Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Artigo em Inglês | MEDLINE | ID: mdl-33318029

RESUMO

BACKGROUND: Evidence for aspirin's chemopreventative properties on colorectal cancer (CRC) is substantial, but its mechanism of action is not well-understood. We combined a proteomic approach with Mendelian randomization (MR) to identify possible new aspirin targets that decrease CRC risk. METHODS: Human colorectal adenoma cells (RG/C2) were treated with aspirin (24 hours) and a stable isotope labeling with amino acids in cell culture (SILAC) based proteomics approach identified altered protein expression. Protein quantitative trait loci (pQTLs) from INTERVAL (N = 3,301) and expression QTLs (eQTLs) from the eQTLGen Consortium (N = 31,684) were used as genetic proxies for protein and mRNA expression levels. Two-sample MR of mRNA/protein expression on CRC risk was performed using eQTL/pQTL data combined with CRC genetic summary data from the Colon Cancer Family Registry (CCFR), Colorectal Transdisciplinary (CORECT), Genetics and Epidemiology of Colorectal Cancer (GECCO) consortia and UK Biobank (55,168 cases and 65,160 controls). RESULTS: Altered expression was detected for 125/5886 proteins. Of these, aspirin decreased MCM6, RRM2, and ARFIP2 expression, and MR analysis showed that a standard deviation increase in mRNA/protein expression was associated with increased CRC risk (OR: 1.08, 95% CI, 1.03-1.13; OR: 3.33, 95% CI, 2.46-4.50; and OR: 1.15, 95% CI, 1.02-1.29, respectively). CONCLUSIONS: MCM6 and RRM2 are involved in DNA repair whereby reduced expression may lead to increased DNA aberrations and ultimately cancer cell death, whereas ARFIP2 is involved in actin cytoskeletal regulation, indicating a possible role in aspirin's reduction of metastasis. IMPACT: Our approach has shown how laboratory experiments and population-based approaches can combine to identify aspirin-targeted proteins possibly affecting CRC risk.

2.
Int J Oncol ; 56(5): 1304-1313, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32319612

RESUMO

First discovered as an oncogene in leukaemia, recent reports highlight an emerging role for the proto­oncogene BCL­3 in solid tumours. Importantly, BCL­3 expression is upregulated in >30% of colorectal cancer cases and is reported to be associated with a poor prognosis. However, the mechanism by which BCL­3 regulates tumorigenesis in the large intestine is yet to be fully elucidated. In the present study, it was shown for the first time that knocking down BCL­3 expression suppressed cyclooxygenase­2 (COX­2)/prostaglandin E2 (PGE2) signalling in colorectal cancer cells, a pathway known to drive several of the hallmarks of cancer. RNAi­mediated suppression of BCL­3 expression decreased COX­2 expression in colorectal cancer cells both at the mRNA and protein level. This reduction in COX­2 expression resulted in a significant and functional reduction (30­50%) in the quantity of pro­tumorigenic PGE2 produced by the cancer cells, as shown by enzyme linked immunoassays and medium exchange experiments. In addition, inhibition of BCL­3 expression also significantly suppressed cytokine­induced (TNF­α or IL­1ß) COX­2 expression. Taken together, the results of the present study identified a novel role for BCL­3 in colorectal cancer and suggested that expression of BCL­3 may be a key determinant in the COX­2­meditated response to inflammatory cytokines in colorectal tumour cells. These results suggest that targeting BCL­3 to suppress PGE2 synthesis may represent an alternative or complementary approach to using non­steroidal anti­inflammatory drugs [(NSAIDs), which inhibit cyclooxygenase activity and suppress the conversion of arachidonic acid to prostaglandin], for prevention and/or recurrence in PGE2­driven tumorigenesis.

3.
Cells ; 9(3)2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32210188

RESUMO

The Wnt and bone morphogenetic protein (BMP) signaling pathways are known to be crucial in the development of neural crest lineages, including the sympathetic nervous system. Surprisingly, their role in paediatric neuroblastoma, the prototypic tumor arising from this lineage, remains relatively uncharacterised. We previously demonstrated that Wnt/b-catenin signaling can have cell-type-specific effects on neuroblastoma phenotypes, including growth inhibition and differentiation, and that BMP4 mRNA and protein were induced by Wnt3a/Rspo2. In this study, we characterised the phenotypic effects of BMP4 on neuroblastoma cells, demonstrating convergent induction of MSX homeobox transcription factors by Wnt and BMP4 signaling and BMP4-induced growth suppression and differentiation. An immunohistochemical analysis of BMP4 expression in primary neuroblastomas confirms a striking absence of BMP4 in poorly differentiated tumors, in contrast to a high expression in ganglion cells. These results are consistent with a tumor suppressive role for BMP4 in neuroblastoma. RNA sequencing following BMP4 treatment revealed induction of Notch signaling, verified by increases of Notch3 and Hes1 proteins. Together, our data demonstrate, for the first time, Wnt-BMP-Notch signaling crosstalk associated with growth suppression of neuroblastoma.

4.
Front Mol Neurosci ; 12: 90, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31040767

RESUMO

The neural crest (NC), which has been referred to as the fourth germ layer, comprises a multipotent cell population which will specify diverse cells and tissues, including craniofacial cartilage and bones, melanocytes, the adrenal medulla and the peripheral nervous system. These cell fates are known to be determined by gene regulatory networks (GRNs) acting at various stages of NC development, such as induction, specification, and migration. Although transcription factor hierarchies and some of their interplay with morphogenetic signaling pathways have been characterized, the full complexity of activities required for regulated development remains uncharted. Deregulation of these pathways may contribute to tumorigenesis, as in the case of neuroblastoma, a frequently lethal embryonic cancer thought to arise from the sympathoadrenal lineage of the NC. In this "Hypothesis and Theory" article, we utilize the next generation sequencing data from neuroblastoma cells and tumors to evaluate the possible influences of Wnt signaling on NC GRNs and on neuroblastoma cell lineages. We propose that Wnt signaling is a major determinant of regulatory networks that underlie mesenchymal/neural crest cell (NCC)-like cell identities through PRRX1 and YAP/TAZ transcription factors. Furthermore, Wnt may also co-operate with Hedgehog signaling in driving proneural differentiation programmes along the adrenergic (ADRN) lineage. Elucidation of Signaling Regulatory Networks can augment and complement GRNs in characterizing cell identities, which may in turn contribute to the design of improved therapeutics tailored to primary and relapsing neuroblastoma.

5.
Dis Model Mech ; 12(3)2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30792270

RESUMO

To decrease bowel cancer incidence and improve survival, we need to understand the mechanisms that drive tumorigenesis. Recently, B-cell lymphoma 3 (BCL-3; a key regulator of NF-κB signalling) has been recognised as an important oncogenic player in solid tumours. Although reported to be overexpressed in a subset of colorectal cancers (CRCs), the role of BCL-3 expression in colorectal tumorigenesis remains poorly understood. Despite evidence in the literature that BCL-3 may interact with ß-catenin, it is perhaps surprising, given the importance of deregulated Wnt/ß-catenin/T-cell factor (TCF) signalling in colorectal carcinogenesis, that the functional significance of this interaction is not known. Here, we show for the first time that BCL-3 acts as a co-activator of ß-catenin/TCF-mediated transcriptional activity in CRC cell lines and that this interaction is important for Wnt-regulated intestinal stem cell gene expression. We demonstrate that targeting BCL-3 expression (using RNA interference) reduced ß-catenin/TCF-dependent transcription and the expression of intestinal stem cell genes LGR5 and ASCL2 In contrast, the expression of canonical Wnt targets Myc and cyclin D1 remained unchanged. Furthermore, we show that BCL-3 increases the functional stem cell phenotype, as shown by colorectal spheroid and tumoursphere formation in 3D culture conditions. We propose that BCL-3 acts as a driver of the stem cell phenotype in CRC cells, potentially promoting tumour cell plasticity and therapeutic resistance. As recent reports highlight the limitations of directly targeting cancer stem cells (CSCs), we believe that identifying and targeting drivers of stem cell plasticity have significant potential as new therapeutic targets.This article has an associated First Person interview with the first author of the paper.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Proteína 3 do Linfoma de Células B , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fenótipo , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fatores de Transcrição TCF/metabolismo , Fatores de Transcrição/genética , beta Catenina/metabolismo
6.
Haematologica ; 104(7): 1365-1377, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30630973

RESUMO

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß- catenin level in human myeloid leukemia.


Assuntos
Núcleo Celular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Síndromes Mielodisplásicas/metabolismo , Proteoma/análise , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Fator 1 de Ligação ao Facilitador Linfoide/antagonistas & inibidores , Fator 1 de Ligação ao Facilitador Linfoide/genética , Síndromes Mielodisplásicas/patologia , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , Ativação Transcricional , Células Tumorais Cultivadas , Proteína Wnt1/genética , beta Catenina/genética
7.
EMBO Mol Med ; 10(11)2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30143543

RESUMO

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 Genética/efeitos dos fármacos , Peixe-Zebra
8.
EMBO J ; 37(13)2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29866703

RESUMO

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.


Assuntos
Macrófagos/fisiologia , Neovascularização Fisiológica , Cicatrização/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Diagnóstico por Imagem , Fibroblastos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos C57BL , Peixe-Zebra/genética
9.
Neoplasia ; 20(4): 335-350, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29505958

RESUMO

Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra- and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues. However, our previous work demonstrated that Wnt3a/Rspo2 treatment of some neuroblastoma cell lines can, paradoxically, decrease c-MYC and MYCN proteins. This prompted us to define the neuroblastoma-specific Wnt3a/Rspo2-driven transcriptome using RNA sequencing, and characterise the accompanying changes in cell biology. Here we report the identification of ninety Wnt target genes, and show that Wnt signalling is upstream of numerous transcription factors and signalling pathways in neuroblastoma. Using live-cell imaging, we show that Wnt signalling can drive differentiation of SK-N-BE(2)-C and SH-SY5Y cell-lines, but, conversely, proliferation of SK-N-AS cells. We show that cell-lines that differentiate show induction of pro-differentiation BMP4 and EPAS1 proteins, which is not apparent in the SK-N-AS cells. In contrast, SK-N-AS cells show increased CCND1, phosphorylated RB and E2F1 in response to Wnt3a/Rspo2, consistent with their proliferative response, and these proteins are not increased in differentiating lines. By meta-analysis of the expression of our 90 genes in primary tumour gene expression databases, we demonstrate discrete expression patterns of our Wnt genes in patient cohorts with different prognosis. Furthermore our analysis reveals interconnectivity within subsets of our Wnt genes, with one subset comprised of novel putative drivers of neuronal differentiation repressed by MYCN. Assessment of ß-catenin immunohistochemistry shows high levels of ß-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.


Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Neuroblastoma/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Genes myc/genética , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética
11.
Gut ; 65(7): 1151-64, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26033966

RESUMO

OBJECTIVE: Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival. DESIGN: Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-κB complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5-aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated. RESULTS: We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-κB p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK-3ß and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells. CONCLUSIONS: Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy.


Assuntos
Neoplasias Colorretais/química , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose , Proteína 3 do Linfoma de Células B , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Colo/química , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Mesalamina/farmacologia , Camundongos , Camundongos Nus , NF-kappa B/análise , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/farmacologia , Reto/química , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Carga Tumoral
12.
Oncotarget ; 6(37): 40053-67, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26517508

RESUMO

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of ß-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neuroblastoma/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Via de Sinalização Wnt/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Criança , Pré-Escolar , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Lactente , Recém-Nascido , Microscopia Confocal , Neuroblastoma/genética , Neuroblastoma/patologia , Interferência de RNA , Receptores Acoplados a Proteínas-G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Catenina/genética , beta Catenina/metabolismo
14.
Mol Oncol ; 9(3): 617-27, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25475372

RESUMO

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.


Assuntos
Neoplasias Encefálicas/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Sequência de Aminoácidos , Apoptose , Neoplasias Encefálicas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/patologia , Proteínas Nucleares/química , Proteínas Oncogênicas/química , Ligação Proteica , Estabilidade Proteica
15.
EMBO J ; 32(13): 1903-16, 2013 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-23736261

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/genética
16.
Carcinogenesis ; 34(5): 1150-7, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23349017

RESUMO

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/metabolismo
17.
Gut ; 61(9): 1306-14, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22082586

RESUMO

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/genética
18.
Carcinogenesis ; 32(11): 1741-7, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21926111

RESUMO

Due to poor tumour-associated vasculature, tumour cells are subjected to a fluctuating microenvironment with periods of limited oxygen and glucose availability. Adaptive mechanisms to adverse microenvironments are important for tumour cell survival. The cyclooxygenase (COX)-2/prostaglandin E(2) (PGE(2)) pathway has key roles in colorectal tumorigenesis. Although glucose is important as an energy source and in maintaining endoplasmic reticulum homeostasis, relatively little is known regarding how tumour cells adapt to the microenvironmental stress of reduced glucose availability. Here, we report the novel findings that glucose deprivation of colorectal tumour cells not only increases COX-2 expression but also decreases 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression, resulting in increased extracellular PGE(2). Furthermore, we have shown that PGE(2) promotes tumour cell survival during glucose deprivation. Glucose deprivation enhances phosphoinositide 3-kinase/Akt activity, which has a role in both the up-regulation of COX-2 and down-regulation of 15-PGDH. Glucose deprivation also activates the unfolded protein response (UPR) resulting in elevated C/EBP-homologous protein (CHOP) expression. Interestingly, inhibiting CHOP expression by small interfering RNA during glucose deprivation attenuates the reduction in 15-PGDH expression. This is the first report linking activation of the UPR with a reduction in expression of tumour-suppressive 15-PGDH and may have implications for tumour cells' ability to survive exposure to therapeutic agents that activate the UPR. Our data suggest that diverse microenvironmental stresses converge to regulate PGE(2) as a common and crucial mediator of cell survival during adaptation to the tumour microenvironment and may lead to novel chemopreventive and therapeutic strategies.


Assuntos
Proliferação de Células , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Glucose/deficiência , Hidroxiprostaglandina Desidrogenases/metabolismo , Western Blotting , Neoplasias do Colo/genética , Ciclo-Oxigenase 2/genética , Elafina/genética , Elafina/metabolismo , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Hipóxia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Microambiente Tumoral , Resposta a Proteínas não Dobradas
19.
Int J Oncol ; 37(1): 187-93, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20514410

RESUMO

Despite recent advances in understanding colorectal tumour biology, there is still a need to improve the 5-year survival rate of patients with colorectal cancer as approximately 40% of patients presenting with advanced disease will remain resistant to therapy. One of the major contributing factors in resistance to therapy is the failure of colorectal tumour cells to undergo apoptosis. Hence there is an urgent need to develop novel therapeutic approaches that can target apoptosis-resistant cells. To this end, we investigated the potential efficacy of the endogenous cannabinoid anandamide to induce cell death in apoptosis-resistant colon cancer cells. Here, for the first time, we show that anandamide can induce cell death in the apoptosis-resistant HCT116 Bax-/- colorectal cell line. Importantly, we provide direct genetic evidence that this induction of cell death is dependent on COX-2 expression. Interestingly, increased COX-2 expression also sensitised the SW480 colorectal cancer cell line (low endogenous COX-2) to anandamide-induced death, whereas COX-2 suppression by RNAi inhibited anandamide-induced cell death in the HCA7 colorectal cancer cell line (high endogenous COX-2 expression). This COX-2-dependent death was independent of cannabinoid receptor engagement (CB1 or CB2), and not a direct consequence of reactive oxygen species (ROS) formation. This study demonstrates a novel utilisation for COX-2 expression, targeting apoptotic defective colorectal cancer cells for destruction by anandamide. As COX-2 is not expressed in the normal colorectal epithelium, but highly expressed in colorectal tumours and apoptosis resistance contributes to treatment failure, these data suggest that anandamide has the potential to be an effective therapeutic in colorectal cancer.


Assuntos
Apoptose/genética , Ácidos Araquidônicos/farmacologia , Carcinoma/patologia , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/fisiologia , Alcamidas Poli-Insaturadas/farmacologia , Apoptose/efeitos dos fármacos , Moduladores de Receptores de Canabinoides/farmacologia , Carcinoma/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Endocanabinoides , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HT29 , Humanos , RNA Interferente Pequeno/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Proteína X Associada a bcl-2/genética
20.
Carcinogenesis ; 30(10): 1796-804, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19638428

RESUMO

Evidence points towards a pivotal role for cyclooxygenase (COX)-2 in promoting colorectal tumorigenesis through increasing prostaglandin E(2) (PGE(2)) levels. PGE(2) signalling is closely associated with the survival, proliferation and invasion of colorectal cancer cells. Recently, a reduction in PGE(2) inactivation, a process mediated by the nicotinamide adenine dinucleotide (NAD+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), has also been shown to promote tumoral PGE(2) accumulation. The hepatocyte growth factor (HGF) receptor, Met, is frequently over-expressed in colorectal tumours and promotes cancer growth, metastasis and resistance to therapy, although the mechanisms for this have not been fully elucidated. Here, we report that HGF/Met signalling can promote PGE(2) biogenesis in colorectal cancer cells via COX-2 up-regulation and 15-PGDH down-regulation at the protein and messenger RNA level. Pharmacological inhibition of MEK and PI3K suggested that both extracellular signal-regulated kinase (ERK) and AKT signalling are required for COX-2 protein up-regulation and 15-PGDH down-regulation downstream of Met. Notably, inhibition of Met with the small molecule inhibitor SU11274 reduced COX-2 expression and increased 15-PGDH expression in high Met-expressing cells. We also show that hypoxia potentiated HGF-driven COX-2 expression and enhanced PGE(2) release. Furthermore, inhibition of COX-2 impeded the growth-promoting effects of HGF, suggesting that the COX-2/PGE(2) pathway is an important mediator of HGF/Met signalling. These data reveal a critical role for HGF/Met signalling in promoting PGE(2) biogenesis in colorectal cancer cells. Targeting the crosstalk between these two important pathways may be useful for therapeutic treatment of colorectal cancer.


Assuntos
Adenoma/genética , Neoplasias Colorretais/genética , Ciclo-Oxigenase 2/genética , Hidroxiprostaglandina Desidrogenases/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/terapia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-met , Transdução de Sinais , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA