RESUMO
The Brn-3b transcription factor has been shown to be overexpressed in human breast cancer cells and contributes toward cell growth regulation. Using micro-arrays, more than 50 cancer-related genes regulated by Brn-3b in human breast cancer cells have been identified. For example, Brn-3b activates the cell cycle regulator CDK4 that provides a mechanism by which Brn-3b controls the growth of breast cancer cells. Here, we show that Brn-3b regulates plakoglobin (gamma-catenin), a member of the catenin family involved in cell-cell adhesion and signal transduction. Brn-3b expression inversely correlates with plakoglobin expression at both mRNA and protein levels in breast cancer cell lines and human breast biopsies. In contrast, no significant correlation was observed between Brn-3b expression and beta-catenin, or between Brn-3b expression and E-cadherin expression. Brn-3b represses the plakoglobin promoter via a Brn-3 consensus binding site contained within the region -965 to -593 relative to the transcriptional start site. Both repression of the promoter and binding of Brn-3b are lost when this site is mutated. To our knowledge, this is the first time that a Brn-3b POU family transcription factor has been shown to regulate a member of the catenin family, which provides insight into the molecular mechanisms by which Brn-3b expression may favour breast cancer progression and tumor invasion.
Assuntos
Neoplasias da Mama/genética , Fator de Transcrição Brn-3B/fisiologia , gama Catenina/biossíntese , Sítios de Ligação , Neoplasias da Mama/patologia , Adesão Celular , Desmoplaquinas , Progressão da Doença , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Invasividade Neoplásica , Regiões Promotoras Genéticas , Transdução de Sinais , Células Tumorais CultivadasRESUMO
In breast cancer, overexpression of the small heat shock protein, HSP-27, is associated with increased anchorage-independent growth, increased invasiveness, and resistance to chemotherapeutic drugs and is associated with poor prognosis and reduced disease-free survival. Therefore, factors that increase the expression of HSP-27 in breast cancer are likely to affect the prognosis and outcome of treatment. In this study, we show a strong correlation between elevated levels of the Brn-3b POU transcription factor and high levels of HSP-27 protein in manipulated MCF-7 breast cancer cells as well as in human breast biopsies. Conversely, HSP-27 is decreased on loss of Brn-3b. In cotransfection assays, Brn-3b can strongly transactivate the HSP-27 promoter, supporting a role for direct regulation of HSP-27 expression. Brn-3b also cooperates with the estrogen receptor (ER) to facilitate maximal stimulation of the HSP-27 promoter, with significantly enhanced activity of this promoter observed on coexpression of Brn-3b and ER compared with either alone. RNA interference and site-directed mutagenesis support the requirement for the Brn-3b binding site on the HSP-27 promoter, which facilitates maximal transactivation either alone or on interaction with the ER. Chromatin immunoprecipitation provides evidence for association of Brn-3b with the HSP-27 promoter in the intact cell. Thus, Brn-3b can, directly and indirectly (via interaction with the ER), activate HSP-27 expression, and this may represent one mechanism by which Brn-3b mediates its effects in breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Choque Térmico/biossíntese , Fatores de Transcrição/biossíntese , Sequência de Bases , Biópsia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Interferência de RNA , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Fator de Transcrição Brn-3 , Fator de Transcrição Brn-3B , Fatores de Transcrição/genética , Ativação Transcricional , TransfecçãoRESUMO
The Brn-3b POU family transcription factor has previously been shown to be over-expressed in human breast cancer and to enhance the growth rate and anchorage independent growth of human breast cancer cells, acting at least in part by inhibiting the expression of the BRCA-1 anti-oncogene. Here we have used gene arrays to identify several other targets for Brn-3b in human breast cancer cells, including cyclin-dependent kinase 4 (CDK4). In particular, we show that levels of CDK4 mRNA and protein correlate with the levels of Brn-3b in breast cancer cell lines manipulated to express different levels of Brn-3b and in human breast cancer biopsies and that Brn-3b can activate the CDK4 promoter. The effect of Brn-3b on the growth regulatory CDK4 protein provides a further mechanism by which Brn-3b can regulate breast cancer cell growth and indicates that it can do this by activating as well as repressing specific target genes.