RESUMO
Despite advancements in treatment options, the overall cure and survival rates for non-small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC. SIGNIFICANCE: Epigenome-wide CRISPR knockout screens identify NPM1 as a novel metabolic vulnerability and demonstrate that targeting NPM1 is a new therapeutic opportunity for patients with NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Técnicas Genéticas , Neoplasias Pulmonares , Proteínas Nucleares/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epigênese Genética , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Nucleares/genética , NucleofosminaRESUMO
Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras G12D/Trp53 -/- LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Chaperonas Moleculares/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Epigênese Genética/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Chaperonas Moleculares/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Guia de Cinetoplastídeos/genética , RNA Interferente Pequeno/metabolismo , RNA-Seq , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/genéticaRESUMO
We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in an H-2b-restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8+ cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Receptores ErbB/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Afatinib/administração & dosagem , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos T CD8-Positivos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Técnicas de Cocultura , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Luciferases de Vaga-Lume/genética , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Linfócitos T Citotóxicos/imunologiaRESUMO
B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel) was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC(50) values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLyS-gel induced cell death was caspase-independent and shown to be at least partially mediated by the "ribotoxic stress response." This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic "payloads" to malignant B cells.
Assuntos
Fator Ativador de Células B/metabolismo , Linfoma de Células B/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Animais , Fator Ativador de Células B/genética , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos SCID , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As a part of an ongoing assessment of its mechanism of action, we evaluated the in vivo pharmacokinetics, tissue distribution, toxicity and antitumor efficacy of VEGF(121)/rGel, a novel fusion protein. Pharmacokinetic studies showed that VEGF(121)/rGel cleared from the circulation in a biphasic manner with calculated half-lives of 0.3 and 6h for the alpha and beta phases, respectively. Pharmacokinetic evaluation of (64)Cu-DOTA-VEGF(121)/rGel showed relatively high blood retention 30 min after injection (26.6 ± 1.73% ID/g), dropping to 11.8 ± 2.83% and 0.82 ± 0.11% ID/g at 60 and 240 min post injection, respectively. Tissue uptake studies showed that kidneys, liver and tumor had the highest drug concentrations 48 h after administration. The maximum tolerated dose (MTD), based on a QOD×5 i.v. administration schedule, was found to be 18 mg/kg with an LD(50) of 25mg/kg. Treatment of BALB/c mice with VEGF(121)/rGel at doses up to the MTD caused no alterations in hematologic parameters. However, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) parameters increased in a dose-related manner. The no-observable-adverse-effect-level (NOAEL) was determined to be 20% of the MTD (3.6 mg/kg). VEGF(121)/rGel treatment of mice bearing orthotopically-placed MDA-MB-231 breast tumors caused increased vascular permeability of tumor tissue by 53% compared to saline-treated controls. Immunohistochemical analysis showed significant tumor hypoxia and necrosis as a consequence of vascular damage. In summary, VEGF(121)/rGel appears to be an effective therapeutic agent causing focused damage to tumor vasculature with minimal toxic effects to normal organs. This agent appears to be an excellent candidate for further clinical development.
Assuntos
Fator A de Crescimento do Endotélio Vascular/farmacocinética , Alanina Transaminase/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Aspartato Aminotransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
IMPORTANCE OF THE FIELD: Agents that activate the TNF-related apoptosis-inducing ligand death receptors, TRAIL-R1 and TRAIL-R2, have attracted substantial attention and investment as potential anti-cancer therapies. Preclinical studies of TRAIL-R agonists indicate that they may be efficacious in a wide range of tumor types, especially when combined with chemotherapeutic agents. AREAS COVERED IN THIS REVIEW: The rationale for clinical development of TRAIL-R agonists is described, including the basis for combining these agents with other agents that modulate the 'checks and balances' of the apoptotic pathways. Accruing data that highlight differences between TRAIL-R1 and TRAIL-R2 that could affect the clinical significance of their specific agonists are described. The clinical experience to date with each of the agonists is summarized. WHAT THE READER WILL GAIN: The reader will gain an understanding of the rationale for the clinical development of TRAIL-R agonists, as well as the current status of clinical trials of these interesting new agents. TAKE HOME MESSAGE: Ongoing clinical trials will provide important information regarding the future development of TRAIL-R agonists.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Animais , HumanosRESUMO
PURPOSE: The vascular targeting antibody bavituximab is being combined with chemotherapy in clinical trials in cancer patients. Bavituximab targets the membrane phospholipid, phosphatidylserine, complexed with beta2-glycoprotein I. Phosphatidylserine is normally intracellular but becomes exposed on the luminal surface of vascular endothelium in tumors. Phosphatidylserine exposure on tumor vessels is increased by chemotherapy and irradiation. Here, we determined whether treatment with the murine equivalent of bavituximab, 2aG4, combined with irradiation can suppress tumor growth in a rat model of glioblastoma. EXPERIMENTAL DESIGN: F98 glioma cells were injected into the brains of syngeneic rats where they grow initially as a solid tumor and then infiltrate throughout the brain. Rats with established tumors were treated with 10 Gy whole brain irradiation and 2aG4. RESULTS: Combination treatment doubled the median survival time of the rats, and 13% of animals were rendered disease free. Neither treatment given individually was as effective. We identified two mechanisms. First, irradiation induced phosphatidylserine exposure on tumor blood vessels and enhanced antibody-mediated destruction of tumor vasculature by monocytes/macrophages. Second, the antibody treatment induced immunity to F98 tumor cells, which are normally weakly immunogenic. Surviving rats were immune to rechallenge with F98 tumor cells. In vitro, 2aG4 enhanced the ability of dendritic cells (DCs) to generate F98-specific cytotoxic T cells. Phosphatidylserine exposure, which is induced on tumor cells by irradiation, likely suppresses tumor antigen presentation, and 2aG4 blocks this tolerogenic effect. CONCLUSION: Bavituximab combined with radiotherapy holds promise as a vascular targeting and immune enhancement strategy for the treatment of human glioblastoma.
Assuntos
Anticorpos Monoclonais/química , Glioblastoma/imunologia , Glioblastoma/terapia , Fosfatidilserinas/química , Animais , Anticorpos Monoclonais/farmacologia , Terapia Combinada/métodos , Células Dendríticas/citologia , Modelos Animais de Doenças , Células Endoteliais/citologia , Glioblastoma/irrigação sanguínea , Glioblastoma/radioterapia , Glioma/patologia , Humanos , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Neovascularização Patológica , Ratos , Ratos Endogâmicos F344RESUMO
Mapatumumab and lexatumumab are fully human monoclonal antibodies that bind and activate human tumor necrosis factor-related apoptosis-inducing ligand receptors 1 and 2, respectively. These antibodies induce apoptosis in various tumor cell types, although the degree of sensitivity can vary from highly sensitive to completely resistant. Importantly, tumor cells that are partially or completely resistant to mapatumumab or lexatumumab can often be sensitized when treated in combination with chemotherapeutic drugs. In this regard, the proteasome inhibitor bortezomib has recently shown synergistic activity against established lymphoma cell lines and primary lymphomas when combined with mapatumumab and lexatumumab. Here, we report similar findings using a panel of human non-small cell lung cancer (NSCLC) cell lines. Specifically, we show that bortezomib rapidly induces sensitivity to mapatumumab and lexatumumab in NSCLC cell lines that are completely resistant to antibody alone and that bortezomib concentrations as low as 25 nmol/L sensitize NSCLC cells to the antibodies. Furthermore, bortezomib at the tested concentration has minimal effect on its own, indicating the combination generates synergistic cytotoxicity. Combination treatment induces activation of the caspase cascade and the effect of the combination is caspase dependent. Bortezomib treatment increases the intracellular levels of several important apoptosis regulators that may mediate enhanced sensitivity to mapatumumab and lexatumumab. These results suggest future evaluation of mapatumumab or lexatumumab in combination with bortezomib is warranted in NSCLC patients.
Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pirazinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Anticorpos/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Bortezomib , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologiaRESUMO
PURPOSE: New treatment strategies aimed at damaging tumor vasculature could potentially improve tumor response to radiation therapy. We recently showed that anionic phospholipids, principally phosphatidylserine, are specifically exposed on the luminal surface of tumor blood vessels. Here we tested the hypothesis that radiation therapy can increase phosphatidylserine exposure on lung tumor vasculature, thereby enhancing the antitumor properties of the anti-phosphatidylserine antibody 2aG4. EXPERIMENTAL DESIGN: The therapeutic efficacy of radiation therapy plus 2aG4 was tested in nude mice bearing radiation-resistant A549 human lung tumors. Radiation-induced phosphatidylserine exposure on endothelial cells and A549 tumor cells was analyzed by immunofluoresence staining. The mechanism of the enhanced antitumor effect was examined by histology and antibody-dependent cell-mediated cytotoxicity experiments. RESULTS: Focal irradiation of A549 human lung cancer xenografts increased the percentage of tumor vessels with exposed phosphatidylserine from 4% to 26%. Treatment of mice bearing A549 tumors with 2aG4 plus focal radiation therapy inhibited tumor growth by 80% and was superior to radiation therapy or 2aG4 alone (P < 0.01). Combination therapy reduced blood vessel density and enhanced monocyte infiltration into the tumor mass beyond that observed with individual treatments. In vitro, 2aG4 enhanced the ability of macrophages to kill endothelial cells with exposed phosphatidylserine in an Fc'-dependent manner. CONCLUSION: These results suggest that 2aG4 enhances the antitumor effects of radiation therapy by increasing antibody-dependent cell-mediated cytotoxicity toward tumor vessels with externalized phosphatidylserine. Bavituximab, a chimeric version of 2aG4 in clinical trials, has the potential to enhance the therapeutic efficacy of radiation therapy in lung cancer patients.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/terapia , Fosfatidilserinas/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Terapia Combinada , Células Endoteliais/citologia , Humanos , Neoplasias Pulmonares/patologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos SCID , Monócitos/fisiologia , Transplante de Neoplasias , Transplante HeterólogoRESUMO
A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane of resting mammalian cells. We have shown previously that PS becomes exposed on the surface of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed. 3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice. A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4 and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein beta-2-glycoprotein 1 (beta2GP1). beta2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show that 3G4 enhances binding of beta2GP1 to EC induced to expose PS. We also show that divalent 3G4-beta2GP1 complexes are required for enhanced binding, since 3G4 Fab' fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial dimeric beta2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by dimerization of beta2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of beta2GP1 for anionic phospholipids through formation of multivalent 3G4-beta2GP1 complexes.
Assuntos
Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Sítios de Ligação de Anticorpos , Endotélio Vascular/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Fosfatidilserinas/metabolismo , beta 2-Glicoproteína I/fisiologia , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Bovinos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/uso terapêutico , Humanos , Camundongos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Fosfatidilserinas/imunologia , beta 2-Glicoproteína I/sangueRESUMO
Pancreatic cancer continues to have a dismal prognosis and novel therapy is needed. In this study, we evaluate a promising new target for therapy, phosphatidylserine (PS). PS is an anionic phospholipid located normally on the inner leaflet of the plasma membrane in mammalian cells. In the tumor microenvironment, PS becomes externalized on vascular endothelium. The monoclonal antibody 3G4 binds PS and promotes an inflammatory response against tumor blood vessels, resulting in reduction of tumor growth. Mice with orthotopic pancreatic tumors were treated with 3G4, gemcitabine or a combination of both drugs. Tumor burden including pancreas weight and metastatic lesions (liver, lymph node and peritoneal) were reduced 3- to 5-fold by the combination therapy as compared with 1.5- to 2-fold with 3G4 and gemcitabine alone, respectively. Treatment of tumor-bearing animals with the combination therapy increased macrophage infiltration into the tumor mass 10-fold and reduced microvessel density in the tumor by 2.5-fold compared with tumors from untreated animals. Gemcitabine alone and 3G4 alone were less effective than the combination of the 2 agents together. The additive therapeutic effect of both agents appears to be because chemotherapy increases PS exposure on tumor vascular endothelium and amplifies the target for attack by 3G4. In conclusion, 3G4 enhanced the anti-tumor and anti-metastatic activity of gemcitabine without contributing to toxicity.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Fosfatidilserinas/imunologia , Animais , Anticorpos Monoclonais/efeitos adversos , Terapia Combinada , Desoxicitidina/uso terapêutico , Camundongos , Camundongos Nus , Metástase Neoplásica/prevenção & controle , Neoplasias Pancreáticas/patologia , Fosfatidilserinas/fisiologia , Transplante Heterólogo , GencitabinaRESUMO
VEGF(121)/rGel, a fusion protein composed of the growth factor VEGF(121) and the recombinant toxin gelonin (rGel), targets the tumor neovasculature and exerts impressive cytotoxic effects by inhibiting protein synthesis. We evaluated the effect of VEGF(121)/rGel on the growth of metastatic MDA-MB-231 tumor cells in SCID mice. VEGF(121)/rGel treatment reduced surface lung tumor foci by 58% compared to controls (means were 22.4 and 53.3, respectively; P < .05) and the mean area of lung colonies by 50% (210 +/- 37 m(2) vs 415 +/- 10 m(2) for VEGF(121)/rGel and control, respectively; P < .01). In addition, the vascularity of metastatic foci was significantly reduced (198 +/- 37 vs 388 +/- 21 vessels/mm(2) for treated and control, respectively). Approximately 62% of metastatic colonies from the VEGF(121)/rGel-treated group had fewer than 10 vessels per colony compared to 23% in the control group. The VEGF receptor Flk-1 was intensely detected on the metastatic vessels in the control but not in the VEGF(121)/rGel-treated group. Metastatic foci present in lungs had a three-fold lower Ki-67 labeling index compared to control tumors. Thus, the antitumor vascular-ablative effect of VEGF(121)/rGel may be utilized not only for treating primary tumors but also for inhibiting metastatic spread and vascularization of metastases.
Assuntos
Neoplasias Pulmonares/secundário , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Imunoprecipitação , Concentração Inibidora 50 , Antígeno Ki-67/biossíntese , Pulmão/patologia , Neoplasias Pulmonares/prevenção & controle , Camundongos , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Neovascularização Patológica , RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Previous studies have shown that there is a strict requirement for fibroblast growth factor-4 (FGF-4) during mammalian embryogenesis, and that FGF-4 expression in embryonic stem (ES) cells and embryonal carcinoma (EC) cells are controlled by a powerful downstream distal enhancer. More recently, mouse ES cells were shown to express significantly more FGF-4 mRNA than human ES cells. In the work reported here, we demonstrate that mouse EC cells also express far more FGF-4 mRNA than human EC cells. Using a panel of FGF-4 promoter/reporter gene constructs, we demonstrate that the enhancer of the mouse FGF-4 gene is approximately tenfold more active than its human counterpart. Moreover, we demonstrate that the critical difference between the mouse and the human FGF-4 enhancer is a 4 bp difference in the sequence of an essential GT box. Importantly, we demonstrate that changing 4 bp in the human enhancer to match the sequence of the mouse GT box elevates the activity of the human FGF-4 enhancer to the same level as that of the mouse enhancer. We extended these studies by examining the roles of Sp1 and Sp3 in FGF-4 expression. Although we demonstrate that Sp3, but not Sp1, can activate the FGF-4 promoter when artificially tethered to the FGF-4 enhancer, we show that Sp3 is not essential for expression of FGF-4 mRNA in mouse ES cells. Finally, our studies with human EC cells suggest that the factor responsible for mediating the effect of the mouse GT box is unlikely to be Sp1 or Sp3, and this factor is either not expressed in human EC cells or it is not sufficiently active in these cells.
Assuntos
Embrião de Mamíferos/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Fatores de Crescimento de Fibroblastos/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Células-Tronco/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Células-Tronco de Carcinoma Embrionário , Elementos Facilitadores Genéticos/genética , Fator 4 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Células-Tronco Neoplásicas/fisiologia , Proteínas Proto-Oncogênicas/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3 , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The exact mechanisms by which enhancers regulate transcription are currently under investigation. For some genes, activation is accomplished by an intricate array of enhancer cis-regulatory elements that direct the assembly of a gene-specific activation complex known as an "enhanceosome". Transcription of the fibroblast growth factor-4 (FGF-4) gene during early development is controlled by a powerful distal enhancer located 3 kb downstream of the transcription start site within the 3' untranslated region of the gene. Previous studies have shown that FGF-4 enhancer function is mediated by at least three critical positive cis-regulatory elements: an HMG, a POU, and a GT-box motif, which bind the transcription factors Sox-2, Oct-3, and Sp1/Sp3, respectively. In this study, we identify a second essential HMG motif within the FGF-4 enhancer that binds the transcription factor Sox-2. Moreover, we demonstrate that spatial alignment of the new HMG motif, relative the other enhancer cis-regulatory elements, is important. Based on findings presented in this report, and work published earlier, we propose that the previously identified core HMG and POU cis-regulatory elements of the FGF-4 enhancer are dependent on one another and function in an enhanceosome-like manner. In contrast, the HMG motif identified in this study is only partially dependent on the other enhancer cis-regulatory elements for its function.
Assuntos
Elementos Facilitadores Genéticos/genética , Fatores de Crescimento de Fibroblastos/genética , Proteínas Proto-Oncogênicas/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Extratos Celulares/química , Linhagem Celular Tumoral , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Fator 4 de Crescimento de Fibroblastos , Regulação da Expressão Gênica , Domínios HMG-Box/genética , Proteínas HMGB , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Plasmídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The transcription factor B-Myb is a cell-cycle regulated phosphoprotein involved in cell cycle progression through the transcriptional regulation of many genes. In this study, we show that the promoter of the fibroblast growth factor-4 (FGF-4) gene is strongly activated by B-Myb in HeLa cells and it can serve as a novel diagnostic tool for assessing B-Myb activity. Specifically, B-Myb deletion mutants were examined and domains of B-Myb required for activation of the FGF-4 promoter were identified. Using phosphorylation-deficient mutant forms of B-Myb, we also show that phosphorylation is essential for B-Myb activity. Moreover, a mutant form of B-Myb, which lacks all identified phosphorylation sites and which has little activity, can function as a dominant-negative and suppress wild-type B-Myb activity. Acetylation is another post-translational modification known to affect the activity of other Myb family members. We show that B-Myb is acetylated by the co-activator p300. We also show that the bromo and histone acetyltransferase domains of p300 are sufficient to interact with and acetylate B-Myb. These data indicate that phosphorylation of B-Myb is an essential modification for activity and that acetylation of B-Myb may play a role in B-Myb activity.