RESUMO
PRKAR1A (R1A)-retinoic acid receptor-alpha (R1A-RARalpha) is the sixth RARalpha-containing fusion protein in acute promyelocytic leukemia (APL). Using the murine bone-marrow retroviral transduction/transformation assay, we showed that R1A-RARalpha fusion protein could transform bone-marrow progenitor/stem cells. In gel-shift assays, R1A-RARalpha was able to bind to a panel of retinoic acid response elements both as a homodimer and as a heterodimer with RXRalpha, and demonstrated distinct DNA-binding characteristics compared with wild-type RARalpha/RXRalpha or other X-RARalpha chimeric proteins. The ratio of R1A-RARalpha to RXRalpha proteins affected the retinoic acid response element interaction pattern of R1A-RARalpha/RXRalpha complexes. Studies comparing R1A-RARalpha with R1A-RARalpha(DeltaRIIa) demonstrated that the RIIa protein interaction domain located within R1A was responsible for R1A-RARalpha homodimeric DNA binding and interaction with wild-type R1A protein. However, the RIIa domain was not required for R1A-RARalpha-mediated transformation because its deletion in R1A-RARalpha(DeltaRIIa) did not compromise its transformation capability. In contrast, introduction of point mutations within the RARalpha portion of either R1A-RARalpha or R1A-RARalpha(DeltaRIIa), previously demonstrated to eliminate RXRalpha interaction or treatment of transduced cells with RXRalpha shRNA or a RXRalpha agonist, reduced transformation capability. Thus, leukemic transformation by APL fusion protein PRKAR1A-RARalpha is critically dependent on RXRalpha, which suggests RXRalpha is a promising target for APL.
Assuntos
Transformação Celular Neoplásica/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptor X Retinoide alfa/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Cultivadas , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Regulação Leucêmica da Expressão Gênica , Células HeLa , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica/fisiologia , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Receptor X Retinoide alfa/fisiologiaRESUMO
Although both heterodimeric subunits of core binding factors (AML1/RUNX1 and CBFbeta) essential for normal hematopoiesis are frequently mutated to form different chimeric fusion proteins in acute leukemia, the underlying molecular mechanisms and structural domains required for cellular transformation remain largely unknown. Despite the critical role of CBFbeta for wild-type AML1 function and its direct involvement in chromosomal translocation, we demonstrate that both the expression and interaction with CBFbeta are superfluous for AML1-ETO (AE)-mediated transformation of primary hematopoietic cells. Similarly, the hetero-oligomeric interaction with transcriptional repressor ETO family proteins and the highly conserved NHR1 domain in AE fusion are also dispensable for transforming activity. In contrast, AE-mediated transformation is critically dependent on the DNA binding and homo-oligomeric properties of the fusion. Abolishment of homo-oligomerization by a small-molecule inhibitor could specifically suppress AML1 fusion-mediated transformation of primary hematopoietic cells. Together, these results not only identify the essential molecular components but also potential avenues for therapeutic targeting of AE-mediated leukemogenesis.
Assuntos
Biopolímeros/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Sequência de Bases , Transformação Celular Neoplásica , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Primers do DNA , Leucemia/genética , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1 , Translocação GenéticaRESUMO
Acute promyelocytic leukemia (APL) is characterized by specific chromosomal translocations, which generate fusion proteins such as promyelocytic leukemia (PML)-retinoic acid receptor (RAR)alpha and promyelocytic leukemia zinc finger (PLZF)-RARalpha (X-RARalpha). In this study, we have applied lac operator array systems to study the effects of X-RARalpha versus wild-type RARalpha on large-scale chromatin structure. The targeting of these enhanced cyan fluorescent protein-lac repressor-tagged RARalpha-containing proteins to the gene-amplification chromosomal region by lac operator repeats led to local chromatin condensation, recruitment of nuclear receptor corepressor, and histone deacetylase complex. The addition of retinoic acid (RA) induced large-scale chromatin decondensation in cells expressing RARalpha; however, cells expressing X-RARalpha, especially PML-RARalpha, demonstrated insensitive response to this effect of all-trans retinoic acid (ATRA). Although we did not reveal differences in RA-dependent colocalization of either silencing mediator for retinoid and thyroid or steroid receptor coactivator (SRC)-1 with RARalpha versus X-RARalpha, the hormone-independent association between SRC-1 and X-RARalpha on the array has been identified. Rather, compared with cells expressing RARalpha, fluorescence recovery after photobleaching of live transfected cells, demonstrated decreased mobility of SRC-1 on the X-RARalpha-bound chromatin. Thus, the impaired ability of APL fusion proteins to activate gene transcription in response to ATRA corresponds to their reduced ability to remodel chromatin, which may link to their ability to impair the mobility of key nuclear receptor coregulators.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Bactérias/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Histona Acetiltransferases/metabolismo , Humanos , Proteínas Luminescentes/metabolismo , Correpressor 2 de Receptor Nuclear , Coativador 1 de Receptor Nuclear , Regiões Operadoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Tretinoína/farmacologiaRESUMO
Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) is the most frequent RARalpha fusion protein in acute promyelocytic leukemia (APL). Our previous study has demonstrated that, compared with RARalpha, PML-RARalpha had reduced intranuclear mobility accompanied with mislocalization. To understand the molecular basis for the altered dynamics of PML-RARalpha fusion protein, we performed FRAP analysis at a single cell level. Results indicated that three known sumoylation site mutated PML-RARalpha had same intracellular localization and reduced mobility as wild-type counterpart. The coiled-coil domain of PML is responsible for the aberrant dynamics of PML-RARalpha. In addition, we revealed that co-repressor SMRT co-localized with PML-RARalpha, resulting in the immobilization of SMRT while ATRA treatment eliminated their association and reversed the immobile effect of SMRT. Furthermore, co-activator CBP, co-localized with PML-RARalpha in an ATRA-independent way, was demonstrated as a high dynamic intranuclear molecule. These results would shed new insights for the molecular mechanisms of PML-RARalpha-associated leukemogenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Rim/química , Rim/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular , Células HeLa , Humanos , Correpressor 2 de Receptor Nuclear , Proteína da Leucemia Promielocítica , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptor alfa de Ácido Retinoico , Relação Estrutura-AtividadeRESUMO
Acute promyelocytic leukemia (APL) cells contain one of five chimeric retinoic acid alpha-receptor (RAR alpha) genes (X-RAR alpha) created by chromosomal translocations or deletion; each generates a fusion protein thought to transcriptionally repress RAR alpha target genes and block myeloid differentiation by an incompletely understood mechanism. To gain spatiotemporal insight into these oncogenic processes, we employed fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Fluorescence microscopy demonstrated that the intracellular localization of each of the X-RAR alpha proteins was distinct from that of RAR alpha and established which portion(s) of each X-RAR alpha protein-X, RAR, or both-contributed to its altered localization. Using FRAP, we demonstrated that the intranuclear mobility of each X-RAR alpha was reduced compared to that of RAR alpha. In addition, the mobility of each X-RAR alpha was reduced further by ligand addition, in contrast to RAR alpha, which showed no change in mobility when ligand was added. Both the reduced baseline mobility of X-RAR alpha and the ligand-induced slowing of X-RAR alpha could be attributed to the protein interaction domain contained within X. RXR alpha aberrantly colocalized within each X-RAR alpha; colocalization of RXR alpha with promyelocytic leukemia (PML)-RAR alpha resulted in reduced mobility of RXR alpha. Thus, X-RAR alpha may interfere with RAR alpha through its aberrant nuclear dynamics, resulting in spatial and temporal sequestration of RXR alpha and perhaps other nuclear receptor coregulators critical for myeloid differentiation.
Assuntos
Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células COS , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Leucemia Promielocítica Aguda/genética , Ligantes , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteína da Leucemia Promielocítica , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Fatores de Transcrição/genética , Proteínas Supressoras de TumorRESUMO
Nuclear mitotic apparatus protein-retinoic acid receptor alpha (NuMA-RARalpha) is the fourth of five fusion proteins identified in acute promyelocytic leukemia (APL) patients. The molecular basis for its oncogenic activity has not been delineated. In gel-shift assays, NuMA-RARalpha bound to retinoic acid response elements (RAREs) both as a homodimer and as a heterodimer with RXRalpha. The binding profile of NuMA-RARalpha to a panel of RAREs was very similar to PML-RARalpha and PLZF-RARalpha. In transient transfection assays using HepG2 cells, NuMA-RARalpha inhibited wild-type RARalpha transcriptional activity, while it augmented STAT3 transcriptional activity. In GST-pull down experiments, NuMA-RARalpha formed a complex with the corepressor SMRT, was released from the NuMA-RARalpha/SMRT complexes by all-trans retinoic acid (ATRA) at 10(-7)-10(-6) M and became associated with the coactivator TRAM-1 at 10(-8) M ATRA. Studies comparing NuMA-RARalpha with NuMA-RARalpha(deltaCC) demonstrated that the dimerization or alpha-helical coiled-coil domain of NuMA was required for homodimer formation, transcriptional repression of wild-type RARalpha, transcriptional activation of STAT3, and stability of the NuMA-RARalpha/SMRT complex. Confocal fluorescent microscopy of HeLa cells was performed following transient expression of cyan fluorescent protein (CFP)-tagged proteins and incubation of cells with or without ATRA. Within the nucleus, CFP-NuMA-RARalpha exhibited a speckled pattern identical to that observed in cells transfected with CFP-NuMA. Furthermore, CFP-NuMA-RARalpha colocalized with yellow fluorescent protein-tagged (YFP)-NuMA. In contrast, CFP-NuMA-RARalpha(deltaCC) exhibited a diffuse granular pattern within the nucleus, similar to RARalpha. These results indicate that the dimerization domain of NuMA-RARalpha is critical for each of the known oncogenic activities of NuMA fusion proteins as well as its sequestration to nuclear sites normally occupied by NuMA and is distinct from RARalpha.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Transativadores/genética , Animais , Células COS , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Estrutura Terciária de Proteína , Fator de Transcrição STAT3 , Transcrição GênicaRESUMO
Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure-activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R(2) of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood-brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26-1.8 µM) against glioma cells with the IDH1 R132H mutation.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Animais , Antineoplásicos/síntese química , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glioma/tratamento farmacológico , Glioma/patologia , Glutaratos/metabolismo , Humanos , Camundongos , Piridonas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Esophageal cancer is the sixth leading cause of cancer-related deaths worldwide. It has been reported that histone demethylases are involved in the carcinogenesis of certain types of tumors. Here, we studied the role of one of the histone lysine demethylases, plant homeodomain finger protein 8 (PHF8), in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). Using short hairpin RNA via lentiviral infection, we established stable ESCC cell lines with constitutive downregulation of PHF8 expression. Knockdown of PHF8 in ESCC cells resulted in inhibition of cell proliferation and an increase of apoptosis. Moreover, there were reductions of both anchorage-dependent and -independent colony formation. In vitro migration and invasion assays showed that knockdown of PHF8 led to a reduction in the number of migratory and invasive cells. Furthermore, downregulation of PHF8 attenuated the tumorigenicity of ESCC cells in vivo. Taken together, our study revealed the oncogenic features of PHF8 in ESCC, suggesting that PHF8 may be a potential diagnostic marker and therapeutic target for ESCC.
Assuntos
Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/genética , Neoplasias Esofágicas/genética , Histona Desmetilases/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histona Desmetilases/metabolismo , Humanos , Camundongos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Carga Tumoral/genética , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute myeloid leukemia (AML) is the most common malignant myeloid disorder of progenitor cells in myeloid hematopoiesis and exemplifies a genetically heterogeneous disease. The patients with AML also show a heterogeneous response to therapy. Although all-trans retinoic acid (ATRA) has been successfully introduced to treat acute promyelocytic leukemia (APL), it is rather ineffective in non-APL AML. In our present study, 1200 off-patent marketed drugs and natural compounds that have been approved by the Food and Drug Administration (FDA) were screened for anti-leukemia activity using the retrovirus transduction/transformation assay (RTTA). Furazolidone (FZD) was shown to inhibit bone marrow transformation mediated by several leukemia fusion proteins, including AML1-ETO. Furazolidone has been used in the treatment of certain bacterial and protozoan infections in human and animals for more than sixty years. We investigated the anti-leukemic activity of FZD in a series of AML cells. FZD displayed potent antiproliferative properties at submicromolar concentrations and induced apoptosis in AML cell lines. Importantly, FZD treatment of certain AML cells induced myeloid cell differentiation by morphology and flow cytometry for CD11b expression. Furthermore, FZD treatment resulted in increased stability of tumor suppressor p53 protein in AML cells. Our in vitro results suggest furazolidone as a novel therapeutic strategy in AML patients.
Assuntos
Antineoplásicos/farmacologia , Antitricômonas/farmacologia , Apoptose/efeitos dos fármacos , Reposicionamento de Medicamentos , Furazolidona/farmacologia , Animais , Bioensaio , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Retroviridae/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
While all-trans retinoic acid (ATRA) treatment in acute promyelocytic leukemia (APL) has been the paradigm of targeted therapy for oncogenic transcription factors, the underlying mechanisms remain largely unknown, and a significant number of patients still relapse and become ATRA resistant. We identified the histone demethylase PHF8 as a coactivator that is specifically recruited by RARα fusions to activate expression of their downstream targets upon ATRA treatment. Forced expression of PHF8 resensitizes ATRA-resistant APL cells, whereas its downregulation confers resistance. ATRA sensitivity depends on the enzymatic activity and phosphorylation status of PHF8, which can be pharmacologically manipulated to resurrect ATRA sensitivity to resistant cells. These findings provide important molecular insights into ATRA response and a promising avenue for overcoming ATRA resistance.
Assuntos
Histona Desmetilases/metabolismo , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Animais , Resistencia a Medicamentos Antineoplásicos , Histona Desmetilases/genética , Histonas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Ácido Okadáico/farmacologia , Proteínas de Fusão Oncogênica/metabolismo , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Recent studies have identified mutations in PHF8, an X-linked gene encoding a JmjC domain-containing protein, as a causal factor for X-linked mental retardation (XLMR) and cleft lip/cleft palate. However, the underlying mechanism is unknown. Here we show that PHF8 is a histone demethylase and coactivator for retinoic acid receptor (RAR). Although activities for both H3K4me3/2/1 and H3K9me2/1 demethylation were detected in cellular-based assays, recombinant PHF8 exhibited only H3K9me2/1 demethylase activity in vitro, suggesting that PHF8 is an H3K9me2/1 demethylase whose specificity may be modulated in vivo. Importantly, a mutant PHF8 (phenylalanine at position 279 to serine) identified in the XLMR patients is defective in enzymatic activity, indicating that the loss of histone demethylase activity is causally linked with the onset of disease. In addition, we show that PHF8 binds specifically to H3K4me3/2 peptides via an N-terminal PHD finger domain. Consistent with a role for PHF8 in neuronal differentiation, knockdown of PHF8 in mouse embryonic carcinoma P19 cells impairs RA-induced neuronal differentiation, whereas overexpression of the wild-type but not the F279S mutant PHF8 drives P19 cells toward neuronal differentiation. Furthermore, we show that PHF8 interacts with RARalpha and functions as a coactivator for RARalpha. Taken together, our results suggest that histone methylation modulated by PHF8 plays a critical role in neuronal differentiation.
Assuntos
Histona Desmetilases/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/genética , Neurônios/enzimologia , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Animais , Diferenciação Celular , Técnicas de Silenciamento de Genes , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/etiologia , Camundongos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição/genéticaRESUMO
Two isoforms of Stat3 (signal transducer and activator of transcription 3) are expressed in cells, alpha (p92) and beta (p83), both derived from a single gene by alternative mRNA splicing. The 55-residue C-terminal transactivation domain of Stat3alpha is deleted in Stat3beta and replaced by seven unique C-terminal residues (CT7) whose function remains uncertain. We subcloned the open reading frames of Stat3alpha and Stat3beta into the C terminus of green fluorescent protein (GFP). Fluorescent microscopic analysis of HEK293T cells transiently transfected with GFP-Stat3alpha or GFP-Stat3beta revealed similar kinetics and cytokine concentration dependence of nuclear accumulation; these findings were confirmed by high throughput microscope analysis of murine embryonic fibroblasts that lacked endogenous Stat3 but stably expressed either GFP-Stat3alpha or GFP-Stat3beta. However, although time to half-maximal cytoplasmic reaccumulation after cytokine withdrawal was 15 min for GFP-Stat3alpha, it was >180 min for GFP-Stat3beta. Furthermore, although the intranuclear mobility of GFP-Stat3alpha was rapid and increased with cytokine stimulation, the intranuclear mobility of GFP-Stat3beta in unstimulated cells was slower than that of GFP-Stat3alpha in unstimulated cells and was slowed further following cytokine stimulation. Deletion of the unique CT7 domain from Stat3beta eliminated prolonged nuclear retention but did not alter its intranuclear mobility. Thus, Stat3alpha and Stat3beta have distinct intracellular dynamics, with Stat3beta exhibiting prolonged nuclear retention and reduced intranuclear mobility especially following ligand stimulation. Prolonged nuclear retention, but not reduced intranuclear mobility, mapped to the CT7 domain of Stat3beta.