RESUMO
Naive CD4+ T cells differentiate into functionally diverse T helper (Th) cell subsets. Th2 cells play a pathogenic role in asthma, yet a clear picture of their transcriptional profile is lacking. We performed single-cell RNA sequencing (scRNA-seq) of T helper cells from lymph node, lung, and airways in the house dust mite (HDM) model of allergic airway disease. scRNA-seq resolved transcriptional profiles of naive CD4+ T, Th1, Th2, regulatory T (Treg) cells, and a CD4+ T cell population responsive to type I interferons. Th2 cells in the airways were enriched for transcription of many genes, including Cd200r1, Il6, Plac8, and Igfbp7, and their mRNA profile was supported by analysis of chromatin accessibility and flow cytometry. Pathways associated with lipid metabolism were enriched in Th2 cells, and experiments with inhibitors of key metabolic pathways supported roles for glucose and lipid metabolism. These findings provide insight into the differentiation of pathogenic Th2 cells in the context of allergy.
Assuntos
Asma/imunologia , Hipersensibilidade Respiratória/imunologia , Sistema Respiratório/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th2/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Modelos Animais de Doenças , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Orexina/genética , Pyroglyphidae/imunologia , Análise de Sequência de RNA , Análise de Célula Única , TranscriptomaRESUMO
Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet-associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Gotículas Lipídicas , Proteínas Proto-Oncogênicas c-myc , Humanos , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glutamina/metabolismo , Neoplasias Renais/patologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Regulação para Cima , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
Immunotherapy has revolutionized cancer treatment, but many cancers are not impacted by currently available immunotherapeutic strategies. Here, we investigated inflammatory signaling pathways in neuroblastoma, a classically "cold" pediatric cancer. By testing the functional response of a panel of 20 diverse neuroblastoma cell lines to three different inflammatory stimuli, we found that all cell lines have intact interferon signaling, and all but one lack functional cytosolic DNA sensing via cGAS-STING. However, double-stranded RNA (dsRNA) sensing via Toll-like receptor 3 (TLR3) was heterogeneous, as was signaling through other dsRNA sensors and TLRs more broadly. Seven cell lines showed robust response to dsRNA, six of which are in the mesenchymal epigenetic state, while all unresponsive cell lines are in the adrenergic state. Genetically switching adrenergic cell lines toward the mesenchymal state fully restored responsiveness. In responsive cells, dsRNA sensing results in the secretion of proinflammatory cytokines, enrichment of inflammatory transcriptomic signatures, and increased tumor killing by T cells in vitro. Using single-cell RNA sequencing data, we show that human neuroblastoma cells with stronger mesenchymal signatures have a higher basal inflammatory state, demonstrating intratumoral heterogeneity in inflammatory signaling that has significant implications for immunotherapeutic strategies in this aggressive childhood cancer.
Assuntos
Epigênese Genética/genética , Inflamação/genética , Neuroblastoma/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/genética , Humanos , Fatores Imunológicos/genética , Imunoterapia/métodos , Masculino , Camundongos , Camundongos SCID , Nucleotidiltransferases/genética , RNA de Cadeia Dupla/genética , Transdução de Sinais/genética , Receptor 3 Toll-Like/genética , Transcriptoma/genéticaRESUMO
Engineering liquid-liquid phase separation (LLPS) of proteins and peptides holds great promise for the development of therapeutic carriers with intracellular delivery capability but requires accurate determination of their assembly properties in vitro, usually with fluorescently labeled cargo. Here, we use mass spectrometry (MS) to investigate redox-sensitive coacervate microdroplets (the dense phase formed during LLPS) assembled from a short His- and Tyr-rich peptide. We can monitor the enrichment of a reduced peptide in dilute phase as the microdroplets dissolve triggered by their redox-sensitive side chain, thus providing a quantitative readout for disassembly. Furthermore, MS can detect the release of a short peptide from coacervates under reducing conditions. In summary, with MS, we can monitor the disassembly and cargo release of engineered coacervates used as therapeutic carriers without the need for additional labels.
Assuntos
Peptídeos , Proteínas , Peptídeos/química , Proteínas/química , Espectrometria de MassasRESUMO
Stemness encompasses the capability of a cell for self-renewal and differentiation. The stem cell maintains a balance between proliferation, quiescence, and regeneration via interactions with the microenvironment. Previously, we showed that ectopic expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to immortalization of primary fibroblasts, accompanied by induction of an embryonic stem cell (ESC) phenotype. Moreover, we demonstrated interaction between S18-2 and the retinoblastoma-associated protein (RB) and hypothesized that the simultaneous expression of RB and S18-2 is essential for maintaining cell stemness. Here, we experimentally investigated the role of S18-2 in cell stemness and differentiation. Concurrent expression of RB and S18-2 resulted in immortalization of Rb1-/- primary mouse embryonic fibroblasts and in aggressive tumor growth in severe combined immunodeficiency mice. These cells, which express both RB and S18-2 at high levels, exhibited the potential to differentiate into various lineages in vitro, including osteogenic, chondrogenic, and adipogenic lineages. Mechanistically, S18-2 formed a multimeric protein complex with prohibitin and the ring finger protein 2 (RNF2). This molecular complex increased the monoubiquitination of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3-ligase activity of RNF2. Furthermore, we found enrichment of KLF4 at the S18-2 promoter region and that the S18-2 expression is positively correlated with KLF4 levels. Importantly, knockdown of S18-2 in zebrafish larvae led to embryonic lethality. Collectively, our findings suggest an important role for S18-2 in cell stemness and differentiation and potentially also in cancerogenesis.
Assuntos
Mitocôndrias/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas Ribossômicas/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Autorrenovação Celular/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/genética , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Mitocôndrias/metabolismo , Complexo Repressor Polycomb 1/genética , Proteínas Ribossômicas/química , Microambiente Tumoral/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Células Matadoras Naturais/imunologia , Melanoma/tratamento farmacológico , Receptores Imunológicos/antagonistas & inibidores , Macrófagos Associados a Tumor/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Células Matadoras Naturais/metabolismo , Masculino , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Knockout , Cultura Primária de Células , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismoRESUMO
BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.
Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Luteolina/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/metabolismo , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Retículo Endoplasmático/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Transdução de SinaisRESUMO
Regulation of ribosomal transcription is under tight control from environmental stimuli, and this control involves changes in the chromatin structure. The underlying mechanism of how chromatin changes in response to nutrient and energy supply in the cell is still unclear. The chromatin-remodeling complex B-WICH is involved in activating the ribosomal transcription, and we show here that knock down of the B-WICH component WSTF results in cells that do not respond to glucose. The promoter is less accessible, and RNA pol I and its transcription factors SL1/TIF-1B and RRN3/TIF-1A, as well as the proto-oncogene c-MYC and the activating deacetylase SIRT7 do not bind upon glucose stimulation. In contrast, the repressive chromatin state that forms after glucose deprivation is reversible, and RNA pol I factors are recruited. WSTF knock down results in an accumulation of the ATPase CHD4, a component of the NuRD chromatin remodeling complex, which is responsible for establishing a repressive poised state at the promoter. The TTF-1, which binds and affect the binding of the chromatin complexes, is important to control the association of activating chromatin component UBF. We suggest that B-WICH is required to allow for a shift to an active chromatin state upon environmental stimulation, by counteracting the repressive state induced by the NuRD complex.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Glucose/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Ribossomos/genética , Transcrição Gênica/genética , Adenosina Trifosfatases/genética , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase I/genética , Sirtuínas/genética , Fatores de Transcrição/genéticaRESUMO
Neuroblastoma (NB) is a remarkably heterogenic childhood tumor of the sympathetic nervous system with clinical behavior ranging from spontaneous regression to poorly differentiated tumors and metastasis. MYCN is amplified in 20% of cases and correlates with an undifferentiated, aggressive phenotype and poor prognosis. Estrogen receptor alpha (ERα) and the nerve growth factor (NGF) receptors TrkA and p75NTR are involved in neuronal differentiation and survival. We have previously shown that MYCN, via miR-18a, targets ERα in NB cells. Here, we demonstrate that interference with miR-18a or overexpression of ERα is sufficient to induce NGF signaling and to modulate both basal and NGF-induced neuronal differentiation in MYCN-amplified NB cells. Proteomic analysis confirmed an increase of neuronal features and showed that processes linked to tumor initiation and progression were inhibited upon ERα overexpression. Indeed, ectopic ERα expression was sufficient to inhibit metabolic activity and tumorigenic processes, including glycolysis, oxidative phosphorylation, cell viability, migration, and anchorage independent growth. Importantly, ERα overexpression reduced tumor burden in NB mouse models and high ERα levels were linked to improved survival in patients. In addition to ERα, several other nuclear hormone receptors (NHRs), including the glucocorticoid and the retinoic acid receptors, correlated with clinical markers for favorable and low-stage NB disease. Our data suggest that MYCN targets ERα and thereby NGF signaling to maintain an undifferentiated and aggressive phenotype. Notably, we identified the estrogen-NGF crosstalk, as well as a set of other NHRs, as potential prognostic markers and targets for therapeutic strategies against NB.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Proto-Oncogênica N-Myc/genética , Fator de Crescimento Neural/farmacologia , Neuroblastoma/patologia , Animais , Diferenciação Celular , Receptor alfa de Estrogênio/genética , Amplificação de Genes , Humanos , Camundongos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fenótipo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The majority of solid tumors are presented with an inflammatory microenvironment. Proinflammatory lipid mediators including prostaglandin E2 (PGE2) contribute to the establishment of inflammation and have been linked to tumor growth and aggressiveness. Here we show that high-risk neuroblastoma with deletion of chromosome 11q represents an inflammatory subset of neuroblastomas. Analysis of enzymes involved in the production of proinflammatory lipid mediators showed that 11q-deleted neuroblastoma tumors express high levels of microsomal prostaglandin E synthase-1 (mPGES-1) and elevated levels of PGE2. High mPGES-1 expression also corresponded to poor survival of neuroblastoma patients. Investigation of the tumor microenvironment showed high infiltration of tumor-promoting macrophages with high expression of the M2-polarization markers CD163 and CD206. mPGES-1-expressing cells in tumors from different subtypes of neuroblastoma showed differential expression of one or several cancer-associated fibroblast markers such as vimentin, fibroblast activation protein α, α smooth muscle actin, and PDGF receptor ß. Importantly, inhibition of PGE2 production with diclofenac, a nonselective COX inhibitor, resulted in reduced tumor growth in an in vivo model of 11q-deleted neuroblastoma. Collectively, these results suggest that PGE2 is involved in the tumor microenvironment of specific neuroblastoma subgroups and indicate that therapeutic strategies using existing anti-inflammatory drugs in combination with current treatment should be considered for certain neuroblastomas.
Assuntos
Dinoprostona/metabolismo , Inflamação/metabolismo , Oxirredutases Intramoleculares/metabolismo , Neuroblastoma/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Deleção Cromossômica , Cromossomos Humanos Par 11 , Modelos Animais de Doenças , Humanos , Inflamação/enzimologia , Inflamação/patologia , Oxirredutases Intramoleculares/genética , Camundongos , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Prostaglandina-E Sintases , RNA Mensageiro/genética , Microambiente TumoralRESUMO
The p53-family member TAp73 is known to function as a tumor suppressor and regulates genomic integrity, cellular proliferation, and apoptosis; however, its role in tumor angiogenesis is poorly understood. Here we demonstrate that TAp73 regulates tumor angiogenesis through repression of proangiogenic and proinflammatory cytokines. Importantly, loss of TAp73 results in highly vascularized tumors, as well as an increase in vessel permeability resulting from disruption of vascular endothelial-cadherin junctions between endothelial cells. In contrast, loss of the oncogenic p73 isoform ΔNp73 leads to reduced blood vessel formation in tumors. Furthermore, we show that up-regulated ΔNp73 levels are associated with increased angiogenesis in human breast cancer and that inhibition of TAp73 results in an accumulation of HIF-1α and up-regulation of HIF-1α target genes. Taken together, our data demonstrate that loss of TAp73 or ΔNp73 up-regulation activates the angiogenic switch that stimulates tumor growth and progression.
Assuntos
Indutores da Angiogênese/metabolismo , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Neoplasias da Mama/patologia , Caderinas/metabolismo , Hipóxia Celular , Linhagem Celular Transformada , Proliferação de Células , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , Neovascularização Patológica/patologia , Neovascularização Fisiológica , Permeabilidade , Isoformas de Proteínas/metabolismo , Proteína Tumoral p73 , Peixe-ZebraRESUMO
Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and DeltaNp73 isoforms. As part of our ongoing program to distinguish the biological functions of these isoforms, we generated mice that are selectively deficient for the DeltaNp73 isoform. Mice lacking DeltaNp73 (DeltaNp73(-/-) mice) are viable and fertile but display signs of neurodegeneration. Cells from DeltaNp73(-/-) mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in DeltaNp73(-/-) cells, we discovered a completely new role for DeltaNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. We found that DeltaNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of DeltaNp73 expression show enhanced resistance to chemotherapy.
Assuntos
Dano ao DNA , Reparo do DNA/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais , Animais , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia , Encéfalo/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Feminino , Fertilidade/genética , Regulação da Expressão Gênica , Células HCT116 , Humanos , Longevidade/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Neurodegenerativas/genética , Fosforilação , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Tumorigenesis is a complex process involving dynamic interactions between malignant cells and their surrounding stroma, including both the cellular and acellular components. Within the stroma, fibroblasts represent not only a predominant cell type, but also a major source of the acellular tissue microenvironment comprising the extracellular matrix (ECM) and soluble factors. Normal fibroblasts can exert diverse suppressive functions against cancer initiating and metastatic cells via direct cell-cell contact, paracrine signaling by soluble factors, and ECM integrity. The loss of such suppressive functions is an inherent step in tumor progression. A tumor cell-induced switch of normal fibroblasts into cancer-associated fibroblasts (CAFs), in turn, triggers a range of pro-tumorigenic signals accompanied by distraction of the normal tissue architecture, thus creating an optimal niche for cancer cells to grow extensively. To further support tumor progression and metastasis, CAFs secrete factors such as ECM remodeling enzymes that further modify the tumor microenvironment in combination with the altered adhesive forces and cell-cell interactions. These paradoxical tumor suppressive and promoting actions of fibroblasts are the focus of this review, highlighting the heterogenic molecular properties of both normal and cancer-associated fibroblasts, as well as their main mechanisms of action, including the emerging impact on immunomodulation and different therapy responses.
Assuntos
Fibroblastos Associados a Câncer/patologia , Neoplasias/patologia , Microambiente Tumoral , Animais , Fibroblastos Associados a Câncer/citologia , Fibroblastos Associados a Câncer/imunologia , Fibroblastos Associados a Câncer/metabolismo , Progressão da Doença , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Imunomodulação , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapiaRESUMO
Neuroblastoma (NB) is a childhood tumor that arises from the sympathoadrenal lineage. MYCN amplification is the most reliable marker for poor prognosis and MYCN overexpression in embryonic mouse sympathetic ganglia results in NB-like tumors. MYCN cooperates with mutational activation of anaplastic lymphoma kinase (ALK), which promotes progression to NB, but the role of MYCN and ALK in tumorigenesis is still poorly understood. Here, we use chick sympathetic neuroblasts to examine the normal function of MYCN and MYC in the control of neuroblast proliferation, as well as effects of overexpression of MYCN, MYC, and activated ALK, alone and in combination. We demonstrate that MYC is more strongly expressed than MYCN during neurogenesis and is important for in vitro neuroblast proliferation. MYC and MYCN overexpression elicits increased proliferation but does not sustain neuroblast survival. Unexpectedly, long-term expression of activated ALKF1174L leads to cell-cycle arrest and promotes differentiation and survival of postmitotic neurons. ALKF1174L induces NEFM, RET, and VACHT and results in decreased expression of proapototic (BMF, BIM), adrenergic (TH), and cell-cycle genes (e.g., CDC25A, CDK1). In contrast, neuroblast proliferation is maintained when MYCN and ALKF1174L are coexpressed. Proliferating MYCN/ALKF1174L neuroblasts display a differentiated phenotype but differ from ALK-expressing neurons by the upregulation of SKP2, CCNA2, E2F8, and DKC1 Inhibition of the ubiquitin ligase SKP2 (S-phase kinase-associated protein 2), which targets the CDK inhibitor p27 for degradation, reduces neuroblast proliferation, implicating SKP2 in the maintained proliferation of MYCN/ALKF1174L neuroblasts. Together, our results characterize MYCN/ALK cooperation leading to neuroblast proliferation and survival that may represent initial steps toward NB development. SIGNIFICANCE STATEMENT: MYCN overexpression combined with activated anaplastic lymphoma kinase (ALK) is sufficient to induce neuroblastoma (NB) in mouse sympathoadrenal cells. To address cellular and molecular effects elicited by MYCN/ALK cooperation, we used cultures of chick sympathetic neuroblasts. We demonstrate that MYCN increases proliferation but not survival, whereas long-term expression of ALKF1174L elicits cell-cycle exit, differentiation, and survival of postmitotic neurons. Combined MYCN/ALKF1174L expression allows long-term proliferation and survival of neuroblasts with differentiated characteristics. In the presence of ALKF1174L signaling, MYCN induces the expression of the ubiquitin ligase SKP2 (S-phase kinase-associated protein 2), which targets p27 for degradation and is also upregulated in high-risk NB. SKP2 inhibition supports a function for SKP2 in the maintained neuroblast proliferation downstream of MYCN/ALK, which may represent an early step toward tumorigenesis.
Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Células-Tronco Neurais , Neuroblastoma/patologia , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Quinase do Linfoma Anaplásico , Animais , Apoptose/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Embrião de Galinha , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neurônios/patologia , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
The transcription factors c-Myc and Mnt regulate gene expression through dimerization with Max and binding to E-boxes in target genes. While c-Myc activates gene expression via recruitment of histone modifying complexes, Mnt acts as a transcriptional repressor. Here, we used the Xenopus leavis oocyte system to address the effect of c-Myc and Mnt on transcription and chromatin remodeling over the E-box region in the human telomerase reverse transcriptase (hTERT) promoter. As expected we found elevated and decreased levels of hTERT transcription upon exogenously expressed c-Myc/Max and Mnt/Max, respectively. In addition, we confirmed binding of these heterodimers to both E-boxes already enriched with H3K9ac and H4K16ac. These chromatin marks were further enhanced upon c-Myc/Max binding followed by increased DNA accessibility in the E-box region. In contrast, Mnt/Max inhibited Myc-induced transcription and mediated repression through complete chromatin condensation and deacetylation of H3K9 and H4K16 across the E-box region. Importantly, Mnt was able to counteract c-Myc mediated activation even when expressed at low levels, suggesting Mnt to act as a strong repressor by closing the chromatin structure. Collectively our data demonstrate that the balance between c-Myc and Mnt activity determines the transcriptional outcome of the hTERT promoter by modulation of the chromatin architecture.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cromatina/metabolismo , Oócitos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Telomerase/genética , Transcrição Gênica/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células Cultivadas , Cromatina/genética , Células HEK293 , Humanos , Oócitos/citologia , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Xenopus laevisRESUMO
Neuroblastoma (NB), an heterogenous pediatric tumor of the sympathetic nervous system, is the most common and deadly extracranial solid malignancy diagnosed in infants. Numerous efforts have been invested in understanding its origin and in development of novel curative targeted therapies. Here, we summarize the recent advances in the identification of the cell of origin and the genetic alterations occurring during development contributing to NB. We discuss current treatment regimens, present and future directions for identification of novel therapeutic metabolic targets, differentiation agents, as well as personalized combinatory therapies as potential approaches for improving survival and quality of life of children with NB.
RESUMO
Therapeutic resistance and recurrence remain core challenges in cancer therapy. How therapy resistance arises is currently not fully understood with tumors surviving via multiple alternative routes. Here, we demonstrate that a subset of cancer cells survives therapeutic stress by entering a transient state characterized by whole-genome doubling. At the onset of the polyploidization program, we identified an upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF2α. We found altered chromatin accessibility, ablated expression of retinoblastoma protein (RB1), and enrichment of AP-1 motif accessibility. We demonstrate that AP-1 and HIF2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF2α reduced the number of surviving cells following chemotherapy treatment. The role of AP-1 and HIF2α in stress response by polyploidy suggests a novel avenue for tackling chemotherapy-induced resistance in cancer. SIGNIFICANCE: In response to cisplatin treatment, some surviving cancer cells undergo whole-genome duplications without mitosis, which represents a mechanism of drug resistance. This study presents mechanistic data to implicate AP-1 and HIF2α signaling in the formation of this surviving cell phenotype. The results open a new avenue for targeting drug-resistant cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fator de Transcrição AP-1/genética , Regulação para Cima , Transdução de Sinais , Neoplasias/tratamento farmacológicoRESUMO
The c-MYC oncogene is activated in over 70% of all human cancers. The intrinsic disorder of the c-MYC transcription factor facilitates molecular interactions that regulate numerous biological pathways, but severely limits efforts to target its function for cancer therapy. Here, we use a reductionist strategy to characterize the dynamic and structural heterogeneity of the c-MYC protein. Using probe-based Molecular Dynamics (MD) simulations and machine learning, we identify a conformational switch in the c-MYC amino-terminal transactivation domain (termed coreMYC) that cycles between a closed, inactive, and an open, active conformation. Using the polyphenol epigallocatechin gallate (EGCG) to modulate the conformational landscape of coreMYC, we show through biophysical and cellular assays that the induction of a closed conformation impedes its interactions with the transformation/transcription domain-associated protein (TRRAP) and the TATA-box binding protein (TBP) which are essential for the transcriptional and oncogenic activities of c-MYC. Together, these findings provide insights into structure-activity relationships of c-MYC, which open avenues towards the development of shape-shifting compounds to target c-MYC as well as other disordered transcription factors for cancer treatment.
Assuntos
Proteínas Proto-Oncogênicas c-myc , Humanos , Ativação Transcricional , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Conformação Molecular , Ligação ProteicaRESUMO
Multimodal contrast agents in biomedical imaging enable the collection of more comprehensive diagnostic information. In the present work, we design hybrid ruthenium-decorated superparamagnetic iron oxide nanoparticles (NPs) as the contrast agents for both magnetic resonance imaging (MRI) and X-ray fluorescence computed tomography (XFCT). The NPs are synthesized via a one-pot polyol hot injection route, in diethylene glycol. In vivo preclinical studies demonstrate the possibility of correlative bioimaging with these contrast agents. The complementarity allows accurate localization, provided by the high contrast of the soft tissues in MRI combined with the elemental selectivity of XFCT, leading to NP detection with high specificity and resolution. We envision that this multimodal imaging could find future applications for early tumor diagnosis, improved long-term treatment monitoring, and enhanced radiotherapy planning.
Assuntos
Meios de Contraste , Tomografia Computadorizada por Raios X , Fluorescência , Tomografia Computadorizada por Raios X/métodos , Imageamento por Ressonância Magnética/métodos , Nanopartículas Magnéticas de Óxido de FerroRESUMO
Aims: To investigate the distribution and toxicity of ruthenium nanoparticles (Ru NPs) injected intravenously in mice. Methods: We synthesized Ru NPs, followed their biodistribution by x-ray fluorescence (XRF) imaging and evaluated organ toxicity by histopathology and gene expression. Results: Ru NPs accumulated, mainly in liver and spleen, where they were phagocyted by tissue macrophages, giving a transient inflammation and oxidative stress response that declined after 2 weeks. Ru NPs gradually accumulated in the skin, which was confirmed by microscopic examination of skin biopsies. Conclusion: Ru NP toxicity in recipient organs is transient. Particles are at least partially excreted by the skin, supporting a role for the skin as a nanoparticle clearing organ.