RESUMO
Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Redes Reguladoras de Genes , Fatores de Transcrição , Transcrição Gênica , Cromatina/metabolismo , Cromatina/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Regulação da Expressão Gênica , Complexo Mediador/metabolismo , Complexo Mediador/genética , Análise de Célula Única , Transcriptoma , Coesinas , Proteínas que Contêm BromodomínioRESUMO
Drug resistance and disease progression are common in multiple myeloma (MM) patients, underscoring the need for new therapeutic combinations. A high-throughput drug screen in 47 MM cell lines and in silico Huber robust regression analysis of drug responses revealed 43 potentially synergistic combinations. We hypothesized that effective combinations would reduce MYC expression and enhance p16 activity. Six combinations cooperatively reduced MYC protein, frequently over-expressed in MM and also cooperatively increased p16 expression, frequently downregulated in MM. Synergistic reductions in viability were observed with top combinations in proteasome inhibitor-resistant and sensitive MM cell lines, while sparing fibroblasts. Three combinations significantly prolonged survival in a transplantable Ras-driven allograft model of advanced MM closely recapitulating high-risk/refractory myeloma in humans and reduced viability of ex vivo treated patient cells. Common genetic pathways similarly downregulated by these combinations promoted cell cycle transition, whereas pathways most upregulated were involved in TGFß/SMAD signaling. These preclinical data identify potentially useful drug combinations for evaluation in drug-resistant MM and reveal potential mechanisms of combined drug sensitivity.
Assuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Ensaios de Triagem em Larga Escala , Sinergismo Farmacológico , Ciclo Celular , Combinação de Medicamentos , Linhagem Celular Tumoral , Resistencia a Medicamentos AntineoplásicosRESUMO
The TGF-ß-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-ß pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.
Assuntos
Neoplasias da Mama , Canais de Cloreto , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Canais de Cloreto/biossíntese , Canais de Cloreto/genética , Feminino , Humanos , Camundongos , Metástase Neoplásica , Proteômica , Fator de Crescimento Transformador beta/metabolismo , Microambiente TumoralRESUMO
Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase and central regulator of cell growth, differentiation, and survival. mTOR is commonly hyperactivated in a diverse number of cancers and critical roles for mTOR in regulating immune cell differentiation and function have been demonstrated. However, there is little work investigating the roles of mTOR in early B-cell development. Here we demonstrate that conditional disruption of mTOR in developing mouse B cells results in reduced pre-B-cell proliferation and survival, as well as a developmental block at the pre-B-cell stage, with a corresponding lack of peripheral B cells. Upon immunization with NP-CGG antigen, mice with Mtor conditional disruption in early B cells lost their ability to form germinal centers and produce specific antibodies. In competitive BM repopulation assays, donor BM cells from conditional knock-out mice were completely impaired in their ability to reconstitute B cells. Our data reveal the essential role of mTOR in early pre-B-cell development and survival.
Assuntos
Transdução de Sinais , Sirolimo , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Ativação Linfocitária , Camundongos , Camundongos Knockout , Serina-Treonina Quinases TOR/metabolismoRESUMO
Unbiased genetic forward screening using retroviral insertional mutagenesis in a genetically engineered mouse model of human multiple myeloma may further our understanding of the genetic pathways that govern neoplastic plasma cell development. To evaluate this hypothesis, we performed a tumor induction study in MYC-transgenic mice infected as neonates with the Moloney-derived murine leukemia virus, MOL4070LTR. Next-generation DNA sequencing of proviral genomic integration sites yielded rank-ordered candidate tumor progression genes that accelerated plasma cell neoplasia in mice. Rigorous clinical and biological validation of these genes led to the discovery of two novel myeloma genes: WDR26 (WD repeat-containing protein 26) and MTF2 (metal response element binding transcription factor 2). WDR26, a core component of the carboxy-terminal to LisH (CTLH) complex, is overexpressed or mutated in solid cancers. MTF2, an ancillary subunit of the polycomb repressive complex 2 (PRC2), is a close functional relative of PHD finger protein 19 (PHF19) which is currently emerging as an important driver of myeloma. These findings underline the utility of genetic forward screens in mice for uncovering novel blood cancer genes and suggest that WDR26-CTLH and MTF2-PRC2 are promising molecular targets for new approaches to myeloma treatment and prevention.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Mieloma Múltiplo/genética , Complexo Repressor Polycomb 2/genética , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Mieloma Múltiplo/terapiaRESUMO
Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.
Assuntos
Células-Tronco Embrionárias , Metabolismo dos Lipídeos , Ácidos Graxos , Oxirredução , Estresse FisiológicoRESUMO
The CBFB gene is frequently mutated in several types of solid tumors. Emerging evidence suggests that CBFB is a tumor suppressor in breast cancer. However, our understanding of the tumor suppressive function of CBFB remains incomplete. Here, we analyze genetic interactions between mutations of CBFB and other highly mutated genes in human breast cancer datasets and find that CBFB and TP53 mutations are mutually exclusive, suggesting a functional association between CBFB and p53. Integrated genomic studies reveal that TAp73 is a common transcriptional target of CBFB and p53. CBFB cooperates with p53 to maintain TAp73 expression, as either CBFB or p53 loss leads to TAp73 depletion. TAp73 re-expression abrogates the tumorigenic effect of CBFB deletion. Although TAp73 loss alone is insufficient for tumorigenesis, it enhances the tumorigenic effect of NOTCH3 overexpression, a downstream event of CBFB loss. Immunohistochemistry shows that p73 loss is coupled with higher proliferation in xenografts. Moreover, TAp73 loss-of-expression is a frequent event in human breast cancer tumors and cell lines. Together, our results significantly advance our understanding of the tumor suppressive functions of CBFB and reveal a mechanism underlying the communication between the two tumor suppressors CBFB and p53.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Subunidade beta de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/genética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/deficiência , Subunidade beta de Fator de Ligação ao Core/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Camundongos , Mutação , Receptor Notch3/genética , Receptor Notch3/metabolismo , Transcrição Gênica , Proteína Tumoral p73/deficiência , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Physiologically, MYC levels must be precisely set to faithfully amplify the transcriptome, but in cancer MYC is quantitatively misregulated. Here, we study the variation of MYC amongst single primary cells (B-cells and murine embryonic fibroblasts, MEFs) for the repercussions of variable cellular MYC-levels and setpoints. Because FUBPs have been proposed to be molecular "cruise controls" that constrain MYC expression, their role in determining basal or activated MYC-levels was also examined. Growing cells remember low and high-MYC setpoints through multiple cell divisions and are limited by the same expression ceiling even after modest MYC-activation. High MYC MEFs are enriched for mRNAs regulating inflammation and immunity. After strong stimulation, many cells break through the ceiling and intensify MYC expression. Lacking FUBPs, unstimulated MEFs express levels otherwise attained only with stimulation and sponsor MYC chromatin changes, revealed by chromatin marks. Thus, the FUBPs enforce epigenetic setpoints that restrict MYC expression.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Epigênese Genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/fisiologia , Animais , Linfócitos B/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Camundongos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Long-term genetic studies utilizing backcross and congenic strain analyses coupled with positional cloning strategies and functional studies identified Cdkn2a, Mtor, and Mndal as mouse plasmacytoma susceptibility/resistance genes. Tumor incidence data in congenic strains carrying the resistance alleles of Cdkn2a and Mtor led us to hypothesize that drug combinations affecting these pathways are likely to have an additive, if not synergistic effect in inhibiting tumor cell growth. Traditional and novel systems-level genomic approaches were used to assess combination activity, disease specificity, and clinical potential of a drug combination involving rapamycin/everolimus, an Mtor inhibitor, with entinostat, an histone deacetylase inhibitor. The combination synergistically repressed oncogenic MYC and activated the Cdkn2a tumor suppressor. The identification of MYC as a primary upstream regulator led to the identification of small molecule binders of the G-quadruplex structure that forms in the NHEIII region of the MYC promoter. These studies highlight the importance of identifying drug combinations which simultaneously upregulate tumor suppressors and downregulate oncogenes.
RESUMO
Specific-pathogen-free (SPF) mice have improved hematopoietic characteristics relative to germ-free mice, however, it is not clear whether improvements in hematopoietic traits will continue when the level of microorganism exposure is further increased. We co-housed SPF C57BL/6 mice in a conventional facility (CVT) and found a significant increase in gut microbiota diversity along with increased levels of myeloid cells and T cells, especially effector memory T cells. Through single cell RNA sequencing of sorted KL (c-Kit+Lin-) cells, we imputed a decline in long-term hematopoietic stem cells and an increase in granulocyte-monocyte progenitors in CVT mice with up-regulation of genes associated with cell survival. Bone marrow transplantation through competitive repopulation revealed a significant increase in KSL (c-Kit+Sca-1+Lin-) cell reconstitution in recipients of CVT donor cells which occurred when donors were co-housed for both one and twelve months. However, there was minimal to no gain in mature blood cell engraftment in recipients of CVT donor cells relative to those receiving SPF donor cells. We conclude that co-housing SPF mice with mice born in a conventional facility increased gut microbiota diversity, augmented myeloid cell production and T cell activation, stimulated KSL cell reconstitution, and altered hematopoietic gene expression.
Assuntos
Bactérias/classificação , Perfilação da Expressão Gênica/métodos , Hematopoese , Células Mieloides/metabolismo , Análise de Sequência de RNA/métodos , Linfócitos T/metabolismo , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Transplante de Medula Óssea , Microbioma Gastrointestinal , Regulação da Expressão Gênica , Abrigo para Animais , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , Análise de Célula Única , Organismos Livres de Patógenos EspecíficosRESUMO
PI3K/AKT/mTOR pathway hyperactivation is frequent in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). To model inhibition of mTOR, pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) tumor development was monitored in mice with T lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) that were either crossed to mTOR knockdown (KD) mice or treated with the mTOR inhibitor everolimus. Lck-MyrAkt2;mTOR KD mice lived significantly longer than Lck-MyrAkt2;mTOR wild-type (WT) mice, although both groups ultimately developed thymic pre-T LBL. An increase in survival was also observed when Lck-MyrAkt2;mTOR WT mice were treated for 8 weeks with everolimus. The transcriptional profiles of WT and KD thymic lymphomas were compared, and Ingenuity Pathway Upstream Regulator Analysis of differentially expressed genes in tumors from mTOR WT versus KD mice identified let-7 and miR-21 as potential regulatory genes. mTOR KD mice had higher levels of let-7a and miR-21 than mTOR WT mice, and rapamycin induced their expression in mTOR WT cells. CDK6 was one of the most downregulated targets of both let-7 and miR21 in mTOR KD tumors. CDK6 overexpression and decreased expression of let-7 in mTOR KD cells rescued a G1 arrest phenotype. Combined mTOR (rapamycin) and CDK4/6 (palbociclib) inhibition decreased tumor size and proliferation in tumor flank transplants, increased survival in an intravenous transplant model of disseminated leukemia compared with single agent treatment, and cooperatively decreased cell viability in human T-ALL/LBL cell lines. Thus, mTOR KD mice provide a model to explore drug combinations synergizing with mTOR inhibitors and can be used to identify downstream targets of inhibition.
Assuntos
Quinase 6 Dependente de Ciclina/metabolismo , Perfilação da Expressão Gênica/métodos , Serina-Treonina Quinases TOR/metabolismo , Animais , Carcinogênese , Regulação para Baixo , Camundongos , Camundongos TransgênicosRESUMO
In humans, bone marrow (BM) failure syndromes, both constitutional and acquired, predispose to myeloid malignancies. We have modeled acquired immune aplastic anemia, the paradigmatic disease of these syndromes, in the mouse by infusing lymph node cells from specific pathogen-free (SPF) CD45.1 congenic C57BL/6 (B6) donors into hybrid CByB6F1 recipients housed either in conventional (CVB) or SPF facilities. The severity of BM damage was reduced in CVB recipients; they also had reduced levels of CD44+ CD62L- effector memory T cells, reduced numbers of donor-type CD44+ T cells, and reduced expansion of donor-type CD8 T cells carrying T-cell receptor ß-variable regions 07, 11, and 17. Analyses of fecal samples through 16S ribosomal RNA amplicon sequencing revealed greater gut microbial alpha diversity in CVB mice relative to that of SPF mice. Thus, the presence of a broader spectrum of gut microorganisms in CVB-housed CByB6F1 could have primed recipient animal's immune system leading to suppression of allogeneic donor T-cell activation and expansion and attenuation of host BM destruction. These results suggest the potential benefit of diverse gut microbiota in patients receiving BM transplants.
Assuntos
Anemia Aplástica/terapia , Transplante de Medula Óssea/métodos , Medula Óssea/imunologia , Microbioma Gastrointestinal/imunologia , Linfócitos T/imunologia , Anemia Aplástica/imunologia , Anemia Aplástica/patologia , Animais , Medula Óssea/patologia , Fezes/microbiologia , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos , Linfócitos T/metabolismo , Linfócitos T/transplante , Imunologia de Transplantes , Transplante HomólogoRESUMO
Mechanistic target of rapamycin (MTOR) is a highly conserved serine/threonine kinase that critically regulates cell growth, proliferation, differentiation, and survival. Previously, we have implicated Mtor as a plasmacytoma-resistance locus, Pctr2, in mice. Here, we report that administration of the tumor-inducing agent pristane decreases Mtor gene expression to a greater extent in mesenteric lymph nodes of BALB/cAnPt mice than of DBA/2N mice. We identified six allelic variants in the Mtor promoter region in BALB/cAnPt and DBA/2N mice. To determine the effects of these variants on Mtor transcription, we constructed a series of luciferase reporters containing these promoter variants and transfected them into mouse plasmacytoma cells. We could attribute the differences in Mtor promoter activity between the two mouse strains to a C â T change at the -6 position relative to the transcriptional start site Tssr 40273; a T at this position in the BALB promoter creates a consensus binding site for the transcription factor MZF1 (myeloid zinc finger 1). Results from electrophoretic mobility shift assays and DNA pulldown assays with ChIP-PCR confirmed that MZF1 binds to the cis-element TGGGGA located in the -6/-1 Mtor promoter region. Of note, MZF1 significantly and differentially down-regulated Mtor promoter activity, with MZF1 overexpression reducing Mtor expression more strongly in BALB mice than in DBA mice. Moreover, MZF1 overexpression reduced Mtor expression in both fibroblasts and mouse plasmacytoma cells, and Mzf1 knockdown increased Mtor expression in BALB3T3 and NIH3T3 fibroblast cells. Our results provide evidence that MZF1 down-regulates Mtor expression in pristane-induced plasmacytomas in mice.
Assuntos
Predisposição Genética para Doença/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação , Plasmocitoma/genética , Regiões Promotoras Genéticas/genética , Serina-Treonina Quinases TOR/genética , Alelos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Regulação para Baixo , Camundongos , Plasmocitoma/patologiaRESUMO
Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1 -/- ) to determine how the enzyme affects PC biology. Although Enpp1 -/- mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1 -/- PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.
Assuntos
Trifosfato de Adenosina/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Plasmócitos/metabolismo , Pirofosfatases/metabolismo , Animais , Formação de Anticorpos/imunologia , Linfócitos B/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Centro Germinativo/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Baço/metabolismo , Regulação para Cima/fisiologiaRESUMO
Mechanistic target of rapamycin (mTOR) enhances immunity in addition to orchestrating metabolism. Here we show that mTOR coordinates immunometabolic reconfiguration of marginal zone (MZ) B cells, a pre-activated lymphocyte subset that mounts antibody responses to T-cell-independent antigens through a Toll-like receptor (TLR)-amplified pathway involving transmembrane activator and CAML interactor (TACI). This receptor interacts with mTOR via the TLR adapter MyD88. The resulting mTOR activation instigates MZ B-cell proliferation, immunoglobulin G (IgG) class switching, and plasmablast differentiation through a rapamycin-sensitive pathway that integrates metabolic and antibody-inducing transcription programs, including NF-κB. Disruption of TACI-mTOR interaction by rapamycin, truncation of the MyD88-binding domain of TACI, or B-cell-conditional mTOR deficiency interrupts TACI signaling via NF-κB and cooperation with TLRs, thereby hampering IgG production to T-cell-independent antigens but not B-cell survival. Thus, mTOR drives innate-like antibody responses by linking proximal TACI signaling events with distal immunometabolic transcription programs.
Assuntos
Linfócitos B/imunologia , Imunoglobulina G/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Serina-Treonina Quinases TOR/imunologia , Proteína Transmembrana Ativadora e Interagente do CAML/imunologia , Animais , Linhagem Celular , Proliferação de Células , Ativação Enzimática , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina G/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Sirolimo/farmacologiaRESUMO
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.
Assuntos
Linfócitos B/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Ativação Linfocitária , Proteínas Proto-Oncogênicas c-myc/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Trifosfato de Adenosina/metabolismo , Animais , Linfócitos B/imunologia , Linhagem Celular , Cromatina/química , Cromatina/genética , Metilação de DNA , Epigênese Genética , Genótipo , Histonas/química , Imunidade Humoral , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Conformação de Ácido Nucleico , Fenótipo , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Imagem Individual de Molécula , Relação Estrutura-Atividade , Fatores de Tempo , Transcrição GênicaRESUMO
Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared with single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared with single agents. Mice bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared with single agents. A set of 37 genes cooperatively affected (34 downregulated; 3 upregulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes downregulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared with normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC-binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivoMol Cancer Ther; 16(9); 2008-21. ©2017 AACR.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Farmacogenética , Variantes Farmacogenômicos , Estabilidade Proteica , Proteólise , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bone marrow-derived mesenchymal stem cells (BMSCs) are proposed as the cells of origin of several subtypes of osteosarcoma (OS). However, signals that direct BMSCs to form different subtypes of OS are unclear. Here we show that the default tumor type from spontaneously transformed p53 knockout (p53_KO) BMSCs is osteoblastic OS. The development of this default tumor type caused by p53 loss can be overridden by various oncogenic signals: RAS reprograms p53_KO BMSCs into undifferentiated sarcoma, AKT enhances osteoblastic OS, while cFOS promotes chondroblastic OS formation. We focus on studying the mechanism of cFOS-induced chondroblastic OS formation. Integrated genome-wide studies reveal a regulatory mechanism whereby cFOS binds to the promoter of a key chondroblastic transcription factor, Sox9, and induces its transcription in BMSCs. Importantly, SOX9 mediates cFOS-induced cartilage formation in chondroblastic OS. In summary, oncogenes determine tumor types derived from BMSCs, and the cFOS-SOX9 axis is critical for chondroblastic OS formation.
Assuntos
Células da Medula Óssea/citologia , Neoplasias Ósseas/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição SOX9/metabolismo , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/terapia , Diferenciação Celular , Reprogramação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Osteogênese , Osteossarcoma/metabolismo , Osteossarcoma/terapia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Fatores de Transcrição SOX9/antagonistas & inibidores , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/metabolismoRESUMO
The receptor tyrosine kinase MET is abundant in many human squamous cell carcinomas (SCCs), but its functional significance in tumorigenesis is not clear. We found that the incidence of carcinogen-induced skin squamous tumors was substantially increased in transgenic MT-HGF (mouse metallothionein-hepatocyte growth factor) mice, which have increased abundance of the MET ligand HGF. Squamous tumors also erupted spontaneously on the skin of MT-HGF mice that were promoted by wounding or the application of 12-O-tetradecanoylphorbol 13-acetate, an activator of protein kinase C. Carcinogen-initiated tumors had Ras mutations, but spontaneous tumors did not. Cultured keratinocytes from MT-HGF mice and oncogenic RAS-transduced keratinocytes shared phenotypic and biochemical features of initiation that were dependent on autocrine activation of epidermal growth factor receptor (EGFR) through increased synthesis and release of EGFR ligands, which was mediated by the kinase SRC, the pseudoproteases iRhom1 and iRhom2, and the metallopeptidase ADAM17. Pharmacological inhibition of EGFR caused the regression of MT-HGF squamous tumors that developed spontaneously in orthografts of MT-HGF keratinocytes combined with dermal fibroblasts and implanted onto syngeneic mice. The global gene expression profile in MET-transformed keratinocytes was highly concordant with that in RAS-transformed keratinocytes, and a core RAS/MET coexpression network was activated in precancerous and cancerous human skin lesions. Tissue arrays revealed that many human skin SCCs have abundant HGF at both the transcript and protein levels. Thus, through the activation of EGFR, MET activation parallels a RAS pathway to contribute to human and mouse cutaneous cancers.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/metabolismo , Queratinócitos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Animais , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/patologia , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
Maintaining genomic integrity is of paramount importance to embryonic stem cells (ESCs), as mutations are readily propagated to daughter cells. ESCs display hypersensitivity to DNA damage-induced apoptosis (DIA) to prevent such propagation, although the molecular mechanisms underlying this apoptotic response are unclear. Here, we report that the regulatory RNA Apela positively regulates p53-mediated DIA. Apela is highly expressed in mouse ESCs and is repressed by p53 activation, and Apela depletion compromises p53-dependent DIA. Although Apela contains a coding region, this coding ability is dispensable for Apela's role in p53-mediated DIA. Instead, Apela functions as a regulatory RNA and interacts with hnRNPL, which prevents the mitochondrial localization and activation of p53. Together, these results describe a tri-element negative feedback loop composed of p53, Apela, and hnRNPL that regulates p53-mediated DIA, and they further demonstrate that regulatory RNAs add a layer of complexity to the apoptotic response of ESCs after DNA damage.