The anti-proliferative function of the TGF-β1 signaling pathway involves the repression of the oncogenic TBX2 by its homologue TBX3

Abstract

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.

Keywords: Cell Cycle; Cell Proliferation; Gene Expression; TBX2; TBX3; Transcription Factor; Transforming Growth Factor β (TGF-β); p21.

FIGURE 1.

TGF-β1 represses TBX2 protein and mRNA expression. TBX2 protein from TGF-β1 (5 ng/ml) treated MCF-12A cells (A) or B16 cells (B) were prepared after indicated times and was examined by Western blot analysis. p38 was used as a loading control. Total RNA extracted from MCF-12A cells (C) or B16 cells (D) after indicated times with TGF-β1 treatment were reverse-transcribed and subjected to qRT-PCR using primers specific to TBX2. mRNA levels were normalized to GUSB. Bars, S.D. **, p < 0.001.

FIGURE 2.

TBX2 is transcriptionally regulated by TGF-β1. A, luciferase assay using MCF-12A cells, which were transfected with full-length human TBX2 promoter (400 ng) constructs and were treated with 5 ng/ml TGF-β1. Mean values (± S.D.) are presented as fold activity over that of an empty firefly luciferase reporter and are representative of at least three independent experiments. MCF-12A (B) and B16 (C) cells were pretreated with vehicle (control) or 30 μg/ml CHX for 1 h and treated with TGF-β1 for 8 h for MCF-12A cells and 4 h for B16 cells. RNA and protein were harvested for use in qRT-PCR and Western blotting analysis. The expression of TBX2 was quantified using UN-SCAN-IT gel 6.1 software and the densitometric values normalized to p38 levels. Bars, S.D. *, p < 0.05; **, p < 0.001.

FIGURE 3.

The down-regulation of TBX2 by TGF-β1 is mediated by TBX3. A, luciferase assay of MCF-12A cells transfected with 400 ng of human TBX2 5′-deletion promoter constructs and treated with 5 ng/ml TGF-β1 or vehicle. B, luciferase assay of MCF-12A cells co-transfected with increasing amounts of Smad3/4 expression vectors and 400 ng of −218 bp TBX2 promoter reporter construct. C, alignment of the T-element on −186 bp of TBX2 promoter of human, chimpanzee, mouse and zebrafish. D and E, upper panels: siTBX3#1 lower panels: siTBX3#2. Serum-starved MCF-12A cells were transfected with either 50 nm siControl or siTBX3 for 48 h, followed by 2 days of TGF-β1 treatment and subjected to Western blotting (D) or qRT-PCR analysis (E). Bars, S.D. *, p < 0.05; **, p < 0.001.

FIGURE 4.

TGF-β1 repression of the TBX2 promoter is mediated by a T-element at −186 bp. A, MCF-12A cells were treated with 5 ng/ml TGF-β1 for 24 h and chromatin immunoprecipitation assays performed with antibodies against TBX3 or IgG (negative control). Immunoprecipitated DNA was assayed by qRT-PCR with primers against the TBX2 proximal promoter. B, biotinylated DNA probes of the TBX2 promoter containing the consensus T-element or homologous WT or MT T-element were immobilized on streptavidin beads, and incubated with nuclear extracts from MCF-12A cells treated with 5 ng/ml TGF-β1 for 24 h. The DNA-bound protein complexes were isolated and analyzed by Western blotting using antibodies to TBX3. C, luciferase assay of MCF-12A cells co-transfected with 100 ng of TBX3 expression vector or empty control vector and 400 ng of either TBX2 WT or T-element mutated (MT) promoter luciferase reporter. D, luciferase assay of MCF-12A cells co-transfected with 400 ng of TBX2 promoter luciferase reporter and 100 ng of an empty control vector or WT TBX3 or DNA binding domain mutant (DBM) or TBX3 N-terminal truncated expression vector. Western blots (C, D, right) show equal expression of TBX3 protein. Bars, S.D. *, p < 0.05; **, p < 0.001.

FIGURE 5.

Ectopic TBX2 expression rescues TGF-β1 induced growth inhibition through down-regulating p21 in breast epithelial cells. A, Western blot analyses with indicated antibodies and B, growth curve assays of MCF-12A control or TBX2-overexpressing cells treated with vehicle or 5 ng/ml TGF-β1 for 3 days. C, flow cytometry assays show relative fold change of the percentage of TGF-β1-treated MCF-12A TBX2 overexpressing and control cells in G1, S, and G2/M phases compared with their corresponding vehicle-treated cells. D, MCF-12A TBX2-overexpressing cells were transiently transfected with either pRc/CMV (TBX2 Control) or pRc/CMV-p21 (TBX2 p21) and treated with vehicle or 5 ng/ml TGF-β1 for 3 days. Replicate wells from growth curve analyses (right) were pooled and protein harvested for Western blot analyses (left). E and F, MCF-12A control or TBX2 cell lines were treated with vehicle or TGF-β1 for 24 h and RNA was harvested for use in qRT-PCR analyses to examine the expression of p21 (E) or p15 (F). Bars, S.D. *, p < 0.05; **, p < 0.001.

FIGURE 6.

The down-regulation of TBX2 by TBX3 mediates the TGF-β1 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-β1 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-β1 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 μg/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 < 0.05; **, p < 0.001.

FIGURE 7.

Proposed model linking TBX2 and TBX3 to the TGF-β1 signaling pathway.