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Cancer Res ; 73(16): 5040-52, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23774208


The DNA repair protein damaged DNA-binding 2 (DDB2) has been implicated in promoting cell-cycle progression by regulating gene expression. DDB2 is selectively overexpressed in breast tumor cells that are noninvasive, but not in those that are invasive. We found that its overexpression in invasive human breast tumor cells limited their motility and invasiveness in vitro and blocked their ability to colonize lungs in vivo, defining a new function for DDB2 in malignant progression. DDB2 overexpression attenuated the activity of NF-κB and the expression of its target matrix metalloprotease 9 (MMP9). Mechanistic investigations indicated that DDB2 decreased NF-κB activity by upregulating expression of IκBα by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IκBα gene expression restored NF-κB activity and MMP9 expression, along with the invasive properties of breast tumor cells overexpressing DDB2. Taken together, our findings enlighten understanding of how breast cancer cells progress to an invasive phenotype and underscore potential clinical interest in DDB2 as a prognostic marker or therapeutic target in this setting.

Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , NF-kappa B/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Células MCF-7 , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Invasividade Neoplásica , Prognóstico , Regiões Promotoras Genéticas , Transcrição Genética , Regulação para Cima/genética
Free Radic Biol Med ; 50(12): 1771-9, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21419216


A high basal expression of manganese superoxide dismutase (MnSOD) has been reported in aggressive breast cancer cells, according to an unknown mechanism, and contributes to their invasive abilities. Here, we report the involvement of Sp1 and nuclear factor-κB (NF-κB) transcription factors in this high basal expression of MnSOD in aggressive breast cancer cells. Suppression or inactivation of Sp1 showed that it plays an essential role in the high MnSOD expression in aggressive breast cancer cells through a unique binding site identified by chromatin immunoprecipitation (ChIP) assay and functional analysis of the MnSOD proximal promoter. Treatment of cells with a specific NF-κB inhibitor peptide decreased significantly high basal MnSOD expression. A ChIP assay showed binding of a constitutive p50/p65 NF-κB complex to the MnSOD intronic enhancer element, associated with hyperacetylation of the H3 histone. Finally, high basal expression of MnSOD resulted in the lack of expression of Damaged DNA binding 2 (DDB2) protein in aggressive breast cancer cells. DDB2 overexpression prevented the binding of Sp1 as well as of NF-κB to their respective elements on the MnSOD gene. These results contribute to a better understanding of MnSOD up-regulation, which may be clinically important in the prediction of breast tumor progression.

Neoplasias da Mama/genética , Proteínas de Ligação a DNA/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fator de Transcrição Sp1/metabolismo , Superóxido Dismutase/genética , Acetilação , Sequência de Bases , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Feminino , Radicais Livres , Histonas/genética , Histonas/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estudos Retrospectivos , Fator de Transcrição Sp1/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
J Biol Chem ; 284(21): 14165-76, 2009 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-19339246


Manganese superoxide dismutase plays a role in breast tumor cell growth, which depends on its constitutive expression. However, the mechanisms responsible for the regulation of constitutive SOD2 gene expression at different malignant phenotype in breast cancers remain to be determined. The present study reports the identification and characterization of a DNA sequence located in the proximal promoter of the SOD2 gene, which forms a complex with a nuclear protein from breast tumor MCF-7 cells. Purification of this complex showed that it contained DDB2 (damaged DNA binding 2), a well known protein involved in nucleotide excision DNA repair and cell cycle regulation. Functional analysis of the proximal promoter of the SOD2 gene or modulation of DDB2 expression allowed us to demonstrate that DDB2 regulates negatively the constitutive expression of the SOD2 gene in breast cancer cells. We demonstrate that the binding of DDB2 was associated with the loss of acetylated H3 histones and the decrease in the binding of Sp1 but not AP-2alpha transcription factors to the SOD2 proximal promoter. In addition, we show that DDB2 exerts, at least in part, a control of breast cancer cell growth through its negative regulation of constitutive expression of the SOD2 gene. For the first time, these data give supporting evidence that DDB2 is a new transcriptional regulator, and they provide insight into the molecular function of breast cancer cell growth, which will have an important clinical interest.

Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Superóxido Dismutase/genética , Transcrição Genética , Sequência de Bases , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Feminino , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Superóxido Dismutase/metabolismo , Fatores de Transcrição/metabolismo
PLoS One ; 3(4): e2002, 2008 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-18431487


The Damaged DNA binding protein 2 (DDB2), is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER)-positive (MCF-7 and T47D), but not in ER-negative breast cancer (MDA-MB231 and SKBR3) or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold) higher in ER-positive than in ER-negative tumor samples (P = 0.0208) from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase). These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.

Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Fase G1 , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Neoplásicas/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptores Estrogênicos/metabolismo , Fase S
Med Sci (Paris) ; 23(5): 515-8, 2007 May.
Artigo em Francês | MEDLINE | ID: mdl-17502068


The peroxisome proliferator-activated receptors (PPARs) are transcription factors and belong to the superfamily of nuclear receptors. They are encoded by three genes located on different chromosomes: PPARalpha, PPARbeta/delta and PPARgamma. PPARalpha plays a key role in the control of lipid metabolism and homeostasis. PPARbeta/delta is expressed ubiquitously and participates in skeletal muscle physiology. PPARbeta/delta and PPARgamma are important factors for placental development and function as well as for embryo implantation. In addition, PPARgamma is mainly involved in adipogenesis. PPARs also participate more or less to cell proliferation, differentiation and apoptosis. Surprisingly, the involvement of these transcription factors in cell-cell and/or cell-matrix interactions has not yet been reviewed except for their role as therapeutic agents in inflammation. Nevertheless, several pioneer reports have recently provided some new insights in this research field, by suggesting that PPARs are involved, directly or indirectly, in these interactions and that their activation by specific ligands may lead to potential therapeutic approaches.

Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Matriz Extracelular/fisiologia , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Apoptose , Diferenciação Celular , Divisão Celular , Humanos , Integrinas/genética , Fígado/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
Biochimie ; 89(3): 329-36, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17070643


Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors and belong to the nuclear hormone receptor superfamily. They form heterodimers with retinoid X receptor (RXR) and bind to specific PPAR-response elements. To identify novel PPAR target genes, we developed an affinity method to isolate human genomic fragments containing binding sites for PPARs. For this, an antibody raised against all PPAR subtypes was used. Immunoselected fragments were amplified and sequenced. One of them, ISF1029, was mapped by BLAT and BLAST searches on different human chromosomes, downstream of several POTE genes. ISF1029 contained three hexamers strongly related to the AGGTCA motif organized according to a DR0/3 motif. The latter was found to bind to PPARalpha in gel mobility shift and supershift assays and to exhibit a downregulation potentiality in transfection experiments under clofibrate treatment. POTE genes were shown to be highly expressed in human Caco-2 colorectal adenocarcinoma cells and downregulated by fenofibrate and 9-cis-retinoic acid, as attested by RT-PCR assays. Microarray analysis confirmed and extended to the human T98-G glioblastoma cells, the downregulation of several POTE genes expression by Wy-14,643, a potent PPARalpha activator. Our data provide new insights about the pleiotropic action of PPARs.

DNA/metabolismo , Genoma Humano , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Elementos de Resposta/genética , Sequência de Bases , Sítios de Ligação/genética , Southern Blotting , Células CACO-2 , Linhagem Celular Tumoral , DNA/imunologia , DNA/isolamento & purificação , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Células HCT116 , Humanos , Imunoprecipitação , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Ativados por Proliferador de Peroxissomo/imunologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa