RESUMO
BACKGROUND: Autophagy is a highly regulated biological process that mediates the degradation of intracellular components. It is required for tumor cell metabolism and homeostasis. Yin-Yang 1 (YY1) has been reported to be involved in autophagy in several carcinomas. However, its role in autophagy in pancreatic cancer, one of the deadliest human malignancies, is unknown. Here, we investigated the function of YY1 in pancreatic cancer cells autophagy and its mechanisms of action. METHODS: The activity of cells undergoing autophagy was assessed using transmission electron microscopy, immunofluorescence, and Western blotting. A luciferase activity assay, real-time quantitative polymerase chain reaction (RT-qPCR), and chromatin immunoprecipitation (ChIP) were also used to identify putative downstream targets of YY1. RESULTS: YY1 was confirmed to regulate autophagy in pancreatic cancer cells. It was found to directly regulate the expression of miR-30a, a known modulator of autophagy-associated genes. Furthermore, overexpression of miR-30a attenuated the pro-autophagic effects of YY1. CONCLUSIONS: Cumulatively, our data suggest that miR-30a acts in a feedback loop to modulate the pro-autophagic activities of YY1. Thus, autophagy in pancreatic cancer cells may be regulated, in part, by a tightly coordinated YY1/miR-30a regulatory circuit. These findings provide a potential druggable target for the development of treatments for pancreatic cancer.
Assuntos
Autofagia/genética , MicroRNAs/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fator de Transcrição YY1/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Sequência de Bases , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Ligação Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The transcription regulator Yin Yang-1 (YY1) serves as a tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). However, the function of YY1 in proliferation of PDAC cells remains to be clarified. In this study, we found that overexpression of YY1 suppressed proliferation and decreased the expression of long non-coding RNA (lncRNA) SOX2OT and its potential target gene SOX2 in PDAC cells. Luciferase reporter, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays revealed binding of YY1 to the SOX2OT promoter. Moreover, YY1 suppressed PDAC cell proliferation through SOX2OT transcriptional inhibition and subsequent decreased SOX2 expression. In addition, YY1 expression was statistically negatively correlated with SOX2OT and SOX2 expression in PDAC tissues and lower level expression of SOX2OT predicted better outcome in PDAC patients. These results confirmed the anti-proliferation effect of YY1 on PDAC cells, which was associated with SOX2 down-regulation in a SOX2OT-dependent mechanism. Although other undiscovered mechanisms may be involved in the YY1-mediated tumor suppression role, the present study suggests that SOX2OT may act as a tumor promotor in PDAC and may represent a valuable diagnostic and therapeutic target.
Assuntos
Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/patologia , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Apoptose , Biomarcadores Tumorais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Imunoprecipitação da Cromatina , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estadiamento de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Prognóstico , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Fatores de Transcrição SOXB1/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Fator de Transcrição YY1/genéticaRESUMO
The transcriptional regulator Yin Yang-1 (YY1) is a tumor suppressor known to be overexpressed in pancreatic cancer. We found that overexpression of YY1 promoted apoptosis and increased the expression and mitochondrial localization of the pro-apoptotic Bax protein in pancreatic cancer cell lines. Luciferase reporter, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays revealed binding of YY1 to the BAX promoter. Moreover, YY1 promoted pancreatic cancer cell apoptosis through Bax transcriptional activation and subsequent translocation of Bax to the mitochondrial membrane, leading to cytochrome c release, and caspase activation.YY1 and BAX are co-expressed in pancreatic cancer tissues and higher BAX expression predicts better outcomes for patients. The ability of YY1 to promote apoptosis in pancreatic cancer cells suggests it may represent a valuable diagnostic and therapeutic target.
Assuntos
Apoptose/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Fator de Transcrição YY1/genética , Proteína X Associada a bcl-2/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos BALB C , Camundongos Nus , Membranas Mitocondriais/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico/genética , Transplante Heterólogo , Fator de Transcrição YY1/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: Increasing evidence indicates an important role of transcription factor Yin Yang-1 (YY1) in human tumorigenesis. However, its function in cancer remains controversial and the relevance of YY1 to pancreatic ductal adenocarcinoma (PDAC) remains to be clarified. METHODS: In this study, we detected YY1 expression in clinical PDAC tissue samples and cell lines using quantitative RT-PCR, immunohistochemistry and western blotting. We also detected MUC4 and MMP10 mRNA levels in 108 PDAC samples using qRT-PCR and analyzed the correlations between YY1 and MUC4 or MMP10 expression. The role of YY1 in the proliferation, invasion and metastatic abilities of PDAC cells in vitro was studied by CCK-8 assay, cell migration and invasion assays. In vivo pancreatic tumor growth and metastasis was studied by a xenogenous subcutaneously implant model and a tail vein metastasis model. The potential mechanisms underlying YY1 mediated tumor progression in PDAC were explored by digital gene expression (DGE) sequencing, signal transduction pathways blockage experiments and luciferase assays. Statistical analysis was performed using the SPSS 15.0 software. RESULTS: We found that the expression of YY1 in PDACs was higher compared with their adjacent non-tumorous tissues and normal pancreas tissues. However, PDAC patients with high level overexpression of YY1 had better outcome than those with low level overexpression. YY1 expression levels were statistically negatively correlated with MMP10 expression levels, but not correlated with MUC4 expression levels. YY1 overexpression suppressed, whereas YY1 knockdown enhanced, the proliferation, invasion and metastatic properties of BXPC-3 cells, both in vitro and in vivo. YY1 suppresses invasion and metastasis of pancreatic cancer cells by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism. CONCLUSIONS: The present study suggested that YY1 plays a negative role, i.e. is a tumor suppressor, in PDAC, and may become a valuable diagnostic and prognostic marker of PDAC.
Assuntos
Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Metaloproteinase 10 da Matriz/genética , Neoplasias Pancreáticas/genética , Fator de Transcrição YY1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/secundário , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Metaloproteinase 10 da Matriz/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mucina-4/genética , Mucina-4/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Fator de Transcrição YY1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The human mucin 4 (MUC4) is aberrantly expressed in pancreatic adenocarcinoma and tumor cell lines, while remaining undetectable in normal pancreas, indicating its important role in pancreatic cancer development. Although its transcriptional regulation has been investigated in considerable detail, some important elements remain unknown. The aim of the present study was to demonstrate the existence of a novel inhibitory element in the MUC4 promoter and characterize some of its binding proteins. By luciferase reporter assay, we located the inhibitory element between nucleotides -2530 and -2521 in the MUC4 promoter using a series of deletion and mutant reporter constructs. Electrophoretic mobility shift assay (EMSA) with Bxpc-3 cell nuclear extracts revealed that one protein or protein complex bind to this element. The proteins binding to this element were purified and identified as Yin Yang 1 (YY1) by mass spectrometry. Supershift assay and chromatin immunoprecipitation (ChIP) assay confirmed that YY1 binds to this element in vitro and in vivo. Moreover, transient YY1 overexpression significantly inhibited MUC4 promoter activity and endogenous MUC4 protein expression. In conclusion, we reported here a novel inhibitory element in the human MUC4 promoter. This provides additional data on MUC4 gene regulation and indicates that YY1 may be a potential target for abnormal MUC4 expression.