TBX2 interacts with heterochromatin protein 1 to recruit a novel repression complex to EGR1-targeted promoters to drive the proliferation of breast cancer cells.

Early Growth Response 1 (EGR1) is a stress response transcription factor with multiple tumour suppressor roles in breast tissue, whose expression is often lost in breast cancers. We have previously shown that the breast cancer oncogene TBX2 (T-BOX2) interacts with EGR1 to co-repress EGR1-target genes including the breast tumour suppressor NDRG1. Here, we show the mechanistic basis of this TBX2 repression complex. We show that siRNA knockdown of TBX2, EGR1, Heterochromatin Protein 1 (HP1) isoforms and the generic HP1-associated corepressor protein KAP1 all resulted in growth inhibition of TBX2-expressing breast cancer cells. We show that TBX2 interacts with HP1 through a conserved HP1-binding motif in its N-terminus, which in turn leads to the recruitment of KAP1 and other associated proteins. Mutation of the TBX2 HP1 binding domain abrogates the TBX2-HP1 interaction and loss of repression of target genes such as NDRG1. Chromatin-immunoprecipitation (ChIP) assays showed that TBX2 establishes a repressive chromatin mark, specifically H3K9me3, around the NDRG1 proximal promoter coincident with the recruitment of the DNA methyltransferase DNMT3B and histone methyltransferase (HMT) complex components (G9A, Enhancer of Zeste 2 (EZH2) and Suppressor of Zeste 12 (SUZ12)). Knockdown of G9A, EZH2 or SUZ12 resulted in upregulation of TBX2/EGR1 co-regulated targets accompanied by a dramatic inhibition of cell proliferation. We show that a generic inhibitor of HMT activity, DzNep, phenocopies expression of an inducible dominant negative TBX2. Knockdown of TBX2, KAP1 or HP1 inhibited NDRG1 promoter decoration specifically with the H3K9me3 repression mark. Correspondingly, treatment with a G9A inhibitor effectively reversed TBX2 repression of NDRG1 and synergistically downregulated cell proliferation following TBX2 functional inhibition. These data demonstrate that TBX2 promotes suppression of normal growth control mechanisms through recruitment of a large repression complex to EGR1-responsive promoters leading to the uncontrolled proliferation of breast cancer cells.

BRCA1 and GATA3 corepress FOXC1 to inhibit the pathogenesis of basal-like breast cancers.

In this study we describe a novel interaction between the breast/ovarian tumor suppressor gene BRCA1 and the transcription factor GATA3, an interaction, which is important for normal breast differentiation. We show that the BRCA1-GATA3 interaction is important for the repression of genes associated with triple-negative and basal-like breast cancer (BLBCs) including FOXC1, and that GATA3 interacts with a C-terminal region of BRCA1. We demonstrate that FOXC1 is an essential survival factor maintaining the proliferation of BLBCs cell lines. We define the mechanistic basis of this corepression and identify the GATA3-binding site within the FOXC1 distal promoter region. We show that BRCA1 and GATA3 interact on the FOXC1 promoter and that BRCA1 requires GATA3 for recruitment to this region. This interaction requires fully functional BRCA1 as a mutant BRCA1 protein is unable to localize to the FOXC1 promoter or repress FOXC1 expression. We demonstrate that this BRCA1-GATA3 repression complex is not a FOXC1-specific phenomenon as a number of other genes associated with BLBCs such as FOXC2, CXCL1 and p-cadherin were also repressed in a similar manner. Finally, we demonstrate the importance of our findings by showing that loss of GATA3 expression or aberrant FOXC1 expression contributes to the drug resistance and epithelial-to-mesenchymal transition-like phenotypes associated with aggressive BLBCs.

T-box 2 represses NDRG1 through an EGR1-dependent mechanism to drive the proliferation of breast cancer cells.

T-box 2 (TBX2) is a transcription factor involved in mammary development and is known to be overexpressed in a subset of aggressive breast cancers. TBX2 has previously been shown to repress growth control genes such as p14(ARF) and p21(WAF1/cip1). In this study we show that TBX2 drives proliferation in breast cancer cells and this is abrogated after TBX2 small interfering RNA (siRNA) knockdown or after the expression of a dominant-negative TBX2 protein. Using microarray analysis we identified a large cohort of novel TBX2-repressed target genes including the breast tumour suppressor NDRG1 (N-myc downregulated gene 1). We show that TBX2 targets NDRG1 through a previously undescribed mechanism involving the recruitment of early growth response 1 (EGR1). We show EGR1 is required for the ability of TBX2 to repress NDRG1 and drive cell proliferation. We show that TBX2 interacts with EGR1 and that TBX2 requires EGR1 to target the NDRG1 proximal promoter. Abrogation of either TBX2 or EGR1 expression is accompanied by the upregulation of cell senescence and apoptotic markers. NDRG1 can recapitulate these effects when transfected into TBX2-expressing cells. Together, these data identify a novel mechanism for TBX2-driven oncogenesis and highlight the importance of NDRG1 as a growth control gene in breast tissue.