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1.
Nucleic Acids Res ; 50(14): 7938-7958, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35871293

RESUMO

Although originally described as transcriptional activator, SPI1/PU.1, a major player in haematopoiesis whose alterations are associated with haematological malignancies, has the ability to repress transcription. Here, we investigated the mechanisms underlying gene repression in the erythroid lineage, in which SPI1 exerts an oncogenic function by blocking differentiation. We show that SPI1 represses genes by binding active enhancers that are located in intergenic or gene body regions. HDAC1 acts as a cooperative mediator of SPI1-induced transcriptional repression by deacetylating SPI1-bound enhancers in a subset of genes, including those involved in erythroid differentiation. Enhancer deacetylation impacts on promoter acetylation, chromatin accessibility and RNA pol II occupancy. In addition to the activities of HDAC1, polycomb repressive complex 2 (PRC2) reinforces gene repression by depositing H3K27me3 at promoter sequences when SPI1 is located at enhancer sequences. Moreover, our study identified a synergistic relationship between PRC2 and HDAC1 complexes in mediating the transcriptional repression activity of SPI1, ultimately inducing synergistic adverse effects on leukaemic cell survival. Our results highlight the importance of the mechanism underlying transcriptional repression in leukemic cells, involving complex functional connections between SPI1 and the epigenetic regulators PRC2 and HDAC1.


Assuntos
Histona Desacetilase 1 , Leucemia Eritroblástica Aguda , Complexo Repressor Polycomb 2 , Proteínas Proto-Oncogênicas , Transativadores , Acetilação , Animais , Cromatina/genética , Histona Desacetilase 1/genética , Leucemia Eritroblástica Aguda/genética , Camundongos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Transativadores/genética
2.
Haematologica ; 102(11): 1850-1860, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28912174

RESUMO

Transcriptional deregulation caused by epigenetic or genetic alterations is a major cause of leukemic transformation. The Spi1/PU.1 transcription factor is a key regulator of many steps of hematopoiesis, and limits self-renewal of hematopoietic stem cells. The deregulation of its expression or activity contributes to leukemia, in which Spi1 can be either an oncogene or a tumor suppressor. Herein we explored whether cellular senescence, an anti-tumoral pathway that restrains cell proliferation, is a mechanism by which Spi1 limits hematopoietic cell expansion, and thus prevents the development of leukemia. We show that Spi1 overexpression triggers cellular senescence both in primary fibroblasts and hematopoietic cells. Erythroid and myeloid lineages are both prone to Spi1-induced senescence. In hematopoietic cells, Spi1-induced senescence requires its DNA-binding activity and a functional p38MAPK14 pathway but is independent of a DNA-damage response. In contrast, in fibroblasts, Spi1-induced senescence is triggered by a DNA-damage response. Importantly, using our well-established Spi1 transgenic leukemia mouse model, we demonstrate that Spi1 overexpression also induces senescence in erythroid progenitors of the bone marrow in vivo before the onset of the pre-leukemic phase of erythroleukemia. Remarkably, the senescence response is lost during the progression of the disease and erythroid blasts do not display a higher expression of Dec1 and CDKN1A, two of the induced senescence markers in young animals. These results bring indirect evidence that leukemia develops from cells which have bypassed Spi1-induced senescence. Overall, our results reveal senescence as a Spi1-induced anti-proliferative mechanism that may be a safeguard against the development of acute myeloid leukemia.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Animais , Biomarcadores , Medula Óssea/metabolismo , Medula Óssea/patologia , Linhagem Celular , Proliferação de Células , Senescência Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Ectópica do Gene , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo
3.
Kidney Int ; 88(2): 299-310, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26039630

RESUMO

The ankyrin repeat and sterile α motif (SAM) domain-containing six gene (Anks6) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.


Assuntos
Proteínas de Transporte/genética , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Rim/metabolismo , Rim/patologia , Proteínas Nucleares/genética , Animais , Repetição de Anquirina , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Feminino , Homozigoto , Humanos , Rim/embriologia , Doenças Renais Císticas/fisiopatologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , Alça do Néfron/metabolismo , Alça do Néfron/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Fenótipo , Podócitos/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos
4.
Oncogene ; 42(37): 2764-2775, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37573408

RESUMO

Leukaemia is caused by the clonal evolution of a cell that accumulates mutations/genomic rearrangements, allowing unrestrained cell growth. However, recent identification of leukaemic mutations in the blood cells of healthy individuals revealed that additional events are required to expand the mutated clones for overt leukaemia. Here, we assessed the functional consequences of deleting the Fanconi anaemia A (Fanca) gene, which encodes a DNA damage response protein, in Spi1 transgenic mice that develop preleukaemic syndrome. FANCA loss increases SPI1-associated disease penetrance and leukaemic progression without increasing the global mutation load of leukaemic clones. However, a high frequency of leukaemic FANCA-depleted cells display heterozygous activating mutations in known oncogenes, such as Kit or Nras, also identified but at low frequency in FANCA-WT mice with preleukaemic syndrome, indicating that FANCA counteracts the emergence of oncogene mutated leukaemic cells. A unique transcriptional signature is associated with the leukaemic status of FANCA-depleted cells, leading to activation of MDM4, NOTCH and Wnt/ß-catenin pathways. We show that NOTCH signalling improves the proliferation capacity of FANCA-deficient leukaemic cells. Collectively, our observations indicate that loss of the FANC pathway, known to control genetic instability, fosters the expansion of leukaemic cells carrying oncogenic mutations rather than mutation formation. FANCA loss may contribute to this leukaemogenic progression by reprogramming transcriptomic landscape of the cells.


Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi , Leucemia , Animais , Camundongos , Heterozigoto , Leucemia/genética , Mutação , Oncogenes/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética
5.
Biochem J ; 437(1): 75-88, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21473742

RESUMO

RhoH is a member of the Rho family of small GTP-binding proteins that lacks GTPase activity. Since RhoH is constantly bound by GTP, it is thought to be constitutively active and controlled predominantly by changes in quantitative expression. RhoH is produced specifically in haematopoietic cells and aberrant expression has been linked to various forms of leukaemia. Transcription of the RHOH gene is the first level at which the quantitative levels of the RhoH protein are regulated. Previous studies have demonstrated that RHOH gene transcription is initiated by three distinct promoter regions designated P1, P2 and P3 that define the 5' end of exons 1, 2 and 4 respectively. In the present study we report that the P3 promoter is largely responsible for RHOH gene transcription in the B-lymphocytic cell line Raji. The P3 promoter contains a minimal promoter region and a repressor region extending from -236 to +67 and +68 to +245 respectively, relative to the 5' end of exon 4. Chromatin immunoprecipitation demonstrated that two AP1 (activator protein 1) sites in the minimal promoter region bind JunD. When JUND is overexpressed, the endogenous RHOH gene is repressed; however, when JUND is inhibited, expression of endogenous RHOH is induced both in the Raji cell line and AML (acute myeloid leukaemia) cells. In the HCL (hairy cell leukaemia) cell line JOK-1, induction of RHOH increases expression of the α isoform of protein kinase C. This downstream target of RHOH is also induced in AML cells by JUND inhibition. Collectively, these data indicate that JunD is an inhibitor of RHOH gene expression.


Assuntos
Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/genética , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Células HeLa , Humanos , Leucemia Mieloide Aguda/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Transfecção , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
6.
Oncotarget ; 8(23): 37104-37114, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28415748

RESUMO

Oncogenes trigger replicative stress that can lead to genetic instability, which participates in cancer progression. Thus, determining how cells cope with replicative stress can help our understanding of oncogenesis and lead to the identification of new antitumor treatment targets. We previously showed that constitutive overexpression of the oncogenic transcription factor Spi1/PU.1 leads to pre-leukemic cells that have a shortened S phase duration with an increased replication fork speed and increased mutability in the absence of DNA breaks. Here, we demonstrate that the S phase checkpoint protein CHK1 is maintained in a low phosphorylation state in Spi1/PU.1-overexpressing cells and provide evidence that this is not due to negative control of its primary kinase ATR. Notably, we found that the expression of the CHK1 phosphatase PP1α is increased in Spi1/PU.1-overexpressing cells. By exogenously modulating its activity, we demonstrate that PP1α is required to maintain CHK1 in a dephosphorylated state and, more importantly, that it is responsible for the accelerated replication fork progression in Spi1/PU.1-overexpressing cells. These results identify a novel pathway by which an oncogene influences replication in the absence of DNA damage.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Replicação do DNA , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Ciclo Celular/genética , Células Cultivadas , Quinase 1 do Ponto de Checagem/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Células K562 , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Camundongos Transgênicos , Fosforilação , Proteína Fosfatase 1/genética , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Transativadores/genética
7.
Anticancer Res ; 35(11): 5983-91, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26504021

RESUMO

BACKGROUND: The combination of irinotecan, a topoisomerase I inhibitor with cetuximab, an antibody against epidermal growth factor receptor, produces synergistic and beneficial effects in patients with irinotecan-refractory colorectal cancer. Our hypothesis was that synergistic effects could be due to anti-angiogenesis and anti-invasion, but not to cytotoxicity. MATERIALS AND METHODS: Cytotoxicity was assessed by viability test and flow cytometry. Anti-angiogenesis, anti-invasion were studied by the endothelial cell capillary-like network formation and transmigration through an extracellular matrix. Protein kinase B (PKB, frequently cited as AKT), and extracellular signal-regulated kinases (ERK) activation was assayed by cell-based enzyme-linked immunosorbent assay (ELISA). RESULTS: Combinations of SN-38 (the active of irinotecan) and cetuximab did not induce any synergistic cytotoxicity confirmed by viability test and cell-cycle analyses. Interestingly, their combination produced synergistic anti-angiogenesis and anti-invasion activities revealed by endothelial cell capillary-like network formation and cell invasion tests. Subsequently, their combination attenuated either expression or phosphorylation of AKT and ERK1/2 using cell-based ELISA. CONCLUSION: SN-38/cetuximab combination has synergistic anti-angiogenesis and anti-invasion activities mediated by down-regulation of phosphatidylinositol-3-kinases/AKT and mitogen-activated protein kinase/ERK pathways.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Sinergismo Farmacológico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cetuximab/administração & dosagem , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Citometria de Fluxo , Humanos , Irinotecano , Invasividade Neoplásica , Neovascularização Patológica/patologia , Fosforilação/efeitos dos fármacos
8.
PLoS One ; 10(9): e0136781, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26327442

RESUMO

Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.


Assuntos
Repetição de Anquirina/genética , Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Rim/metabolismo , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia , Vasopressinas/metabolismo , Motivos de Aminoácidos/genética , Animais , Apoptose/genética , Proteínas de Transporte/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Cílios/genética , Regulação para Baixo/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Ligação Proteica/genética , Transdução de Sinais/genética , Vasopressinas/genética
9.
Cancer Res ; 68(12): 4531-40, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18559497

RESUMO

The cause of hairy cell leukemia (HCL) is unknown. Current treatments seem effective only for a limited period of time. In addition, a significant proportion of patients remain refractive to all treatment options. These considerations indicate the need to develop alternative therapeutic strategies for HCL. Here, we report that HCL is characterized by underexpression of RhoH. In vitro reconstitution of RhoH expression inhibits the aberrant adhesion and transendothelial migration that drives disease pathogenesis. In an in vivo model of HCL, RhoH reconstitution limits malignant progression and protects against mortality. These findings provide the proof of principle that RhoH reconstitution represents a potential new approach to the treatment of HCL.


Assuntos
Leucemia de Células Pilosas/metabolismo , Neoplasias Esplênicas/metabolismo , Trombocitemia Essencial/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Western Blotting , Antígeno CD11c/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Doença Crônica , Citometria de Fluxo , Humanos , Leucemia de Células Pilosas/genética , Leucemia de Células Pilosas/patologia , Leucócitos , Linfócitos/metabolismo , Linfócitos/patologia , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Masculino , Camundongos , Camundongos SCID , Regiões Promotoras Genéticas , Baço/metabolismo , Baço/patologia , Neoplasias Esplênicas/genética , Neoplasias Esplênicas/patologia , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologia , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rho de Ligação ao GTP/genética
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