RESUMO
The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistable-like behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Motivos de Aminoácidos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Progressão da Doença , Retroalimentação Fisiológica , Feminino , Variação Genética , Humanos , Células MCF-7 , Modelos Teóricos , Metástase Neoplásica , Estresse Oxidativo , Regiões Promotoras Genéticas , Fatores de Tempo , Transcrição GênicaRESUMO
The ten-eleven translocation (TET) family of methylcytosine dioxygenases initiates demethylation of DNA and is associated with tumorigenesis in many cancers; however, the mechanism is mostly unknown. Here we identify upstream activators and downstream effectors of TET1 in breast cancer using human breast cancer cells and a genetically engineered mouse model. We show that depleting the architectural transcription factor high mobility group AT-hook 2 (HMGA2) induces TET1. TET1 binds and demethylates its own promoter and the promoter of homeobox A (HOXA) genes, enhancing its own expression and stimulating expression of HOXA genes including HOXA7 and HOXA9. Both TET1 and HOXA9 suppress breast tumor growth and metastasis in mouse xenografts. The genes comprising the HMGA2-TET1-HOXA9 pathway are coordinately regulated in breast cancer and together encompass a prognostic signature for patient survival. These results implicate the HMGA2-TET1-HOX signaling pathway in the epigenetic regulation of human breast cancer and highlight the importance of targeting methylation in specific subpopulations as a potential therapeutic strategy.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Proteína HMGA2/genética , Proteínas de Homeodomínio/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteína HMGA2/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Estimativa de Kaplan-Meier , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxigenases de Função Mista , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let-7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let-7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let-7-regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Carcinoma/diagnóstico , Carcinoma/genética , MicroRNAs/fisiologia , Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/fisiologia , Neoplasias da Mama/patologia , Carcinoma/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Análise em Microsséries , Modelos Biológicos , Metástase Neoplásica , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Prognóstico , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
In the past decade cancer research has recognized the importance of tumorstroma interactions for the progression of primary tumors to an aggressive and invasive phenotype and for colonization of new organs in the context of metastasis. The dialogue between tumor cells and the surrounding stroma is a complex and dynamic phenomenon, as many cell types and soluble factors are involved. While the function of many of the players involved in this cross talk have been studied, the regulatory mechanisms and signaling pathways that control their expression haven't been investigated in depth. By using a novel, interdisciplinary approach applied to the mechanism of action of the metastasis suppressor, Raf kinase inhibitory protein (RKIP), we identified a signaling pathway that suppresses invasion and metastasis through regulation of stroma-associated genes. Conceptually, the approach we developed uses a master regulator and expression arrays from breast cancer patients to formulate hypotheses based on clinical data. Experimental validation is followed by further bioinformatic analysis to establish the clinical significance of discoveries. Using RKIP as an example we show here that this multi-step approach can be used to identify gene regulatory mechanisms that affect tumor-stroma interactions that in turn influence metastasis to the bone or other organs.