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1.
Cell ; 150(3): 563-74, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22863009

RESUMO

Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis.


Assuntos
Genes Supressores de Tumor , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tristetraprolina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estabilidade de RNA , RNA Mensageiro/química
2.
Proc Natl Acad Sci U S A ; 111(32): E3260-8, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25071166

RESUMO

Chimeric oncoproteins created by chromosomal translocations are among the most common genetic mutations associated with tumorigenesis. Malignant mucoepidermoid salivary gland tumors, as well as a growing number of solid epithelial-derived tumors, can arise from a recurrent t (11, 19)(q21;p13.1) translocation that generates an unusual chimeric cAMP response element binding protein (CREB)-regulated transcriptional coactivator 1 (CRTC1)/mastermind-like 2 (MAML2) (C1/M2) oncoprotein comprised of two transcriptional coactivators, the CRTC1 and the NOTCH/RBPJ coactivator MAML2. Accordingly, the C1/M2 oncoprotein induces aberrant expression of CREB and NOTCH target genes. Surprisingly, here we report a gain-of-function activity of the C1/M2 oncoprotein that directs its interactions with myelocytomatosis oncogene (MYC) proteins and the activation of MYC transcription targets, including those involved in cell growth and metabolism, survival, and tumorigenesis. These results were validated in human mucoepidermoid tumor cells that harbor the t (11, 19)(q21;p13.1) translocation and express the C1/M2 oncoprotein. Notably, the C1/M2-MYC interaction is necessary for C1/M2-driven cell transformation, and the C1/M2 transcriptional signature predicts other human malignancies having combined involvement of MYC and CREB. These findings suggest that such gain-of-function properties may also be manifest in other oncoprotein fusions found in human cancer and that agents targeting the C1/M2-MYC interface represent an attractive strategy for the development of effective and safe anticancer therapeutics in tumors harboring the t (11, 19) translocation.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 19/genética , Proteínas de Ligação a DNA/química , Redes Reguladoras de Genes , Genes myc , Células HEK293 , Humanos , Camundongos , Tumor Mucoepidermoide/genética , Tumor Mucoepidermoide/metabolismo , Células NIH 3T3 , Proteínas Nucleares/química , Proteínas de Fusão Oncogênica/química , Domínios e Motivos de Interação entre Proteínas , Ratos , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/metabolismo , Transativadores , Fatores de Transcrição/química , Translocação Genética
3.
Blood ; 124(13): 2081-90, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25143484

RESUMO

Myc oncogenic transcription factors (c-Myc, N-Myc, and L-Myc) coordinate the control of cell growth, division, and metabolism. In cancer, Myc overexpression is often associated with aggressive disease, which is in part due to the destruction of select targets by the ubiquitin-proteasome system (eg, SCF(Skp2)-directed destruction of the Cdk inhibitor p27(Kip1)). We reasoned that Myc would also regulate SUMOylation, a related means of posttranslational modification of proteins, and that this circuit would play essential roles in Myc-dependent tumorigenesis. Here, we report marked increases in the expression of genes that encode regulators and components of the SUMOylation machinery in mouse and human Myc-driven lymphomas, resulting in hyper-SUMOylation in these tumors. Further, inhibition of SUMOylation by genetic means disables Myc-induced proliferation, triggering G2/M cell-cycle arrest, polyploidy, and apoptosis. Using genetically defined cell models and conditional expression systems, this response was shown to be Myc specific. Finally, in vivo loss-of-function and pharmacologic studies demonstrated that inhibition of SUMOylation provokes rapid regression of Myc-driven lymphoma. Thus, targeting SUMOylation represents an attractive therapeutic option for lymphomas with MYC involvement.


Assuntos
Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ácidos Anacárdicos/farmacologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma de Células B/genética , Camundongos , Camundongos Transgênicos , Poliploidia , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais , Sumoilação/efeitos dos fármacos , Transcrição Gênica , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo
4.
Blood ; 115(10): 2003-7, 2010 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-20061559

RESUMO

Somatic mutations in TET2 occur in patients with myeloproliferative neoplasms and other hematologic malignancies. It has been suggested that TET2 is a tumor suppressor gene and mutations in TET2 precede the acquisition of JAK2-V617F. To examine the order of events, we performed colony assays and genotyped TET2 and JAK2 in individual colonies. In 4 of 8 myeloproliferative neoplasm patients, we found that some colonies with mutated TET2 carried wild-type JAK2, whereas others were JAK2-V617F positive, indicating that TET2 occurred before JAK2-V617F. One of these patients carried a germline TET2 mutation. However, in 2 other patients, we obtained data compatible with the opposite order of events, with JAK2 exon 12 mutation preceding TET2 mutation in one case. Finally, in 2 of 8 patients, the TET2 and JAK2-V617F mutations defined 2 separate clones. The lack of a strict temporal order of occurrence makes it unlikely that mutations in TET2 represent a predisposing event for acquiring mutations in JAK2.


Assuntos
Células Clonais/patologia , Proteínas de Ligação a DNA/genética , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas/genética , Idoso , Separação Celular/métodos , Células Clonais/metabolismo , Análise Mutacional de DNA , Dioxigenases , Progressão da Doença , Feminino , Predisposição Genética para Doença , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Transtornos Mieloproliferativos/patologia , Polimorfismo de Nucleotídeo Único/fisiologia , Células Tumorais Cultivadas
5.
Cancer Res ; 82(7): 1234-1250, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35149590

RESUMO

MYC family oncoproteins are regulators of metabolic reprogramming that sustains cancer cell anabolism. Normal cells adapt to nutrient-limiting conditions by activating autophagy, which is required for amino acid (AA) homeostasis. Here we report that the autophagy pathway is suppressed by Myc in normal B cells, in premalignant and neoplastic B cells of Eµ-Myc transgenic mice, and in human MYC-driven Burkitt lymphoma. Myc suppresses autophagy by antagonizing the expression and function of transcription factor EB (TFEB), a master regulator of autophagy. Mechanisms that sustained AA pools in MYC-expressing B cells include coordinated induction of the proteasome and increases in AA transport. Reactivation of the autophagy-lysosomal pathway by TFEB disabled the malignant state by disrupting mitochondrial functions, proteasome activity, AA transport, and AA and nucleotide metabolism, leading to metabolic anergy, growth arrest, and apoptosis. This phenotype provides therapeutic opportunities to disable MYC-driven malignancies, including AA restriction and treatment with proteasome inhibitors. SIGNIFICANCE: MYC suppresses TFEB and autophagy and controls amino acid homeostasis by upregulating amino acid transport and the proteasome, and reactivation of TFEB disables the metabolism of MYC-driven tumors.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Lisossomos , Proteínas Proto-Oncogênicas c-myc , Aminoácidos/metabolismo , Animais , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Homeostase , Humanos , Lisossomos/metabolismo , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética
6.
Blood ; 113(9): 2022-7, 2009 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-19047681

RESUMO

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q), screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders, and identified 19 patients with del20q (2.9%), of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F, we performed colony assays in methylcellulose, picked individual burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte (CFU-G) colonies, and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients, we found that some colonies with del20q carried only wild-type JAK2, whereas other del20q colonies were JAK2-V617F positive, indicating that del20q occurred before the acquisition of JAK2-V617F. However, in colonies from 3 of 9 patients, we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L, microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events, such as del20q or 9pLOH, occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 20 , Janus Quinase 2/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Cromossomos Humanos Par 20/fisiologia , Células Clonais/patologia , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Fenilalanina/genética , Valina/genética
7.
Blood Cancer Discov ; 2(2): 162-185, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33860275

RESUMO

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML) and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Isocitrato Desidrogenase , Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-myc , Transdução de Sinais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Diferenciação Celular/genética , Epigênese Genética , Humanos , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Proto-Oncogênicas c-myc/genética
8.
EBioMedicine ; 60: 102988, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32927276

RESUMO

BACKGROUND: Homologous recombination deficiencies (HRD) are present in approximately half of epithelial ovarian cancers, for which PARP inhibitors (PARPi) are becoming a preferred treatment option. However, a considerable proportion of these carcinomas acquire resistance or harbour de novo resistance, posing a significant challenge to treatment. METHODS: To identify new combinatorial therapeutics to overcome resistance to PARPi, we employed high-throughput conditional RNAi and drug screening of patient-derived ovarian cancer cells. To prioritise clinically relevant drug combinations, we integrated empirical validation with analysis of The Cancer Genome Atlas (TCGA) and Genomics of Drug Sensitivity in Cancer (GDSC) datasets to nominate candidate targets and drugs, reaching three main findings. FINDINGS: Firstly, we found that the PARPi rucaparib enhanced the effect of BET inhibitors (CPI-203 & CPI-0610) irrespective of clinical subtype or HRD status. Additional drug combination screens identified that dasatinib, a non-receptor tyrosine kinase inhibitor, augmented the effects of rucaparib and BET inhibitors, proposing a potential broadly applicable triple-drug combination for high-grade serous and clear cell ovarian carcinomas. Secondly, rucaparib synergised with the BCL2 family inhibitor navitoclax, with preferential activity in ovarian carcinomas that harbour alterations in BRCA1/2, BARD1, or MSH2/6. Thirdly, we identified potentially antagonistic drug combinations between the PARPi rucaparib and vinca alkaloids, anthracyclines, and antimetabolites, cautioning their use in the clinic. INTERPRETATION: These findings propose therapeutic strategies to address PARP inhibitor resistance using agents that are already approved or are in clinical development, with the potential for rapid translation to benefit a broad population of ovarian cancer patients.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidores
10.
Cell Syst ; 6(3): 282-300.e2, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29596783

RESUMO

Although the MYC oncogene has been implicated in cancer, a systematic assessment of alterations of MYC, related transcription factors, and co-regulatory proteins, forming the proximal MYC network (PMN), across human cancers is lacking. Using computational approaches, we define genomic and proteomic features associated with MYC and the PMN across the 33 cancers of The Cancer Genome Atlas. Pan-cancer, 28% of all samples had at least one of the MYC paralogs amplified. In contrast, the MYC antagonists MGA and MNT were the most frequently mutated or deleted members, proposing a role as tumor suppressors. MYC alterations were mutually exclusive with PIK3CA, PTEN, APC, or BRAF alterations, suggesting that MYC is a distinct oncogenic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such as immune response and growth factor signaling; chromatin, translation, and DNA replication/repair were conserved pan-cancer. This analysis reveals insights into MYC biology and is a reference for biomarkers and therapeutics for cancers with alterations of MYC or the PMN.


Assuntos
Genes myc/genética , Genes myc/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores Tumorais/genética , Carcinogênese/genética , Cromatina , Biologia Computacional/métodos , Genômica , Humanos , Neoplasias/genética , Neoplasias/fisiopatologia , Oncogenes , Proteômica , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética
11.
Clin Cancer Res ; 24(12): 2828-2843, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29599409

RESUMO

Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low-passage tumor cells derived from a patient with treatment-resistant HPV-negative HNSCC.Experimental Design: A tumor cell culture was established and subjected to whole-exome sequencing, RNA sequencing, comparative genome hybridization, and high-throughput phenotyping with a siRNA library covering the druggable genome and an oncology drug library. Secondary screens of candidate target genes were performed on the primary tumor cells and two nontumorigenic keratinocyte cell cultures for validation and to assess cancer specificity. siRNA screens of the kinome on two isogenic pairs of p53-mutated HNSCC cell lines were used to determine generalizability. Clinical utility was addressed by performing drug screens on two additional HNSCC cell cultures derived from patients enrolled in a clinical trial.Results: Many of the identified copy number aberrations and somatic mutations in the primary tumor were typical of HPV(-) HNSCC, but none pointed to obvious therapeutic choices. In contrast, siRNA profiling identified 391 candidate target genes, 35 of which were preferentially lethal to cancer cells, most of which were not genomically altered. Chemotherapies and targeted agents with strong tumor-specific activities corroborated the siRNA profiling results and included drugs that targeted the mitotic spindle, the proteasome, and G2-M kinases WEE1 and CHK1 We also show the feasibility of ex vivo drug profiling for patients enrolled in a clinical trial.Conclusions: High-throughput phenotyping with siRNA and drug libraries using patient-derived tumor cells prioritizes mutated driver genes and identifies novel drug targets not revealed by genomic profiling. Functional profiling is a promising adjunct to DNA sequencing for precision oncology. Clin Cancer Res; 24(12); 2828-43. ©2018 AACR.


Assuntos
Biomarcadores Tumorais , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Terapia de Alvo Molecular , Medicina de Precisão , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Hibridização Genômica Comparativa , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Genômica/métodos , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Mutação , Tomografia por Emissão de Pósitrons , Medicina de Precisão/métodos , RNA Interferente Pequeno/genética , Tomografia Computadorizada por Raios X , Transcriptoma , Sequenciamento do Exoma
12.
Oncotarget ; 7(50): 83462-83475, 2016 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-27825143

RESUMO

Tristetraprolin (TTP) is an RNA-binding protein that post-transcriptionally suppresses gene expression by delivering mRNA cargo to processing bodies (P-bodies) where the mRNA is degraded. TTP functions as a tumor suppressor in a mouse model of B cell lymphoma, and in some human malignancies low TTP expression correlates with reduced survival. Here we report important prognostic and functional roles for TTP in human prostate cancer. First, gene expression analysis of prostate tumors revealed low TTP expression correlates with patients having high-risk Gleason scores and increased biochemical recurrence. Second, in prostate cancer cells with low levels of endogenous TTP, inducible TTP expression inhibits their growth and proliferation, as well as their clonogenic growth. Third, TTP functions as a tumor suppressor in prostate cancer, as forced TTP expression markedly impairs the tumorigenic potential of prostate cancer cells in a mouse xenograft model. Finally, pathway analysis of gene expression data suggested metabolism is altered by TTP expression in prostate tumor cells, and metabolic analyses revealed that such processes are impaired by TTP, including mitochondrial respiration. Collectively, these findings suggest that TTP is an important prognostic indicator for prostate cancer, and augmenting TTP function would effectively disable the metabolism and proliferation of aggressive prostate tumors.


Assuntos
Proliferação de Células , Metabolismo Energético , Neoplasias da Próstata/metabolismo , Tristetraprolina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Intervalo Livre de Doença , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Calicreínas/sangue , Masculino , Camundongos Nus , Gradação de Tumores , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdução de Sinais , Fatores de Tempo , Tristetraprolina/genética , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cancer Res ; 75(23): 5023-33, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26424696

RESUMO

Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep-sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity, and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, noninvasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.


Assuntos
Carcinogênese/química , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/química , Luciferases/química , Substâncias Luminescentes/química , Imagem Óptica/métodos , Proteínas Recombinantes de Fusão/química , Animais , Linfoma de Burkitt/química , Linfoma de Burkitt/patologia , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/patologia , Decápodes/enzimologia , Proteínas de Fluorescência Verde/genética , Células HEK293 , Xenoenxertos , Humanos , Luciferases/genética , Neoplasias Pulmonares/química , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/genética
14.
J Cell Biol ; 208(6): 745-59, 2015 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-25778921

RESUMO

Casein kinase 1δ/ε (CK1δ/ε) and their yeast homologue Hrr25 are essential for cell growth. Further, CK1δ is overexpressed in several malignancies, and CK1δ inhibitors have shown promise in several preclinical animal studies. However, the substrates of Hrr25 and CK1δ/ε that are necessary for cell growth and survival are unknown. We show that Hrr25 is essential for ribosome assembly, where it phosphorylates the assembly factor Ltv1, which causes its release from nascent 40S subunits and allows subunit maturation. Hrr25 inactivation or expression of a nonphosphorylatable Ltv1 variant blocked Ltv1 release in vitro and in vivo, and prevented entry into the translation-like quality control cycle. Conversely, phosphomimetic Ltv1 variants rescued viability after Hrr25 depletion. Finally, Ltv1 knockdown in human breast cancer cells impaired apoptosis induced by CK1δ/ε inhibitors, establishing that the antiproliferative activity of these inhibitors is due, at least in part, to disruption of ribosome assembly. These findings validate the ribosome assembly pathway as a novel target for the development of anticancer therapeutics.


Assuntos
Caseína Quinase I/fisiologia , Caseína Quinase Idelta/fisiologia , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Linhagem Celular Tumoral , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/citologia
15.
EMBO Mol Med ; 6(12): 1509-11, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25368331

RESUMO

MYC family oncoproteins (MYC, N­MYC and L­MYC) function as basic helix­loop­helix­leucine zipper (bHLH­Zip) transcription factors that are activated (i.e., overexpressed) in well over half of all human malignancies (Boxer & Dang, 2001; Beroukhim et al, 2010). In this issue of EMBO Molecular Medicine, Eilers and colleagues (Peter et al, 2014) describe a novel approach to disable MYC, whereby inhibition of the ubiquitin ligase HUWE1 stabilizes MIZ1 and leads to the selective repression of MYC­activated target genes.


Assuntos
Neoplasias Colorretais/enzimologia , Proteína Oncogênica p55(v-myc)/antagonistas & inibidores , Proteína Oncogênica p55(v-myc)/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Humanos
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