RESUMO
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.
Assuntos
Neoplasias da Mama/patologia , Proliferação de Células/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regiões Promotoras Genéticas , Proteínas com Domínio T/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Cromatina/genética , Imunoprecipitação da Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Feminino , Técnicas de Silenciamento de Genes , Histona Metiltransferases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ligação Proteica , Proteínas Repressoras/genética , Proteínas com Domínio T/genética , Proteína 28 com Motivo Tripartido/genéticaRESUMO
Here, we show for the first time that the familial breast/ovarian cancer susceptibility gene, BRCA1, along with interacting ΔNp63 proteins, transcriptionally upregulate the putative tumour suppressor protein, S100A2. Both BRCA1 and ΔNp63 proteins are required for S100A2 expression. BRCA1 requires ΔNp63 proteins for recruitment to the S100A2 proximal promoter region, while exogenous expression of individual ΔNp63 proteins cannot activate S100A2 transcription in the absence of a functional BRCA1. Consequently, mutation of the ΔNp63/p53 response element within the S100A2 promoter completely abrogates the ability of BRCA1 to upregulate S100A2. S100A2 shows growth control features in a range of cell models. Transient or stable exogenous S100A2 expression inhibits the growth of BRCA1 mutant and basal-like breast cancer cell lines, while short interfering RNA (siRNA) knockdown of S100A2 in non-tumorigenic cells results in enhanced proliferation. S100A2 modulates binding of mutant p53 to HSP90, which is required for efficient folding of mutant p53 proteins, by competing for binding to HSP70/HSP90 organising protein (HOP). HOP is a cochaperone that is required for the efficient transfer of proteins from HSP70 to HSP90. Loss of S100A2 leads to an HSP90-dependent stabilisation of mutant p53 with a concomitant loss of p63. Accordingly, S100A2-deficient cells are more sensitive to the HSP-90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, potentially representing a novel therapeutic strategy for S100A2- and BRCA1-deficient cancers. Taken together, these data demonstrate the importance of S100A2 downstream of the BRCA1/ΔNp63 signalling axis in modulating transcriptional responses and enforcing growth control mechanisms through destabilisation of mutant p53.
Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Fatores Quimiotáticos/metabolismo , Proteínas S100/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Fatores Quimiotáticos/genética , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Células MCF-7 , Mutação , Regiões Promotoras Genéticas , Estabilidade Proteica , Interferência de RNA , Proteínas S100/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/genética , Transcrição Gênica , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
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.