: siTBX2#1, right: siTBX2#2. C, Western blot analyses of MCF-12A cells transduced with adeno-TBX3 tet-off virus, which allows TBX3 expression to be regulated by 10 g/ml doxycycline (Dox). Cells were treated as indicated, and protein harvested was subjected to Western blot analyses using antibodies specific to TBX3, TBX2, and p21, and p38 was used as a loading control. Bars, S.D. *, p Ͻ 0.05; **, p Ͻ 0.001. DECEMBER 19, 2014 • VOLUME 289 • NUMBER 51 interplay between TBX2, TBX3, and TGF-␤1 signaling in normal epithelial cells (Fig. 7). Briefly, in this context TGF-␤1 signaling activates TBX3 expression in a Smad/JunB dependent manner and TBX3 then represses TBX2 transcription, which allows for the de-repression of p21 and a G1 cell cycle arrest. Interestingly, malignant cells acquire immortalizing mutations that enable them to evade the anti-proliferative effects of TGF-␤1 signaling. For example, one of the mechanisms by which TGF-␤1 exerts its anti-proliferative effects is through inhibiting expression of MYC that leads to the de-repression of p21. Many malignant cells however have mutations that result in the constitutive expression of MYC and hence these cells are resistant to the anti-proliferative effects of TGF-␤1. It is therefore possible that cancers in which TBX2 is overexpressed will similarly disregard the TGF-␤1 anti-proliferative signals. Intriguingly, the TBX3 promoter has a full consensus MYC binding site, raising the possibility that MYC will impact the TBX3-TBX2 axis identified here. The expression of MYC is usually down-regulated by TGF-␤1 treatment, and ectopic expression of MYC in mouse keratinocytes desensitized the cells to TGF-␤1 mediated growth inhibition (33)(34)(35). Furthermore, sustained MYC expression in ovarian cancers coincides with resistance to the anti-proliferative effects of TGF-␤1 (36).

TBX2 Is Repressed by TBX3 in the TGF-␤1 Signaling Pathway
Although, the precise relationship between MYC and TBX2 and TBX3 expression remains to be determined, there is no doubt that TBX2 and TBX3 are likely to represent key effectors of TGF-␤1 signaling in multiple cell types.