RESUMO
The Wnt/ß-catenin signalling and autophagy pathways each play important roles during development, adult tissue homeostasis and tumorigenesis. Here we identify the Wnt/ß-catenin signalling pathway as a negative regulator of both basal and stress-induced autophagy. Manipulation of ß-catenin expression levels in vitro and in vivo revealed that ß-catenin suppresses autophagosome formation and directly represses p62/SQSTM1 (encoding the autophagy adaptor p62) via TCF4. Furthermore, we show that during nutrient deprivation ß-catenin is selectively degraded via the formation of a ß-catenin-LC3 complex, attenuating ß-catenin/TCF-driven transcription and proliferation to favour adaptation during metabolic stress. Formation of the ß-catenin-LC3 complex is mediated by a W/YXXI/L motif and LC3-interacting region (LIR) in ß-catenin, which is required for interaction with LC3 and non-proteasomal degradation of ß-catenin. Thus, Wnt/ß-catenin represses autophagy and p62 expression, while ß-catenin is itself targeted for autophagic clearance in autolysosomes upon autophagy induction. These findings reveal a regulatory feedback mechanism that place ß-catenin at a key cellular integration point coordinating proliferation with autophagy, with implications for targeting these pathways for cancer therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias do Colo/patologia , Lisossomos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Western Blotting , Imunoprecipitação da Cromatina , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Sequestossoma-1 , Fator de Transcrição 4 , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Proteínas Wnt/genética , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
Cyclooxygenase-2 is overexpressed in the majority of colorectal tumours leading to elevated levels of prostaglandin E2 (PGE2), promoting many hallmarks of cancer. Importantly, PGE2 is reported to enhance Wnt/ß-catenin signalling in colorectal carcinoma cells and in normal haematopoietic stem cells where it promotes stem cell function. Although Wnt signalling plays a crucial role in intestinal stem cells, the relationship between PGE2 and intestinal stem cells is unclear. Given that the key intestinal cancer stem cell marker LGR5 (leucine-rich G-protein coupled receptor 5) is a Wnt target and PGE2 enhances Wnt signalling, the focus of this study was to investigate whether PGE2 regulated LGR5 expression in colorectal adenoma cells and whether LGR5 was important for tumour cell survival. PGE2 upregulated LGR5 protein in adenoma (RG/C2) and carcinoma (DLD-1) cell lines. LGR5 knockdown induced cell death in RG/C2 and AA/C1 adenoma cells, suggesting that LGR5 has an important survival-promoting role in adenoma cells. Indeed, we detected LGR5 protein expression in 4 of 4 human adenoma cell lines. Furthermore, LGR5 small interfering RNA inhibited the survival-promoting effects of PGE2 in RG/C2, suggesting that PGE2 promotes adenoma cell survival, at least in part, by increasing LGR5 expression. These studies, therefore, show the first link between PGE2 and LGR5 in human colorectal adenoma and carcinoma cells and demonstrate a survival-promoting role of LGR5. As non-steroidal anti-inflammatory drugs (NSAIDs) cause adenomas to regress in FAP patients, these studies could have important implications for the mechanism by which NSAIDs are chemopreventive, as lowering PGE2 levels could reduce LGR5 expression and survival of LGR5(+) adenoma stem cells.
Assuntos
Adenoma/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Neoplasias Colorretais/metabolismo , Dinoprostona/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenoma/genética , Adenoma/patologia , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Dinoprostona/genética , Feminino , Humanos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Regulação para Cima , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression in colorectal cancer increases levels of its pro-tumorigenic product prostaglandin E2 (PGE(2)). The recently identified colorectal tumour suppressor 15-prostaglandin dehydrogenase (15-PGDH) catalyses prostaglandin turnover and is downregulated at a very early stage in colorectal tumorigenesis; however, the mechanism responsible remains unclear. As Wnt/ß-catenin signalling is also deregulated early in colorectal neoplasia, a study was undertaken to determine whether ß-catenin represses 15-PGDH expression. METHODS: The effect of modulating Wnt/ß-catenin signalling (using ß-catenin siRNA, mutant TCF4, Wnt3A or GSK3 inhibition) on 15-PGDH mRNA, protein expression and promoter activity was determined in colorectal cell lines by immunoblotting, qRT-PCR and reporter assays. The effect of ß-catenin deletion in vivo was addressed by 15-PGDH immunostaining of ß-catenin(-/lox)-villin-creERT2 mouse tissue. 15-PGDH promoter occupancy was determined using chromatin immunoprecipitation and PGE(2) levels by ELISA. RESULTS: The study shows for the first time that ß-catenin knockdown upregulates 15-PGDH in colorectal adenoma and carcinoma cells without affecting COX-2 protein levels. A dominant negative mutant form of TCF4 (dnTCF4), unable to bind ß-catenin, also upregulated 15-PGDH; conversely, increasing ß-catenin activity using Wnt3A or GSK3 inhibition downregulated 15-PGDH. Importantly, inducible ß-catenin deletion in vivo also upregulated intestinal epithelial 15-PGDH. 15-PGDH regulation occurred at the protein, mRNA and promoter activity levels and chromatin immunoprecipitation indicated ß-catenin/TCF4 binding to the 15-PGDH promoter. ß-catenin knockdown decreased PGE(2) levels, and this was significantly rescued by 15-PGDH siRNA. CONCLUSION: These data suggest a novel role for ß-catenin in promoting colorectal tumorigenesis through very early 15-PGDH suppression leading to increased PGE(2) levels, possibly even before COX-2 upregulation.
Assuntos
Adenoma/enzimologia , Neoplasias Colorretais/enzimologia , Hidroxiprostaglandina Desidrogenases/metabolismo , Mucosa Intestinal/enzimologia , beta Catenina/fisiologia , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Regulação para Baixo , Repressão Enzimática , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hidroxiprostaglandina Desidrogenases/biossíntese , Hidroxiprostaglandina Desidrogenases/genética , Immunoblotting , Imuno-Histoquímica , Camundongos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Regulação para Cima , beta Catenina/genéticaRESUMO
Hypoxia is a hallmark of solid tumours and a key physiological feature distinguishing cancer from normal tissue. However, a major challenge remains in identifying tractable molecular targets that hypoxic cancer cells depend on for survival. Here, we used SILAC-based proteomics to identify the orphan G protein-coupled receptor GPRC5A as a novel hypoxia-induced protein that functions to protect cancer cells from apoptosis during oxygen deprivation. Using genetic approaches in vitro and in vivo, we reveal HIFs as direct activators of GPRC5A transcription. Furthermore, we find that GPRC5A is upregulated in the colonic epithelium of patients with mesenteric ischaemia, and in colorectal cancers high GPRC5A correlates with hypoxia gene signatures and poor clinical outcomes. Mechanistically, we show that GPRC5A enables hypoxic cell survival by activating the Hippo pathway effector YAP and its anti-apoptotic target gene BCL2L1 Importantly, we show that the apoptosis induced by GPRC5A depletion in hypoxia can be rescued by constitutively active YAP. Our study identifies a novel HIF-GPRC5A-YAP axis as a critical mediator of the hypoxia-induced adaptive response and a potential target for cancer therapy.
Assuntos
Adaptação Fisiológica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/patologia , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Adaptação Fisiológica/efeitos dos fármacos , Animais , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição , Transcrição Gênica/efeitos dos fármacos , Proteínas de Sinalização YAP , Peixe-ZebraRESUMO
The insulin-like growth factor binding protein 3 (IGFBP-3) is the major circulating IGF binding protein, its function regulated by proteolytic cleavage. The fragments generated have recently been suggested to have IGF-independent biological activity. We have previously established that IGFBP-3 can potentiate apoptosis in colorectal epithelial cells, although its use as a therapeutic reagent may be limited by the fact that it is cleaved in the circulation. Therefore the aim of these experiments was to determine whether the 16-kDa proteolytic fragment (1-95IGFBP-3) would have IGF-independent pro-apoptotic activity in human colonic carcinoma derived cells. We report that the enforced expression of 1-95IGFBP-3 increased the induction of apoptosis by the naturally occurring short chain fatty acid sodium butyrate (NaBt) in the IGF non-responsive HT29 human colorectal carcinoma cell line. Furthermore, the addition of condition medium containing the secreted 1-95IGFBP-3 was as effective as the intact IGFBP-3 protein at potentiating apoptosis. Although not associated with changes in Bcl-2, Bcl-XL, Bax, Bad or Bak expression levels, we report that the expression of the pro-apoptotic 1-95IGFBP-3 fragment is associated with the inhibition of TNFalpha-induced NF-kappaB activity, similar to that reported for the full length IGFBP-3 protein. These results suggest that the 16-kDa 1-95IGFBP-3 fragment is as effective as an intact recombinant protein when used in combination with apoptosis inducing agents, and due to its relative stability in the circulation, it may be important for use as an adjuvant in the treatment of colorectal cancer.
Assuntos
Apoptose , Carcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Meios de Cultivo Condicionados/farmacologia , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , NF-kappa B/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Oxibato de Sódio/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide range of human tumours, overexpression in colonic carcinomas has been linked to the antiapoptotic function of the protein. In the current study we show that the Retinoblastoma susceptibility protein (Rb) protein interacts with Bag-1, an apoptotic regulator, in human colonic adenoma- and carcinoma-derived cell lines. Coimmunoprecipitation demonstrated that endogenous Rb and Bag-1 interact in both adenoma- and carcinoma-derived cell lines. The specificity of the interaction was demonstrated by expression of human Papillomavirus E7 oncoprotein, an inhibitor of Rb protein interactions, which disrupted the Rb/Bag-1 complex. We report that Bag-1 is predominantly localised in the nucleus of colorectal adenoma- and carcinoma-derived epithelial cells. Disruption of the Rb/Bag-1 complex through expression of E7 changes the subcellular distribution of Bag-1, decreasing nuclear localised Bag-1. Our work establishes that the Rb protein interacts with the Bag-1 apoptotic regulator protein, and introduces a novel function for Rb, involving modulation of the subcellular localisation of Bag-1 in human colonic epithelial cells.