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1.
Nucleic Acids Res ; 47(3): 1141-1163, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30544251

RESUMO

Temporal data on gene expression and context-specific open chromatin states can improve identification of key transcription factors (TFs) and the gene regulatory networks (GRNs) controlling cellular differentiation. However, their integration remains challenging. Here, we delineate a general approach for data-driven and unbiased identification of key TFs and dynamic GRNs, called EPIC-DREM. We generated time-series transcriptomic and epigenomic profiles during differentiation of mouse multipotent bone marrow stromal cell line (ST2) toward adipocytes and osteoblasts. Using our novel approach we constructed time-resolved GRNs for both lineages and identifed the shared TFs involved in both differentiation processes. To take an alternative approach to prioritize the identified shared regulators, we mapped dynamic super-enhancers in both lineages and associated them to target genes with correlated expression profiles. The combination of the two approaches identified aryl hydrocarbon receptor (AHR) and Glis family zinc finger 1 (GLIS1) as mesenchymal key TFs controlled by dynamic cell type-specific super-enhancers that become repressed in both lineages. AHR and GLIS1 control differentiation-induced genes and their overexpression can inhibit the lineage commitment of the multipotent bone marrow-derived ST2 cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Mesenquimais/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição/metabolismo , Adipócitos/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Redes Reguladoras de Genes , Células-Tronco Mesenquimais/citologia , Camundongos , Osteoblastos/metabolismo
2.
Br J Cancer ; 117(11): 1689-1701, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29024942

RESUMO

BACKGROUND: Selecting the most beneficial treatment regimens for colorectal cancer (CRC) patients remains challenging due to a lack of prognostic markers. Members of the Myosin family, proteins recognised to have a major role in trafficking and polarisation of cells, have recently been reported to be closely associated with several types of cancer and might thus serve as potential prognostic markers in the context of CRC. METHODS: We used a previously established meta-analysis of publicly available gene expression data to analyse the expression of different members of the Myosin V family, namely MYO5A, 5B, and 5C, in CRC. Using laser-microdissected material as well as tissue microarrays from paired human CRC samples, we validated both RNA and protein expression of Myosin Vb (MYO5B) and its known adapter proteins (RAB8A and RAB25) in an independent patient cohort. Finally, we assessed the prognostic value of both MYO5B and its adapter-coupled combinatorial gene expression signatures. RESULTS: The meta-analysis as well as an independent patient cohort study revealed a methylation-independent loss of MYO5B expression in CRC that matched disease progression. Although MYO5B mutations were identified in a small number of patients, these cannot be solely responsible for the common downregulation observed in CRC patients. Significantly, CRC patients with low MYO5B expression displayed shorter overall, disease-, and metastasis-free survival, a trend that was further reinforced when RAB8A expression was also taken into account. CONCLUSIONS: Our data identify MYO5B as a powerful prognostic biomarker in CRC, especially in early stages (stages I and II), which might help stratifying patients with stage II for adjuvant chemotherapy.


Assuntos
Neoplasias Colorretais/genética , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Recidiva Local de Neoplasia/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Biologia Computacional , Metilação de DNA , Transição Epitelial-Mesenquimal , Humanos , Mutação , Cadeias Pesadas de Miosina/análise , Miosina Tipo V/análise , Prognóstico , Análise Serial de Tecidos , Proteínas rab de Ligação ao GTP/genética
3.
Nat Metab ; 4(4): 458-475, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35437333

RESUMO

The gut microbiome is a key player in the immunomodulatory and protumorigenic microenvironment during colorectal cancer (CRC), as different gut-derived bacteria can induce tumour growth. However, the crosstalk between the gut microbiome and the host in relation to tumour cell metabolism remains largely unexplored. Here we show that formate, a metabolite produced by the CRC-associated bacterium Fusobacterium nucleatum, promotes CRC development. We describe molecular signatures linking CRC phenotypes with Fusobacterium abundance. Cocultures of F. nucleatum with patient-derived CRC cells display protumorigenic effects, along with a metabolic shift towards increased formate secretion and cancer glutamine metabolism. We further show that microbiome-derived formate drives CRC tumour invasion by triggering AhR signalling, while increasing cancer stemness. Finally, F. nucleatum or formate treatment in mice leads to increased tumour incidence or size, and Th17 cell expansion, which can favour proinflammatory profiles. Moving beyond observational studies, we identify formate as a gut-derived oncometabolite that is relevant for CRC progression.


Assuntos
Neoplasias Colorretais , Microbioma Gastrointestinal , Animais , Bactérias , Neoplasias Colorretais/metabolismo , Formiatos , Fusobacterium nucleatum , Humanos , Camundongos , Microambiente Tumoral
4.
Autophagy ; 16(8): 1436-1452, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31775562

RESUMO

In solid tumors, cancer stem cells (CSCs) or tumor-initiating cells (TICs) are often found in hypoxic niches. Nevertheless, the influence of hypoxia on TICs is poorly understood. Using previously established, TIC-enrichedpatient-derived colorectal cancer (CRC) cultures, we show that hypoxia increases the self-renewal capacity of TICs while inducing proliferation arrest in their more differentiated counterpart cultures. Gene expression data revealed macroautophagy/autophagy as one of the major pathways induced by hypoxia in TICs. Interestingly, hypoxia-induced autophagy was found to induce phosphorylation of EZR (ezrin) at Thr567 residue, which could be reversed by knocking down ATG5, BNIP3, BNIP3L, or BECN1. Furthermore, we identified PRKCA/PKCα as a potential kinase involved in hypoxia-induced autophagy-mediated TIC self-renewal. Genetic targeting of autophagy or pharmacological inhibition of PRKC/PKC and EZR resulted in decreased tumor-initiating potential of TICs. In addition, we observed significantly reduced in vivo tumor initiation and growth after a stable knockdown of ATG5. Analysis of human CRC samples showed that p-EZR is often present in TICs located in the hypoxic and autophagic regions of the tumor. Altogether, our results establish the hypoxia-autophagy-PKC-EZR signaling axis as a novel regulatory mechanism of TIC self-renewal and CRC progression. Autophagy inhibition might thus represent a promising therapeutic strategy for cancer patients. ABBREVIATIONS: ATG: autophagy related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CQ: chloroquine; CSC: cancer stem cells; CRC: colorectal cancer; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PRKC/PKC: protein kinase C; SQSTM1/p62: sequestosome 1; TICs: tumor-initiating cells.


Assuntos
Carcinogênese/patologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Proteínas do Citoesqueleto/metabolismo , Progressão da Doença , Hipóxia/complicações , Proteína Quinase C/metabolismo , Transdução de Sinais , Animais , Autofagossomos/metabolismo , Autofagia , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/metabolismo , Autorrenovação Celular , Colo/patologia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Fosforilação
5.
Cancer Lett ; 450: 32-41, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-30790680

RESUMO

Cancer stem cells, also known as tumor-initiating cells (TICs), are a population of aggressive and self-renewing cells that are responsible for the initiation and progression of many cancers, including colorectal carcinoma. Intratumoral hypoxia, i.e. reduced oxygen supply following uncontrolled proliferation of cancer cells, is thought to support TIC activity by inducing specific hypoxia-responsive mechanisms that are not yet entirely understood. Using previously established and fully characterized patient-derived TIC cultures, we could observe increased sphere and colony formation under hypoxic conditions. Mechanistically, microRNA (miRNA)-profiling experiments allowed us to identify miR-215 as one of the main hypoxia-induced miRNAs in primary colon TICs. Through stable overexpression of miR-215, followed by a set of functional in vitro and in vivo investigations, miR-215 was pinpointed as a negative feedback regulator, working against the TIC-promoting effects of hypoxia. Furthermore, we could single out LGR5, a bona fide marker of non-neoplastic intestinal stem cells, as a downstream target of hypoxia/miR-215 signaling. The strong tumor- and TIC-suppressor potential of miR-215 and the regulatory role of the hypoxia/miR-215/LGR5 axis may thus represent interesting points of attack for the development of innovative anti-CSC therapy approaches.


Assuntos
Hipóxia Celular/fisiologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Neoplasias do Colo/genética , Genes Supressores de Tumor , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Esferoides Celulares , Células Tumorais Cultivadas , Regulação para Cima
6.
Cancer Res ; 78(14): 3793-3808, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29748374

RESUMO

The vast majority of colorectal cancer-related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different colorectal cancer cell lines and recently established primary cultures enriched in colon cancer stem cells, also known as tumor-initiating cells (TIC), to identify genes and miRNAs with regulatory functions in colorectal cancer progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, TGFß signaling activity, and reduced expression of the miR-371∼373 cluster compared with nonmetastatic cultures. TGFß receptor 2 (TGFBR2) and aldehyde dehydrogenase A1 (ALDH1A1) were identified as important target genes of the miR-371∼373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371∼373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced in vitro self-renewal activity as well as lowered tumor initiation and metastatic outgrowth capacity in vivo following stable overexpression of the miR-371∼373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371∼373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371∼373 and ID1. Altogether, our results establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization.Significance: These findings establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel mechanism regulating self-renewal of tumor-initiating cell and metastatic colonization, potentially opening new concepts for therapeutic targeting of cancer metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg Cancer Res; 78(14); 3793-808. ©2018 AACR.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteína 1 Inibidora de Diferenciação/genética , MicroRNAs/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais/genética , Animais , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/patologia
8.
Cell Signal ; 29: 138-149, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27777072

RESUMO

Mutations in KIT or PDGFRA are responsible for >85% of gastrointestinal stromal tumors. The introduction of imatinib in the GIST therapy scheme revolutionized the patient outcome. Unfortunately, the therapy allows the disease stabilization instead of curation. Furthermore the resistance to the inhibitor arises in most cases within two first years of therapy. A thorough investigation of the signalling pathways activated by the major PDGFRA and KIT mutants encountered in the GIST landscape allowed to identify striking differences between the two receptor tyrosine kinases. PDGFRA mutants were not responsive to their ligand, PDGFAA, and displayed a high constitutive kinase activity. In contrast, all KIT mutants retained, in addition to their constitutive activation, the ability to be stimulated by their ligand. Kit mutants displayed a lower intrinsic kinase activity relative to PDGFRA mutants, while the KIT Exon 11 deletion mutant exhibited the highest intrinsic kinase activity among KIT mutants. At the transcriptomic level, the MAPK pathway was established as the most prominent activated pathway, which is commonly up-regulated by all PDGFRA and KIT mutants. Inhibition of this pathway, using the MEK inhibitor PD0325901, reduced the proliferation of GIST primary cells at nanomolar concentrations. Altogether, our data demonstrate the high value of MEK inhibitors for combination therapy in GIST treatment and more importantly the interest of evaluating the SCF expression profile in GIST patients presenting KIT mutations.


Assuntos
Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Transdução de Sinais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Tumores do Estroma Gastrointestinal/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Simulação de Dinâmica Molecular , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
9.
Artigo em Inglês | MEDLINE | ID: mdl-16869093

RESUMO

Prostate cancer is one of the most common cancers among men and has long been recognized to occur in familial clusters. Identification of genetic susceptibility loci for prostate cancer has however been extremely difficult, and only in 1996 was the first prostate cancer susceptibility locus HPC1 mapped to chromosome 1q24-25. Since, several additional putative loci have been identified by genetic linkage analysis on chromosome 1, 17, 20 and X (reviewed in). For three of these loci, family-based studies have identified three genes associated with inherited prostate cancer: the 3' processing endoribonuclease ELAC2/HPC2 gene, the macrophage scavenger receptor 1 gene (MSR1), and the endoribonuclease RNase L gene (RNAse L/HPC1). Here we will focus our review on the RNAse L gene and its involvement in prostate cancer and other diseases.


Assuntos
Endorribonucleases/genética , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Predisposição Genética para Doença , Humanos , Masculino
10.
Data Brief ; 9: 828-838, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27872880

RESUMO

The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.

11.
Int J Oncol ; 26(6): 1549-58, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15870868

RESUMO

A major technical challenge related to gene expression profiling of tissue samples is the difficulty of procuring selected cell populations from tissues that by nature are heterogeneous, such as prostate tissue. In this study we have examined the expression of integrin-linked kinase (ILK) mRNA in prostate adenocarcinoma cells versus normal prostate epithelial cells in order to determine whether ILK could be used as a reference marker gene for prostate adenocarcinoma cell mRNA isolation. Using laser microdissection (LMD) technology and real-time PCR, together with immunohistochemistry, we have analyzed ILK mRNA expression in epithelial cells isolated from frozen prostate biopsy specimens as well as 4 prostate cell lines (RWPE-1, LNCaP, PC-3 and DU 145) and correlated ILK mRNA expression with ILK protein expression. We demonstrate that quantitative upregulation of ILK mRNA expression in prostate epithelial cells derived from prostate tissue correlated with ILK protein expression and with the histopathology diagnosis of prostate adenocarcinoma. We further show that the level of ILK overexpression was directly influenced by the method used to isolate prostate adenocarcinoma cells (bulk tissue versus LMD dissected cells). These data provide evidence that ILK mRNA is quantitatively upregulated in prostate adenocarcinoma cells versus normal epithelial cells and is therefore a useful internal reference gene marker to evaluate the quality of prostate adenocarcinoma cell derived mRNA used for large scale prostate cancer cDNA gene profiling.


Assuntos
Adenocarcinoma/genética , Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/análise , Adenocarcinoma/química , Biomarcadores Tumorais , Biópsia , Western Blotting , Linhagem Celular Tumoral , Secções Congeladas , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Microdissecção , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/química , Proteínas Serina-Treonina Quinases/análise , Controle de Qualidade
12.
Biochem Pharmacol ; 66(8): 1475-83, 2003 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-14555224

RESUMO

Expression of glutathione S-transferase P1-1 (GSTP1-1) is correlated to carcinogenesis and resistance of cancer cells against chemotherapeutic agents. Curcumin, a natural compound extracted from Curcuma longa, has shown strong antioxidant and anticancer properties and also the ability to regulate a wide variety of genes that require activating protein 1 and nuclear factor kappaB (NF-kappaB) activation. In the present study, we examined the inhibitory effect of curcumin on the expression of GSTP1-1 mRNA as well as protein, and we correlated this inhibition with the apoptotic effect of curcumin on K562 leukemia cells. Curcumin efficiently inhibited the tumour necrosis factor alpha- and phorbol ester-induced binding of AP-1 and NF-kappaB transcription factors to sites located on the GSTP1-1 gene promoter. TNFalpha-induced GSTP1-1 promoter activity was also inhibited by curcumin as shown by reporter gene assay. In parallel, curcumin induced pro-caspases 8 and 9 as well as poly ADP ribose polymerase cleavage and thus leading to apoptosis in K562 cells. Our results overall add a novel role for curcumin as this chemoprotective compound could contribute to induce apoptosis by its ability to inhibit the GSTP1-1 expression at the level of transcription.


Assuntos
Apoptose , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Interações Medicamentosas , Glutationa S-Transferase pi , Humanos , Células K562 , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
13.
Chem Commun (Camb) ; (10): 1086-7, 2002 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-12122677

RESUMO

Under visible irradiation, [Ru(TAP)2(phen)]2+(Cl-)2, [Ru(TAP)2(POQ-Nmet)]2+(Cl-)2 and [Ru(bpy)2(phen)]2+(Cl-)2 were able to dramatically reduce the in vitro transcription of a plasmid DNA template by a bacteriophage RNA polymerase. This photoactivity is related to two different mechanisms of reactivity towards DNA exhibited by these complexes under illumination.


Assuntos
Compostos Aza/farmacologia , Compostos de Rutênio/farmacologia , Transcrição Gênica/efeitos dos fármacos , Compostos Aza/química , DNA/química , Plasmídeos , Compostos de Rutênio/química
14.
Int J Cancer ; 120(6): 1284-92, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17163422

RESUMO

The EGF/IGF growth factors are potent mitogens that regulate cell proliferation and cell survival and are involved in prostate cancer development. Using laser microdissection technology and real-time PCR, together with immunohistochemistry, we have explored the growth factor and integrin dependent PI3-kinase/PTEN/Akt signalling pathway in prostate cell lines and tumour samples by analysing EGF-R, IGF1-R, ILK, beta3 integrin, PTEN and p-Akt protein expression. We provide evidence that loss of PTEN expression rather than upregulated EGF/IGF1 receptor expression was responsible for increased p-Akt in neoplastic prostate cells. We therefore compared PTEN expression in patient biopsies at first time diagnosis recruited prospectively (Study I, 112 patients) and patients with confirmed metastasis recruited retrospectively from the Luxembourg cancer registry (Study II, 42 patients). In Study I, loss of PTEN expression at first time diagnosis was found in 26 of 112 patients (23%). In Study II, 25 of the 42 patients (59%) with lymph node metastasis had complete loss of PTEN expression in both the neoplastic glands of the prostate and the invasive prostate cancer cells in the lymph node, and of these 13 (52%) exhibited already loss of PTEN expression at first diagnosis. These findings demonstrate that loss of PTEN expression is an important factor in progression towards metastatic disease and could potentially serve as an early prognostic marker for prostate cancer metastasis.


Assuntos
Adenocarcinoma/secundário , Biomarcadores Tumorais/análise , PTEN Fosfo-Hidrolase/análise , Próstata/patologia , Neoplasias da Próstata/patologia , Adenocarcinoma/química , Linhagem Celular Tumoral , Receptores ErbB/genética , Humanos , Imuno-Histoquímica , Integrina beta3/genética , Masculino , Fosfatidilinositol 3-Quinases/análise , Prognóstico , Próstata/química , Neoplasias da Próstata/química , Proteínas Proto-Oncogênicas c-akt/análise , RNA Mensageiro/análise , Receptor IGF Tipo 1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
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