RESUMO
The mitotic checkpoint maintains genomic stability by ensuring that chromosomes are accurately segregated during mitosis. When the checkpoint is activated, the mitotic checkpoint complex (MCC), assembled from BUBR1, BUB3, CDC20, and MAD2, directly binds and inhibits the anaphase-promoting complex/cyclosome (APC/C) until all chromosomes are properly attached and aligned. The mechanisms underlying MCC assembly and MCC-APC/C interaction are not well characterized. Here, we show that a novel interaction between BUBR1 and closed MAD2 (C-MAD2) is essential for MCC-mediated inhibition of APC/C. Intriguingly, Arg(133) and Gln(134) in C-MAD2 are required for BUBR1 interaction. The same residues are also critical for MAD2 dimerization and MAD2 binding to p31(comet), a mitotic checkpoint silencing protein. Along with previously characterized BUBR1-CDC20 and C-MAD2-CDC20 interactions, our results underscore the integrity of the MCC for its activity and suggest the fundamental importance of the MAD2 αC helix in modulating mitotic checkpoint activation and silencing.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Anáfase , Ciclossomo-Complexo Promotor de Anáfase , Dimerização , Inativação Gênica , Células HeLa , Humanos , Proteínas Mad2 , Masculino , Próstata/metabolismo , Fuso Acromático/metabolismo , Transfecção , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In order to better understand the process of breast cancer metastasis, we have generated a mammary epithelial progression series of increasingly aggressive cell lines that metastasize to lung. Here we demonstrate that upregulation of an endoplasmic reticulum (ER) to Golgi trafficking gene signature in metastatic cells enhances transport kinetics, which promotes malignant progression. We observe increased ER-Golgi trafficking, an altered secretome and sensitivity to the retrograde transport inhibitor brefeldin A (BFA) in cells that metastasize to lung. CREB3 was identified as a transcriptional regulator of upregulated ER-Golgi trafficking genes ARF4, COPB1, and USO1, and silencing of these genes attenuated the metastatic phenotype in vitro and lung colonization in vivo. Furthermore, high trafficking gene expression significantly correlated with increased risk of distant metastasis and reduced relapse-free and overall survival in breast cancer patients, suggesting that modulation of ER-Golgi trafficking plays an important role in metastatic progression.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Animais , Neoplasias da Mama/mortalidade , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Análise em Microsséries , Metástase Neoplásica , Transporte Proteico/genética , Transcriptoma/genética , Células Tumorais CultivadasRESUMO
The contribution of lncRNAs to tumour progression and the regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleocytoplasmic translocation or in response to TGFß, allows alternative splicing and generates a non-coding isoform of PNUTS. Functionally the lncRNA-PNUTS serves as a competitive sponge for miR-205 during epithelial-mesenchymal transition (EMT). In mesenchymal breast tumour cells and in breast tumour samples, the expression of lncRNA-PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS mRNA and lncRNA-PNUTS, each eliciting distinct biological functions. While PNUTS mRNA is ubiquitously expressed, lncRNA-PNUTS appears to be tightly regulated dependent on the status of hnRNP E1 and tumour context.
Assuntos
Processamento Alternativo , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células A549 , Animais , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células CACO-2 , Movimento Celular , Proteínas de Ligação a DNA/genética , Éxons , Feminino , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Ligação Proteica , Interferência de RNA , Precursores de RNA/química , Precursores de RNA/genética , Sítios de Splice de RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Relação Estrutura-Atividade , Transcrição Gênica , Transfecção , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
This corrects the article DOI: 10.1038/ncb3595.
RESUMO
UNLABELLED: CDC27 is a core component of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, whose oscillatory activity is responsible for the metaphase-to-anaphase transition and mitotic exit. Here, in normal murine mammary gland epithelial cells (NMuMG), CDC27 expression is controlled posttranscriptionally through the RNA binding protein poly(rC) binding protein 1 (PCBP1)/heterogeneous nuclear ribonucleoprotein E1 (HNRNP E1). shRNA-mediated knockdown of HNRNP E1 abrogates translational silencing of the Cdc27 transcript, resulting in constitutive expression of CDC27. Dysregulated expression of CDC27 leads to premature activation of the G2-M-APC/C-CDC20 complex, resulting in the aberrant degradation of FZR1/CDH1, a cofactor of the G1 and late G2-M-APC/C and a substrate normally reserved for the SCF-ßTRCP ligase. Loss of CDH1 expression and of APC/C-CDH1 activity, upon constitutive expression of CDC27, results in mitotic aberrations and aneuploidy in NMuMG cells. Furthermore, tissue microarray of breast cancer patient tumor samples reveals high CDC27 levels compared with nonneoplastic breast tissue and a significant correlation between disease recurrence and CDC27 expression. These results suggest that dysregulation of HNRNP E1-mediated translational regulation of Cdc27 leads to chromosomal instability and aneuploidy and that CDC27 expression represents a significant predictor of breast cancer recurrence. IMPLICATIONS: The RNA-binding protein HNRNP E1 mediates translational regulation of the cell-cycle regulator CDC27 and that dysregulation of CDC27 leads to aneuploidy. In addition, high CDC27 expression in breast cancer patient tumor specimens significantly predicts disease recurrence, suggesting a novel role for CDC27 as a predictor of relapse. Mol Cancer Res; 14(7); 634-46. ©2016 AACR.
Assuntos
Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/biossíntese , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/genética , Instabilidade Cromossômica , Ribonucleoproteínas Nucleares Heterogêneas/biossíntese , Ribonucleoproteínas Nucleares Heterogêneas/genética , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular , Proteínas de Ligação a DNA , Feminino , Células HEK293 , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/fisiologia , Biossíntese de Proteínas , Proteínas de Ligação a RNA , TransfecçãoRESUMO
A major challenge in the clinical management of human cancers is to accurately stratify patients according to risk and likelihood of a favorable response. Stratification is confounded by significant phenotypic heterogeneity in some tumor types, often without obvious criteria for subdivision. Despite intensive transcriptional array analyses, the identity and validation of cancer specific 'signature genes' remains elusive, partially because the transcriptome does not mirror the proteome. The simplification associated with transcriptomic profiling does not take into consideration changes in the relative expression among transcripts that arise due to post-transcriptional regulatory events. We have previously shown that TGFß post-transcriptionally regulates epithelial-mesenchymal transition (EMT) by causing increased expression of two transcripts, Dab2 and ILEI, by modulating hnRNP E1 phosphorylation. Using a genome-wide combinatorial approach involving expression profiling and RIP-Chip analysis, we have identified a cohort of translationally regulated mRNAs that are induced during TGFß-mediated EMT. Coordinated translational regulation by hnRNP E1 constitutes a post-transcriptional regulon inhibiting the expression of related EMT-facilitating genes, thus enabling the cell to rapidly and coordinately regulate multiple EMT-facilitating genes.