RESUMO
BACKGROUND & AIMS: The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. The epigenetic mechanisms regulating CSCs are currently insufficiently understood, which hampers the development of novel strategies for eliminating CSCs. METHODS: By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodeling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signaling pathway. RESULTS: Inhibition and genetic ablation of BRD9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumors from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. CONCLUSIONS: Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Proteínas que Contêm Bromodomínio , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Gencitabina , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteína Smad2/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.
Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Mutação de Sentido Incorreto , Proteínas Circadianas Period/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Caseína Quinase 1 épsilon/genética , Caseína Quinase 1 épsilon/metabolismo , Chlorocebus aethiops , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Atividade Motora/genética , Atividade Motora/fisiologia , Proteínas Circadianas Period/química , Proteínas Circadianas Period/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiopatologiaRESUMO
Tissue formation and organ homeostasis is achieved by precise coordination of proliferation and differentiation of stem cells and progenitors. While deregulation of these processes can result in degenerative disease or cancer, their molecular interplays remain unclear. Here we show that the switch of human pluripotent stem cell (hPSC) self-renewal to differentiation is associated with the induction of distinct cyclin dependent kinase inhibitors (CDKIs). In hPSCs, Activin/Nodal/TGFß signalling maintains CDKIs in a poised state via SMAD2/3-NANOG-OCT4-EZH2-SNON transcriptional complex. Upon gradual differentiation, CDKIs are induced by successive transcriptional complexes between SMAD2/3-SMYD2 and developmental regulators such as EOMES, thereby lengthening the G1 phase. This, in turn, induces SMAD2/3 transcriptional activity by blocking its linker phosphorylation. Such SMAD2/3-CDKI positive feedback loops drive the exit from pluripotency and stepwise cell fate specification that could be harnessed for producing cells for therapeutic applications. Our study uncovers fundamental mechanisms how cell fate specification is interconnected to cell cycle dynamics and provides insight to autonomous circuitries governing tissue self-formation.
RESUMO
The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.
Assuntos
Fator de Transcrição E2F1 , Fator de Transcrição E2F4 , Células-Tronco Neoplásicas , Neoplasias Pancreáticas , Comunicação Parácrina , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F1/genética , Linhagem Celular Tumoral , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Fator de Transcrição E2F4/metabolismo , Fator de Transcrição E2F4/genética , Animais , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Proteína do Retinoblastoma/metabolismo , Proteína do Retinoblastoma/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fatores de Transcrição de p300-CBP/metabolismo , Fatores de Transcrição de p300-CBP/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Feminino , Proliferação de Células , Camundongos , Transdução de Sinais , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
The retinoblastoma family proteins (RBs) and E2F transcription factors are cell-autonomous regulators of cell-cycle progression, but they also impact fate choice in addition to tumor suppression. The range of mechanisms involved remains to be uncovered. Here, we show that RBs, particularly RBL2/p130, repress WNT ligands such as WNT4 and WNT8A, thereby directing ectoderm specification between neural crest to neuroepithelium. RBL2 achieves this function through cell-cycle-dependent cooperation with E2Fs and GCN5 on the regulatory regions of WNT loci, which direct neuroepithelial versus neural crest specification by temporal fluctuations of WNT/ß-catenin and DLL/NOTCH signaling activity. Thus, the RB-E2F bona fide cell-autonomous axis controls cell fate decisions, and RBL2 regulates field effects via WNT ligands. This reveals a non-cell-autonomous function of RBL2-E2F in stem cell and tissue progenitor differentiation that has broader implications for cell-cycle-dependent cell fate specification in organogenesis, adult stem cells, tissue homeostasis, and tumorigenesis.
Assuntos
Padronização Corporal , Proteína do Retinoblastoma , Transdução de Sinais , Humanos , Ciclo Celular , Diferenciação Celular , Divisão Celular , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismoRESUMO
Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.
Assuntos
Células-Tronco Pluripotentes , Humanos , Diferenciação Celular/genética , Regulação da Expressão Gênica , Antígenos de Diferenciação/metabolismo , Epigênese Genética , EndodermaRESUMO
Pancreatic cancer (PC), one of the most aggressive and life-threatening human malignancies, is known for its resistance to cytotoxic therapies. This is increasingly ascribed to the subpopulation of undifferentiated cells, known as pancreatic cancer stem cells (PCSCs), which display greater evolutionary fitness than other tumor cells to evade the cytotoxic effects of chemotherapy. PCSCs are crucial for tumor relapse as they possess 'stem cell-like' features that are characterized by self-renewal and differentiation. However, the molecular mechanisms that maintain the unique characteristics of PCSCs are poorly understood. Here, we identify the histone methyltransferase KMT2A as a physical binding partner of an RNA polymerase-associated PHF5A-PHF14-HMG20A-RAI1 protein subcomplex and an epigenetic regulator of PCSC properties and functions. Targeting the protein subcomplex in PCSCs with a KMT2A-WDR5 inhibitor attenuates their self-renewal capacity, cell viability, and in vivo tumorigenicity.
Assuntos
Pâncreas , Neoplasias Pancreáticas , Humanos , Células-Tronco Neoplásicas , Neoplasias Pancreáticas/genética , Pesquisadores , Histona Metiltransferases , Proteínas de Grupo de Alta Mobilidade , Transativadores , Proteínas de Ligação a RNA , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.
RESUMO
We investigated the expression of the PLZF gene in purified human hematopoietic progenitors induced to unilineage erythroid, granulocytic or megakaryocytic differentiation and maturation in serum-free culture. PLZF is expressed in quiescent progenitors: the expression level progressively rises through megakaryocytic development, whereas it gradually declines in erythroid and granulopoietic culture. To investigate the role of PLZF in megakaryopoiesis, we transduced the PLZF gene into the erythro-megakaryocytic TF1 cell line. PLZF overexpression upmodulates the megakaryocytic specific markers (CD42a, CD42b, CD61, PF4) and induces the thrombopoietin receptor (TpoR). The proximal promoter of the TpoR gene is activated in PLZF-expressing TF1 cells: in this promoter region, a PLZF DNA-binding site was identified by deletion constructs studies. Interestingly, PLZF and GATA1 proteins coimmunoprecipitate in PLZF-expressing TF1 cells: enforced expression of both PLZF and GATA1 in TF1 cells results in increased upregulation of megakaryocytic markers, as compared to exogenous PLZF or GATA1 alone, suggesting a functional role for the PLZF/GATA1 complex. Our data indicate that PLZF plays a significant stimulatory role in megakaryocytic development, seemingly mediated in part by induction of TpoR expression at transcriptional level. This stimulatory effect is potentiated by physical interaction of PLZF and GATA1, which are possibly assembled in a multiprotein transcriptional complex.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Megacariócitos/fisiologia , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/genética , Receptores de Citocinas , Fatores de Transcrição/fisiologia , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Humanos , Fatores de Transcrição Kruppel-Like , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Mensageiro/análise , Receptores de Trombopoetina , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 5 (IL-5) exert their biological activities through interaction with cell-surface receptors that consist of two subunits, a specific a subunit and a common beta transducing subunit (beta c). We have evaluated the effect of growth factors on the expression of beta c in normal monocytes. Addition of either GM-CSF or M-CSF to monocytes elicited a marked increase of beta c chain expression, a phenomenon seemingly related to a stimulation of the transcriptional activity of this gene mediated through an enhancement of the PU.1 DNA binding activity. Interestingly, during the activation of beta c chain expression by growth factors a switch from the synthesis of the truncated betaIT to the full-length beta c was observed. Similar observations have been made also in the growth factor-dependent erythroleukemic cell line TF-1, showing that GM-CSF deprivation elicited a marked decrease of beta c chain expression.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Interleucina-3/genética , Células Cultivadas , Humanos , Regiões Promotoras Genéticas , Subunidades Proteicas , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismoRESUMO
PURPOSE: This work investigates the hypothesis that genetic background plays a significant role in the signalling mechanisms underlying induction and perpetuation of genomic instability following radiation exposure. MATERIALS AND METHODS: Bone marrow from two strains of mice (CBA and C57) were exposed to a range of X-ray doses (0, 0.01, 0.1, 1 and 3 Gy). Different cellular signalling endpoints: Apoptosis, cytokine levels and calcium flux, were evaluated at 2 h, 24 h and 7 d post-irradiation to assess immediate and delayed effects. RESULTS: In CBA (radiosensitive) elevated apoptosis levels were observed at 24 h post X-irradiation, and transforming growth factor-ß (TGF-ß) levels which increased with time and dose. C57 showed a higher background level of apoptosis, and sustained apoptotic levels 7 days after radiation exposure. Levels of tumor necrosis factor-α (TNF-α were increased in C57 at day 7 for higher X-ray doses. TGF-ß levels were higher in CBA, whilst C57 exhibited a greater TNF-α response. Calcium flux was induced in reporter cells on exposure to conditioned media from both strains. CONCLUSIONS: These results show genetic and dose specific differences in radiation-induced signalling in the initiation and perpetuation of the instability process, which have potential implications on evaluation of non-targeted effects in radiation risk assessment.
Assuntos
Efeito Espectador/genética , Efeito Espectador/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos da radiação , Cálcio/metabolismo , Linhagem Celular , Relação Dose-Resposta à Radiação , Predisposição Genética para Doença , Transferência Linear de Energia , Masculino , Camundongos , Tolerância a Radiação/genética , Ratos , Especificidade da Espécie , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Raios X/efeitos adversosRESUMO
Interleukin-6 (IL-6) is a growth and survival factor in Epstein-Barr virus (EBV)-infected B lymphoma cells and IL-6 antagonists have been used in clinical practice for this pathology. We thus wanted to investigate the effect of the IL-6 receptor antagonist Sant7 on proliferative and anti-apoptotic signals in the IL-6-secreting LCL41 B lymphoid cells, taken from a patient with EBV-induced lymphoproliferative disorder. Results show efficient inhibition of constitutive Stat3 activation by Sant7. However, this inhibition is associated with marginal induction of apoptosis and with minor decrease of cell proliferation, contrary to the effect of the Jak kinase inhibitor AG490, which down-regulates both proliferation and Stat3 activation. Anti-apoptotic markers such as Bcl-xL or Mcl-1 are constitutively expressed in these cells, and their expression is not affected by Sant7 treatment. Inhibition of Stat3 activation is therefore not sufficient to prevent proliferation and to induce apoptosis in these cells. In addition, low cell density is a condition favouring inhibition of cell clustering and anti-proliferative Sant7 activity. A marked inhibition of cell cluster formation and proliferation is achieved by antibody treatment against the CD23 mature B cell surface marker expressed in LCL41 cells. These findings may thus contribute to the identification of possible resistance mechanisms to growth arrest in B cell lymphoproliferative conditions.
Assuntos
Apoptose/fisiologia , Divisão Celular/fisiologia , Interleucina-6/fisiologia , Linfoma de Células B/patologia , Receptores de IgE/fisiologia , Western Blotting , Linhagem Celular Tumoral , Citometria de Fluxo , HumanosRESUMO
We have investigated the expression of interleukin-3 receptor alpha (IL-3Ralpha) chain in primary blasts from 79 patients with acute myeloid leukemia (AML), 25 patients with B-acute lymphoid leukemia (B-ALL), and 7 patients with T-acute lymphoid leukemia (T-ALL) to evaluate a linkage between the expression of this receptor chain, blast proliferative status, and disease prognosis. Although IL-3Ralpha chain was scarcely expressed in most patients with T-ALL, it was overexpressed in 40% and 45% of patients with B-ALL and AML, respectively, compared with the levels observed in normal CD34(+) progenitors. The biological and clinical significance of this overexpression pattern was investigated in AML. At the biological level, elevated IL-3Ralpha expression was associated with peculiar properties of leukemic blasts, specifically in 3 areas. First, in all patients the blasts expressing elevated IL-3Ralpha levels exhibited higher cycling activity and increased resistance to apoptosis triggered by growth factor deprivation. Second, spontaneous signal transducer and activator of transcription 5 (Stat5) phosphorylation was observed in 13% of AML patients, all pertaining to the group of patients exhibiting high IL-3Ralpha expression. Third, following IL-3 treatment, Stat5 was activated at higher levels in blasts with elevated IL-3Ralpha expression. At the clinical level, a significant correlation was observed between the level of IL-3Ralpha expression and the number of leukemic blasts at diagnosis, and patients exhibiting elevated IL-3Ralpha levels had a lower complete remission rate and survival duration than those showing normal IL-3Ralpha levels. These findings suggest that in AML, deregulated expression of IL-3Ralpha may contribute to the proliferative advantage of the leukemic blasts and, hence, to a poor prognosis.
Assuntos
Leucemia Mieloide Aguda/genética , Receptores de Interleucina-3/genética , Adulto , Idoso , Antígenos de Superfície/análise , Apoptose , Crise Blástica , Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa de Receptor de Interleucina-3 , Leucemia Eritroblástica Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Recidiva , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
The morphologic, immunophenotypic, genotypic, genomic, and functional features of an undifferentiated acute leukemia with stem cell features are reported. At light and electron microscopy, the leukemic population was represented by primitive progenitor cells with no evidence of differentiation. The blasts were CD34(+), AC133(+), CD71(-), HLA-DR(-), CD38(-/dim+), CD90(+), CD117(dim+), flt3(+); did not express B, T, or myeloid-associated antigens; and showed a germline configuration of the immunoglobulin and T-cell receptor. Genomic profiling documented the expression of early stem cell and myeloid-associated genes. Receptors for early-acting hemopoietic growth factors (HGFs) were detected, while receptors for unilineage HGF were not expressed. Incubation with the flt3 or Kit ligand induced the expression of unilineage HGF receptors, allowing these cells to respond to their respective ligands. Growth without differentiation was sustained only in the presence of early-acting HGF, namely flt3 ligand, while early and unilineage HGF gave rise to all types of hemopoietic colonies.