RESUMO
Erythropoietin (EPO) provides the major survival signal to maturing erythroid precursors (EPs) and is essential for terminal erythropoiesis. Nonetheless, progenitor cells can irreversibly commit to an erythroid fate well before EPO acts, risking inefficiency if these progenitors are unneeded to maintain red blood cell (RBC) counts. We identified a new modular organization of erythropoiesis and, for the first time, demonstrate that the pre-EPO module is coupled to late EPO-dependent erythropoiesis by megakaryocyte (Mk) signals. Disrupting megakaryocytic transforming growth factor ß1 (Tgfb1) disorganized hematopoiesis by expanding the pre-EPO pool of progenitor cells and consequently triggering significant apoptosis of EPO-dependent EPs. Similarly, pharmacologic blockade of TGFß signaling in normal mice boosted the pre-EPO module, leading to apoptosis of EPO-sensitive EPs. Subsequent treatment with low-dose EPO triggered robust RBC production in both models. This work reveals modular regulation of erythropoiesis and offers a new strategy for overcoming chronic anemias.
Assuntos
Células Precursoras Eritroides/citologia , Eritropoese/fisiologia , Megacariócitos/citologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Células Precursoras Eritroides/metabolismo , Eritropoetina/farmacologia , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quimera por Radiação , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow-derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.
Assuntos
Hepcidinas/metabolismo , Macrófagos Alveolares/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumar/metabolismo , Animais , Medula Óssea/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Fumar Cigarros/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Eritropoetina/metabolismo , Humanos , Ferro/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , FumaçaRESUMO
Trabectedin is a marine natural product, approved in Europe for the treatment of soft tissue sarcoma and relapsed ovarian cancer. Clinical and experimental evidence indicates that trabectedin is particularly effective against myxoid liposarcomas where response is associated to regression of capillary networks. Here, we investigated the mechanism of the antiangiogenic activity of trabectedin in myxoid liposarcomas. Trabectedin directly targeted endothelial cells, impairing functions relying on extracellular matrix remodeling (invasion and branching morphogenesis) through the upregulation of the inhibitors of matrix metalloproteinases TIMP-1 and TIMP-2. Increased TIMPs synthesis by the tumor microenvironment following trabectedin treatment was confirmed in xenograft models of myxoid liposarcoma. In addition, trabectedin upregulated tumor cell expression of the endogenous inhibitor thrombospondin-1 (TSP-1, a key regulator of angiogenesis-dependent dormancy in sarcoma), in in vivo models of myxoid liposarcomas, in vitro cell lines and primary cell cultures from patients' myxoid liposarcomas. Chromatin Immunoprecipitation analysis showed that trabectedin displaced the master regulator of adipogenesis C/EBPß from the TSP-1 promoter, indicating an association between the up-regulation of TSP-1 and induction of adipocytic differentiation program by trabectedin. We conclude that trabectedin inhibits angiogenesis through multiple mechanisms, including directly affecting endothelial cells in the tumor microenvironment--with a potentially widespread activity--and targeting tumor cells' angiogenic activity, linked to a tumor-specific molecular alteration.
Assuntos
Inibidores da Angiogênese/farmacologia , Dioxóis/farmacologia , Lipossarcoma Mixoide/tratamento farmacológico , Tetra-Hidroisoquinolinas/farmacologia , Trombospondina 1/fisiologia , Inibidor Tecidual de Metaloproteinase-1/fisiologia , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Feminino , Humanos , Lipossarcoma Mixoide/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , TrabectedinaRESUMO
We recently defined a critical role for p53 in regulating the quiescence of adult hematopoietic stem cells (HSCs) and identified necdin as a candidate p53 target gene. Necdin is a growth-suppressing protein and the gene encoding it is one of several that are deleted in patients with Prader-Willi syndrome. To define the intrinsic role of necdin in adult hematopoiesis, in the present study, we transplanted necdin-null fetal liver cells into lethally irradiated recipients. We show that necdin-null adult HSCs are less quiescent and more proliferative than normal HSCs, demonstrating the similar role of necdin and p53 in promoting HSC quiescence during steady-state conditions. However, wild-type recipients repopulated with necdin-null hematopoietic stem/progenitor cells show enhanced sensitivity to irradiation and chemotherapy, with increased p53-dependent apoptosis, myelosuppression, and mortality. Necdin controls the HSC response to genotoxic stress via both cell-cycle-dependent and cell-cycle-independent mechanisms, with the latter occurring in a Gas2L3-dependent manner. We conclude that necdin functions as a molecular switch in adult hematopoiesis, acting in a p53-like manner to promote HSC quiescence in the steady state, but suppressing p53-dependent apoptosis in response to genotoxic stress.
Assuntos
Dano ao DNA , Hematopoese , Células-Tronco Hematopoéticas/citologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Tratamento Farmacológico , Deleção de Genes , Genes p53 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/efeitos da radiação , Fígado/citologia , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
PURPOSE: Goal of this study was to identify mechanisms that limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma (EWS), so as to develop a clinical applicable combination therapy. EXPERIMENTAL DESIGN: By chromatin immunoprecipitation, we analyzed EWS-FLI1 binding to the promoters of several target genes, such as TGFßR2, CD99, insulin-like growth factor receptor 1 (IGF1R), and IGF1, both in vitro and in xenografts treated with trabectedin or doxorubicin. Combined therapy with trabectedin and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in vitro and in xenografts. RESULTS: We confirm that both trabectedin and doxorubicin were able to strongly reduce EWS-FLI1 (both type I and type II) binding to two representative target genes (TGFßR2 and CD99), both in vitro and in xenografts. However, trabectedin, but not doxorubicin, was also able to increase the occupancy of EWS-FLI1 to IGF1R promoters, leading to IGF1R upregulation. Inhibition of IGF1R either by the specific AVE1642 human antibody or by the dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) greatly potentiate the efficacy of trabectedin in the 13 EWS cell lines here considered as well as in TC-71 and 6647 xenografts. Combined therapy induced synergistic cytotoxic effects. Trabectedin and OSI-906 deliver complementary messages that likely converge on DNA-damage response and repair pathways. CONCLUSIONS: We showed that trabectedin may not only inhibit but also enhance the binding of EWS-FLI1 to certain target genes, leading to upregulation of IGF1R. We here provide the rationale for combining trabectedin to anti-IGF1R inhibitors.
Assuntos
Dioxóis/farmacologia , Doxorrubicina/farmacologia , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Receptor IGF Tipo 1/antagonistas & inibidores , Sarcoma de Ewing/tratamento farmacológico , Tetra-Hidroisoquinolinas/farmacologia , Antígeno 12E7 , Animais , Antígenos CD/genética , Antineoplásicos Alquilantes/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Pirazinas/farmacologia , Receptor IGF Tipo 1/biossíntese , Receptor IGF Tipo 1/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , TrabectedinaRESUMO
BACKGROUND: The present study is aimed to identify genetic pathways correlated with chemoresistance in epithelial ovarian cancer (EOC). METHODS: We compared the molecular profiles of 23 tumour biopsies of stage III-IV (training set) at primary surgery, before chemotherapy, to the profile from the same patients at second surgery, after several lines of platinum (Pt)-based chemotherapy when the tumours were resistant. In the hypothesis that identified markers were related to Pt-resistance and to prognosis, we validated this signature in 52 EOC taken at primary surgery (validation set) selected to be either very sensitive to the first line therapy, i.e. not relapsing before one year from the end of therapy, or resistant, i.e. relapsing within 6 months from the end of therapy. RESULTS: In the training set, we identified a resistance signature indicative of the activation of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta pathway. We then validated this signature in 52 EOC taken at primary surgery (validation set). Some genes involved in EMT, such as BMP and activin membrane-bound inhibitor (BAMBI), and mir-141 resulted in association with overall or progression free survival. CONCLUSION: Some genes involved in EMT were associated to overall or progression free survival, suggesting EMT as vital to the resistance mechanisms.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Transição Epitelial-Mesenquimal/genética , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Adulto , Idoso , Biópsia , Carcinoma Epitelial do Ovário , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/genética , Compostos Organoplatínicos/administração & dosagem , Neoplasias Ovarianas/genética , Transdução de Sinais , Resultado do TratamentoRESUMO
The Polycomb group protein Bmi1 is a transcriptional silencer of the Ink4a-Arf locus, which encodes the cell cycle regulator p16(Ink4a) and the tumor suppressor p19(Arf). Bmi1 plays a key role in oncogenesis and stem cell self-renewal. We report that phosphorylation of human Bmi1 at Ser³¹6 by Akt impaired its function by triggering its dissociation from the Ink4a-Arf locus, which resulted in decreased ubiquitylation of histone H2A and the inability of Bmi1 to promote cellular proliferation and tumor growth. Moreover, Akt-mediated phosphorylation of Bmi1 also inhibited its ability to promote self-renewal of hematopoietic stem and progenitor cells. Our study provides a mechanism for the increased abundance of p16(Ink4a) and p19(Arf) seen in cancer cells with an activated phosphoinositide 3-kinase to Akt signaling pathway and identifies crosstalk between phosphorylation events and chromatin structure.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Loci Gênicos , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Cromatina/genética , Cromatina/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inativação Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Ubiquitinação/genéticaRESUMO
The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway is a major target for cancer therapy. As a strategy to induce the maximal inhibition of this pathway in cancer cells, we combined allosteric mTOR inhibitors (rapamycin and RAD001) with a dual PI3K/mTOR kinase inhibitor (PI-103). Both in vitro and in vivo, the combination exhibited more activity than single agents in human ovarian and prostate cancer cells that harbor alterations in the pathway. At the molecular level, combined inhibition of mTOR prevented the rebound activation of Akt that is seen after treatment with rapamycin and its analogues and caused more sustained inhibition of Akt phosphorylation. Furthermore, the combination strongly inhibited the expression of PI3K/Akt/mTOR downstream proteins. In particular, it showed greater activity than the single agents in inhibiting the phosphorylation of 4EBP1, both in vitro and in vivo, resulting in selective inhibition of CAP-dependent translation. A proteomic approach was used to confirm the identification of c-Myc as the key regulator for the reduction in downstream proteins affected by the combined inhibition of mTOR. In conclusion, the combination of a catalytic and an allosteric inhibitor of mTOR shows greater activity, without a concomitant increase in toxicity, than either drug alone, and this may have therapeutic implications for inhibiting this pathway in the clinical setting.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Esquema de Medicação , Sinergismo Farmacológico , Feminino , Furanos/administração & dosagem , Furanos/farmacologia , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Nus , Complexos Multiproteicos , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/administração & dosagem , Piridinas/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Sirolimo/administração & dosagem , Sirolimo/farmacologiaRESUMO
The ATM kinase plays a critical role in initiating the DNA damage response that is triggered by genotoxic stresses capable of inducing DNA double-strand breaks. Here, we show that ELF4/MEF, a member of the ETS family of transcription factors, contributes to the persistence of γH2AX DNA damage foci and promotes the DNA damage response leading to the induction of apoptosis. Conversely, the absence of ELF4 promotes the faster repair of damaged DNA and more rapid disappearance of γH2AX foci in response to γ-irradiation, leading to a radio-resistant phenotype despite normal ATM phosphorylation. Following γ-irradiation, ATM phosphorylates ELF4, leading to its degradation; a mutant form of ELF4 that cannot be phosphorylated by ATM persists following γ-irradiation, delaying the resolution of γH2AX foci and triggering an excessive DNA damage response. Thus, although ELF4 promotes the phosphorylation of H2AX by ATM, its activity must be dampened by ATM-dependent phosphorylation and degradation to avoid an excessive DNA damage response.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Ativação Enzimática , Raios gama , Células HEK293 , Histonas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células NIH 3T3 , Fosforilação/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/efeitos da radiação , Proteínas Supressoras de Tumor/metabolismoRESUMO
In response to diverse stresses, the tumor suppressor p53 differentially regulates its target genes, variably inducing cell-cycle arrest, apoptosis or senescence. Emerging evidence indicates that p53 plays an important role in regulating hematopoietic stem cell (HSC) quiescence, self-renewal, apoptosis and aging. The p53 pathway is activated by DNA damage, defects in ribosome biogenesis, oxidative stress and oncogene induced p19 ARF upregulation. We present an overview of the current state of knowledge about p53 (and its target genes) in regulating HSC behavior, with the hope that understanding the molecular mechanisms that control p53 activity in HSCs and how p53 mutations affect its role in these events may facilitate the development of therapeutic strategies for eliminating leukemia (and cancer) propagating cells.
Assuntos
Células-Tronco Hematopoéticas/citologia , Proteína Supressora de Tumor p53/fisiologia , Apoptose , Ciclo Celular , Senescência Celular , Dano ao DNA , Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The importance of the p53 protein in the cellular response to DNA damage is well known, but its function during steady-state hematopoiesis has not been established. We have defined a critical role of p53 in regulating hematopoietic stem cell quiescence, especially in promoting the enhanced quiescence seen in HSCs that lack the MEF/ELF4 transcription factor. Transcription profiling of HSCs isolated from wild-type and p53 null mice identified Gfi-1 and Necdin as p53 target genes, and using lentiviral vectors to upregulate or knockdown the expression of these genes, we show their importance in regulating HSC quiescence. Establishing the role of p53 (and its target genes) in controlling the cell-cycle entry of HSCs may lead to therapeutic strategies capable of eliminating quiescent cancer (stem) cells.