RESUMO
The role of polarity signaling in cancer metastasis is ill defined. Using two three-dimensional culture models of mammary epithelial cells and an orthotopic mouse model of breast cancer, we reveal that Par6 signaling, which is regulated directly by TGFbeta, plays a role in breast cancer metastasis. Interference with Par6 signaling blocked TGFbeta-dependent loss of polarity in acini-like structures formed by non-transformed mammary cells grown in three-dimensional structures and suppressed the protrusive morphology of mesenchymal-like invasive mammary tumor cells without rescuing E-cadherin expression. Moreover, blockade of Par6 signaling in an in vivo orthotopic model of metastatic breast cancer induced the formation of ZO-1-positive epithelium-like structures in the primary tumor and suppressed metastasis to the lungs. Analysis of the pathway in tissue microarrays of human breast tumors further revealed that Par6 activation correlated with markers of the basal carcinoma subtype in BRCA1-associated tumors. These studies thus reveal a key role for polarity signaling and the control of morphologic transformation in breast cancer metastasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Progressão da Doença , Feminino , Genes BRCA1 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência/métodos , Metástase Neoplásica , Transdução de SinaisRESUMO
The anterior heart field (AHF) mediates formation of the outflow tract (OFT) and right ventricle (RV) during looping morphogenesis of the heart. Foxh1 is a forkhead DNA binding transcription factor in the TGFbeta-Smad pathway. Here we demonstrate that Foxh1-/- mutant mouse embryos form a primitive heart tube, but fail to form OFT and RV and display loss of outer curvature markers of the future working myocardium, similar to the phenotype of Mef2c-/- mutant hearts. Further, we show that Mef2c is a direct target of Foxh1, which physically and functionally interacts with Nkx2-5 to mediate strong Smad-dependent activation of a TGFbeta response element in the Mef2c gene. This element directs transgene expression to the presumptive AHF, as well as the RV and OFT, a pattern that closely parallels endogenous Mef2c expression in the heart. Thus, Foxh1 and Nkx2-5 functionally interact and are essential for development of the AHF and its derivatives, the RV and OFT, in response to TGFbeta-like signals.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra , Animais , Sequência de Bases , Células COS , Diferenciação Celular , Clonagem Molecular , DNA/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Embrião não Mamífero , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead , Glutationa Transferase/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Íntrons , Fatores de Transcrição MEF2 , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Fatores de Regulação Miogênica/biossíntese , Fatores de Regulação Miogênica/genética , Fenótipo , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células-Tronco/citologia , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Transgenes/genética , Peixe-ZebraRESUMO
C1D is a gamma-irradiation inducible nuclear matrix protein that interacts with and activates the DNA-dependent protein kinase (DNA-PK) that is essential for the repair of the DNA double-strand breaks and V(D)J recombination. Recently, it was demonstrated that C1D can also interact with TRAX and prevent the association of TRAX with Translin, a factor known to bind DNA break-point junctions, and that over expression of C1D can induce p53-dependent apoptosis. Taken together, these findings suggest that mammalian C1D could be involved in maintenance of genome integrity by regulating the activity of proteins involved in DNA repair and recombination. To obtain direct evidence for the biological function of C1D that we show is highly conserved between diverse species, we have analysed the Saccharomyces cerevisiae C1D homologue. We report that the disruption of the YC1D gene results in a temperature sensitivity and that yc1d mutant strains exhibit defects in non-homologous DNA end joining (NHEJ) and accurate DNA repair. In addition, using a novel plasmid-based in vivo recombination assay, we show that yc1d mutant strains are also defective in homologous recombination. These results indicate that YC1D is implicated in both homologous recombination and NHEJ pathways for the repair of DNA double-strand breaks.
Assuntos
DNA Fúngico/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , DNA/genética , DNA/metabolismo , Reparo do DNA/genética , DNA Fúngico/genética , Deleção de Genes , Dados de Sequência Molecular , Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , TemperaturaRESUMO
The nuclear matrix protein C1D is an activator of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DSBs) and V(D)J recombination. C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon gamma-irradiation, suggesting that C1D may play a role in repair of DSBs in vivo. In an attempt to identify the biological function of C1D, we have employed the yeast two-hybrid system and found that C1D interacts specifically with Translin-associated factor X, TRAX. Although the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins, including Translin, into the nucleus. We show that C1D and TRAX interact specifically in both yeast and mammalian cells. Interestingly, however, interaction of these two proteins in mammalian cells only occur following gamma-irradiation, raising the possibility of involvement of TRAX in DNA double-strand break repair and providing evidence for biological functions of the nuclear matrix protein C1D and TRAX. Moreover, we show, using fluorescently tagged proteins, that the relative expression levels of TRAX and Translin affect their subcellular localization. These results suggest that one role for C1D may be to regulate TRAX/Translin complex formation.