RESUMO
Sepsis is the culmination of hyperinflammation and immune suppression in response to severe infection. Neutrophils are critical early responders to bacterial infection but can become highly dysfunctional during sepsis and other inflammatory disorders. The transmembrane protease ADAM17 (a disintegrin and metalloproteinase 17) is expressed by leukocytes and most other cells and has many substrates that regulate inflammation. We have reported that conditional knockout mice lacking ADAM17 in all leukocytes had a survival advantage during sepsis, which was associated with improved neutrophil effector functions. These and other findings indicate aberrant ADAM17 activity during sepsis. For this study, we evaluated for the first time the effects of an ADAM17 function blocking monoclonal antibody (mAb) on the pathogenesis of polymicrobial sepsis. Mice treated with the ADAM17 mAb MEDI3622 prior to sepsis induction exhibited significantly decreased mortality. When the ADAM17 mAb was combined with antibiotic administration, sepsis survival was markedly enhanced compared to either intervention alone, which was associated with a significant reduction in plasma levels of various inflammation-related factors. MEDI3622 and antibiotic administration after sepsis induction also significantly improved survival. Our results indicate that the combination of blocking ADAM17 as an immune modulator and appropriate antibiotics may provide a new therapeutic avenue for sepsis treatment.
Assuntos
Proteína ADAM17/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Sepse/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacosRESUMO
Several clinically successful tumor-targeting mAbs induce NK cell effector functions. Human NK cells exclusively recognize tumor-bound IgG by the FcR CD16A (FcγRIIIA). Unlike other NK cell activating receptors, the cell surface density of CD16A can be rapidly downregulated in a cis manner by the metalloproteinase ADAM17 following NK cell stimulation in various manners. CD16A downregulation takes place in cancer patients and this may affect the efficacy of tumor-targeting mAbs. We examined the effects of MEDI3622, a human mAb and potent ADAM17 inhibitor, on NK cell activation by antibody-bound tumor cells. MEDI3622 effectively blocked ADAM17 function in NK cells and caused a marked increase in their production of IFNγ. This was observed for NK cells exposed to different tumor cell lines and therapeutic antibodies, and over a range of effector/target ratios. The augmented release of IFNγ by NK cells was reversed by a function-blocking CD16A mAb. In addition, NK92 cells, a human NK cell line that lacks endogenous FcγRs, expressing a recombinant non-cleavable version of CD16A released significantly higher levels of IFNγ than NK92 cells expressing equivalent levels of wildtype CD16A. Taken together, our data show that MEDI3622 enhances the release of IFNγ by NK cells engaging antibody-bound tumor cells by blocking the shedding of CD16A. These findings support ADAM17 as a dynamic inhibitory checkpoint of the potent activating receptor CD16A, which can be targeted by MEDI3622 to potentially increase the efficacy of anti-tumor therapeutic antibodies.
Assuntos
Proteína ADAM17/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Interferon gama/biossíntese , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Proteína ADAM17/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Humanos , Interferon gama/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/imunologiaRESUMO
INTRODUCTION: Anti-ASCT2 antibody drug conjugate (ADC) MEDI7247 has been under development as a potential anti-cancer therapy for patients with selected relapsed/refractory hematological malignancies and advanced solid tumors by MedImmune. Although promising efficacy was observed in the clinic, pharmacokinetic (PK) analyses observed low exposure of MEDI7247 in phase I hematological patients. To investigate the biodistribution properties of MEDI7247, MEDI7247 and control antibodies were radiolabeled with zirconium-89 and in vitro and in vivo properties characterized. METHODS: MEDI7247 (human anti-ASCT2 antibody conjugated with pyrrolobenzodiazepine (PBD)) and MEDI7519 (MEDI7247 without PBD drug conjugate) and an isotype control antibody drug conjugate construct were conjugated with p-isothiocyanatobenzyl-deferoxamine (Df) and radiolabeled with zirconium-89. In vitro studies included determining the radiochemical purity, protein integrity, immunoreactivity (Lindmo analysis), apparent antigen binding affinity for ASCT2-positive cells by Scatchard analysis and serum stability of the radiolabeled immunoconjugates. In vivo studies included biodistribution and PET/MRI imaging studies of the radiolabeled immunoconjugates in an ASCT2-positive tumor model, HT-29 colorectal carcinoma xenografts. RESULTS: Conditions for the Df-conjugation and radiolabeling of antibody constructs were determined to produce active radioimmunoconjugates. In vivo biodistribution and whole body PET/MRI imaging studies of [89Zr]Zr-Df-MEDI7519 and [89Zr]Zr-Df-MEDI7247 radioimmunoconjugates in HT-29 colon carcinoma xenografts in BALB/c nude mice demonstrated specific tumor localization. However, more rapid blood clearance and non-specific localization in liver was observed for [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 compared to isotype control ADC. Except for liver and bone, other normal tissues demonstrated clearance reflecting the blood clearance for all three constructs and no other abnormal tissue uptake. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: Preclinical biodistribution analyses of [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 showed the biodistribution pattern of anti-ASCT2 ADC MEDI7247 was similar to parental MEDI7519, and both antibodies showed specific tumor uptake compared to an isotype control ADC. This study highlights an important role nuclear medicine imaging techniques can play in early preclinical assessment of drug specificity as part of the drug development pipeline.
Assuntos
Neoplasias do Colo , Imunoconjugados , Camundongos , Animais , Humanos , Distribuição Tecidual , Imunoconjugados/farmacocinética , Camundongos Nus , Tomografia por Emissão de Pósitrons/métodos , Zircônio/química , Linhagem Celular TumoralRESUMO
Natural killer (NK) cells are innate cytotoxic lymphocytes that can recognize assorted determinants on tumor cells and rapidly kill these cells. Due to their anti-tumor effector functions and potential for allogeneic use, various NK cell platforms are being examined for adoptive cell therapies. However, their limited in vivo persistence is a current challenge. Cytokine-mediated activation of these cells is under extensive investigation and interleukin-15 (IL-15) is a particular focus since it drives their activation and proliferation. IL-15 efficacy though is limited in part by its induction of regulatory checkpoints. A disintegrin and metalloproteinase-17 (ADAM17) is broadly expressed by leukocytes, including NK cells, and it plays a central role in cleaving cell surface receptors, a process that regulates cell activation and cell-cell interactions. We report that ADAM17 blockade with a monoclonal antibody markedly increased human NK cell proliferation by IL-15 both in vitro and in a xenograft mouse model. Blocking ADAM17 resulted in a significant increase in surface levels of the homing receptor CD62L on proliferating NK cells. We show that NK cell proliferation in vivo by IL-15 and the augmentation of this process upon blocking ADAM17 are dependent on CD62L. Hence, our findings reveal for the first time that ADAM17 activation in NK cells by IL-15 limits their proliferation, presumably functioning as a feedback system, and that its substrate CD62L has a key role in this process in vivo. ADAM17 blockade in combination with IL-15 may provide a new approach to improve NK cell persistence and function in cancer patients.
Assuntos
Proteína ADAM17/metabolismo , Interleucina-15/farmacologia , Células Matadoras Naturais/citologia , Proteína ADAM17/antagonistas & inibidores , Proteína ADAM17/imunologia , Transferência Adotiva , Animais , Divisão Celular , Ativação Enzimática , Feminino , Xenoenxertos , Humanos , Interleucina-15/metabolismo , Células Matadoras Naturais/enzimologia , Selectina L/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Mutations in the STK11 (LKB1) gene regulate resistance to PD-1/PD-L1 blockade. This study evaluated this association in patients with nonsquamous non-small cell lung cancer (NSCLC) enrolled in three phase I/II trials. STK11 mutations were associated with resistance to the anti-PD-L1 antibody durvalumab (alone/with the anti-CTLA4 antibody tremelimumab) independently of KRAS mutational status, highlighting STK11 as a potential driver of resistance to checkpoint blockade. Retrospective assessments of tumor tissue, whole blood, and serum revealed a unique immune phenotype in patients with STK11 mutations, with increased expression of markers associated with neutrophils (i.e., CXCL2, IL6), Th17 contexture (i.e., IL17A), and immune checkpoints. Associated changes were observed in the periphery. Reduction of STAT3 in the tumor microenvironment using an antisense oligonucleotide reversed immunotherapy resistance in preclinical STK11 knockout models. These results suggest that STK11 mutations may hinder response to checkpoint blockade through mechanisms including suppressive myeloid cell biology, which could be reversed by STAT3-targeted therapy. SIGNIFICANCE: Patients with nonsquamous STK11-mutant (STK11mut) NSCLC are less likely than STK11 wild-type (STK11wt) patients to respond to anti-PD-L1 ± anti-CTLA4 immunotherapies, and their tumors show increased expression of genes and cytokines that activate STAT3 signaling. Preclinically, STAT3 modulation reverses this resistance, suggesting STAT3-targeted agents as potential combination partners for immunotherapies in STK11mut NSCLC.This article is highlighted in the In This Issue feature, p. 2659.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Quinases Proteína-Quinases Ativadas por AMP , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Proteínas Serina-Treonina Quinases/genética , Estudos Retrospectivos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Microambiente TumoralRESUMO
Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.
Assuntos
Regulação Enzimológica da Expressão Gênica , Histona Desacetilases/genética , Proteínas Repressoras/genética , Fator de Transcrição Sp1/fisiologia , Fator de Transcrição Sp3/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Sequência Consenso/fisiologia , Drosophila/citologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Histona Desacetilases/efeitos dos fármacos , Histona Desacetilases/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Distribuição TecidualRESUMO
Glioblastomas are malignant brain tumors that are very difficult to cure, even with aggressive therapy consisting of surgery, chemotherapy, and radiation. Glioblastomas frequently have loss of the phosphatase and tensin homologue (PTEN), leading to the activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway. We examined whether PTEN deficiency leads to radioresistance and whether this can be reversed by nelfinavir, a protease inhibitor that decreases Akt signaling. Nelfinavir decreased Akt phosphorylation and enhanced radiosensitization in U251MG and U87MG glioblastoma cells, both of which are PTEN deficient. In the derivative line U251MG-PTEN, induction of wild-type PTEN with doxycycline decreased P-Akt expression and increased radiosensitivity to a similar extent as nelfinavir. Combining these two approaches had no greater effect on radiosensitivity than either alone. This epistasis-type analysis suggests that the nelfinavir acts along the Akt pathway to radiosensitize cells. However, nelfinavir neither decreased Akt phosphorylation in immortalized human astrocytes nor radiosensitized them. Radiosensitization was also assessed in vivo using a tumor regrowth delay assay in nude mice implanted with U87MG xenografts. The mean time to reach 1,000 mm(3) in the radiation + nelfinavir group was 71 days, as compared with 41, 34, or 45 days for control, nelfinavir alone, or radiation alone groups, respectively. A significant synergistic effect on tumor regrowth was detected between radiation and nelfinavir. (P = 0.01). Nelfinavir also increased the sensitivity of U251MG cells to temozolomide. These results support the clinical investigation of nelfinavir in combination with radiation and temozolomide in future clinical trials for patients with glioblastomas.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/terapia , Dacarbazina/análogos & derivados , Glioblastoma/terapia , Nelfinavir/farmacologia , PTEN Fosfo-Hidrolase/deficiência , Inibidores de Proteases/farmacologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Dacarbazina/farmacologia , Interações Medicamentosas , Resistencia a Medicamentos Antineoplásicos , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Glioblastoma/radioterapia , Humanos , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação , TemozolomidaRESUMO
Epidermal growth factor receptor (EGFR) inhibitors can decrease vascular endothelial growth factor (VEGF) expression and tumor angiogenesis. In the current study, we investigate the molecular pathways by which this occurs using two drugs that have been used in the clinic, gefitinib (Iressa) and erlotinib (Tarceva). The decrease in VEGF expression by gefitinib in SQ20B squamous cell carcinoma cells was opposed by adenoviral expression of Akt in these cells. The hypoxia-inducible factor-1 (HIF-1) binding site located at approximately -1 kbp in the VEGF promoter was not required for down-regulation of promoter activity by gefitinib under normoxia. Furthermore, the drug decreased activity of a reporter containing the -88/+54 region. In a gel shift assay, gefitinib led to decreased retardation of a labeled DNA oligonucleotide probe corresponding to the -88/-66 region of the VEGF promoter, which contains Sp1 binding sites. These effects of gefitinib on VEGF promoter activity and DNA binding were both reversed by Akt expression. Phosphorylation of Sp1 was decreased in the presence of gefitinib. Gefitinib also decreases VEGF expression by decreasing HIF-1alpha expression. This occurs due to decreased protein translation without any change in the level of HIF-1alpha mRNA. Together, these results suggest that gefitinib decreases VEGF expression both by decreasing Sp1 binding to the proximal core VEGF promoter and by down-regulating HIF-1alpha expression. Similar results were obtained with erlotinib in SQ20B and gefitinib in HSC3 squamous carcinoma cells. These results indicate that there are at least two separate mechanisms by which EGFR inhibitors decrease VEGF expression.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Sítios de Ligação , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/enzimologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Cloridrato de Erlotinib , Gefitinibe , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/enzimologia , Humanos , Regiões Promotoras Genéticas , Quinazolinas/farmacologia , Fator de Transcrição Sp1/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. We examined the effect of nelfinavir, an HIV protease inhibitor that inhibits Akt signaling, on VEGF and HIF-1alpha expression and on angiogenesis, tumor oxygenation, and radiosensitization. Nelfinavir decreases VEGF expression under normoxia via the transcription factor Sp1, which regulates the proximal core VEGF promoter. Nelfinavir decreased Sp1 phosphorylation and decreased Sp1 binding to a probe corresponding to the proximal VEGF promoter in a gel shift assay. Nelfinavir also decreased the hypoxic induction of HIF-1alpha, which also regulates the VEGF promoter, most likely by decreasing its translation. The effect of nelfinavir on VEGF expression had the functional consequence of decreasing angiogenesis in an in vivo Matrigel plug assay. To determine the effect this might have on tumor radiosensitization, we did tumor regrowth assays with xenografts in nude mice. The combination of nelfinavir and radiation increased time to regrowth compared with radiation alone whereas nelfinavir alone had little effect on tumor regrowth. This radiosensitizing effect was greater than suggested by in vitro clonogenic survival assays. One possible explanation for the discordance is that nelfinavir has an effect on tumor oxygenation. Therefore, we examined this with the hypoxia marker EF5 and found that nelfinavir leads to increased oxygenation within tumor xenografts. Our results suggest that nelfinavir decreases HIF-1alpha/VEGF expression and tumor hypoxia, which could play a role in its in vivo radiosensitizing effect. These data support the use of nelfinavir in combination with radiation in future clinical trials.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Neoplasias Pulmonares/metabolismo , Nelfinavir/farmacologia , Oxigênio/metabolismo , Inibidores de Proteases/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Distribuição Aleatória , Fator de Transcrição Sp1/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The phosphoinositide 3-kinase (PI3K)/Akt pathway is commonly activated in cancer; therefore, we investigated its role in hypoxia-inducible factor-1alpha (HIF-1alpha) regulation. Inhibition of PI3K in U87MG glioblastoma cells, which have activated PI3K/Akt activity secondary to phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation, with LY294002 blunted the induction of HIF-1alpha protein and its targets vascular endothelial growth factor and glut1 mRNA in response to hypoxia. Introduction of wild-type PTEN into these cells also blunted HIF-1alpha induction in response to hypoxia and decreased HIF-1alpha accumulation in the presence of the proteasomal inhibitor MG132. Akt small interfering RNA (siRNA) also decreased HIF-1alpha induction under hypoxia and its accumulation in normoxia in the presence of dimethyloxallyl glycine, a prolyl hydroxylase inhibitor that prevents HIF-1alpha degradation. Metabolic labeling studies showed that Akt siRNA decreased HIF-1alpha translation in normoxia in the presence of dimethyloxallyl glycine and in hypoxia. Inhibition of mammalian target of rapamycin (mTOR) with rapamycin (10-100 nmol/L) had no significant effect on HIF-1alpha induction in a variety of cell lines, a finding that was confirmed using mTOR siRNA. Furthermore, neither mTOR siRNA nor rapamycin decreased HIF-1alpha translation as determined by metabolic labeling studies. Therefore, our results indicate that Akt can augment HIF-1alpha expression by increasing its translation under both normoxic and hypoxic conditions; however, the pathway we are investigating seems to be rapamycin insensitive and mTOR independent. These observations, which were made on cells grown in standard tissue culture medium (10% serum), were confirmed in PC3 prostate carcinoma cells. We did find that rapamycin could decrease HIF-1alpha expression when cells were cultured in low serum, but this seems to represent a different pathway.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Cromonas/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Morfolinas/farmacologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , RNA Interferente Pequeno/genética , Serina-Treonina Quinases TOR , Transfecção , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas Nucleares/biossíntese , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adenoviridae/genética , Fosfatase Alcalina/metabolismo , Sequência de Bases , Sítios de Ligação , Northern Blotting , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Colágeno/farmacologia , Densitometria , Combinação de Medicamentos , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hipóxia , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Laminina/farmacologia , Dados de Sequência Molecular , Neovascularização Patológica , Fosfatos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Plasmídeos/metabolismo , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteoglicanas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Transfecção , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Our previous work showed that, compared with parental U87MG human glioblastoma cells, vascular endothelial growth factor (VEGF) mRNA levels are decreased in U87/T691, a derivative line in which epidermal growth factor receptor (EGFR) signaling is inhibited by introduction of a truncated p185(Neu) protein (A. Maity et al., Cancer Res., 60: 5879-5886, 2000). The effect of EGFR activation on VEGF was mediated at the level of transcription via a phosphatidylinositol 3'-kinase (PI3K)-dependent pathway. In the current study we investigated the effect of PTEN, a negative regulator of PI3K signaling commonly mutated in glioblastoma cells, on VEGF expression. Several glioblastoma cell lines containing mutant PTEN, including U87MG, U87/T691, and U251MG, were infected with adenovirus expressing wild-type PTEN. This led to a decrease in the levels of both VEGF mRNA and phosphorylated Akt, a marker for PI3K activation. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing wild-type PTEN decreased VEGF promoter activity using reporters containing either 1.5 kb of the promoter or a fragment extending from -88 to +54 bp. Activity of the -88/+54 VEGF promoter was down-regulated by dominant negative Akt and up-regulated by constitutively active myristoylated Akt. Introduction of wild-type PTEN and pharmacological inhibition of EGFR decreased VEGF mRNA expression and VEGF promoter activity in U87MG cells to a greater extent that did either manipulation by itself. Therefore, in human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally up-regulating the proximal VEGF promoter via the PI3K/Akt pathway.
Assuntos
Fatores de Crescimento Endotelial/genética , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linfocinas/genética , Monoéster Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Proteínas Supressoras de Tumor/genética , Genes Supressores de Tumor , Glioblastoma/genética , Humanos , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/metabolismo , Plasmídeos , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Reprogramming of energy metabolism is one of the emerging hallmarks of cancer. Up-regulation of energy metabolism pathways fuels cell growth and division, a key characteristic of neoplastic disease, and can lead to dependency on specific metabolic pathways. Thus, targeting energy metabolism pathways might offer the opportunity for novel therapeutics. Here, we describe the application of a novel in vivo screening approach for the identification of genes involved in cancer metabolism using a patient-derived pancreatic xenograft model. Lentiviruses expressing short hairpin RNAs (shRNAs) targeting 12 different cell surface protein transporters were separately transduced into the primary pancreatic tumor cells. Transduced cells were pooled and implanted into mice. Tumors were harvested at different times, and the frequency of each shRNA was determined as a measure of which ones prevented tumor growth. Several targets including carbonic anhydrase IX (CAIX), monocarboxylate transporter 4, and anionic amino acid transporter light chain, xc- system (xCT) were identified in these studies and shown to be required for tumor initiation and growth. Interestingly, CAIX was overexpressed in the tumor initiating cell population. CAIX expression alone correlated with a highly tumorigenic subpopulation of cells. Furthermore, CAIX expression was essential for tumor initiation because shRNA knockdown eliminated the ability of cells to grow in vivo. To the best of our knowledge, this is the first parallel in vivo assessment of multiple novel oncology target genes using a patient-derived pancreatic tumor model.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno/genética , Animais , Anidrase Carbônica IX , Proteínas de Ligação a DNA/fisiologia , Citometria de Fluxo , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR) inhibitors have shown only modest clinical activity when used as single agents to treat cancers. They decrease tumor cell expression of hypoxia-inducible factor 1-alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). Hypothesizing that this might normalize tumor vasculature, we examined the effects of the EGFR inhibitor erlotinib on tumor vascular function, tumor microenvironment (TME) and chemotherapy and radiotherapy sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: Erlotinib treatment of human tumor cells in vitro and mice bearing xenografts in vivo led to decreased HIF-1alpha and VEGF expression. Treatment altered xenograft vessel morphology assessed by confocal microscopy (following tomato lectin injection) and decreased vessel permeability (measured by Evan's blue extravasation), suggesting vascular normalization. Erlotinib increased tumor blood flow measured by Power Doppler ultrasound and decreased hypoxia measured by EF5 immunohistochemistry and tumor O(2) saturation measured by optical spectroscopy. Predicting that these changes would improve drug delivery and increase response to chemotherapy and radiation, we performed tumor regrowth studies in nude mice with xenografts treated with erlotinib and either radiotherapy or the chemotherapeutic agent cisplatin. Erlotinib therapy followed by cisplatin led to synergistic inhibition of tumor growth compared with either treatment by itself (p<0.001). Treatment with erlotinib before cisplatin led to greater tumor growth inhibition than did treatment with cisplatin before erlotinib (p = 0.006). Erlotinib followed by radiation inhibited tumor regrowth to a greater degree than did radiation alone, although the interaction between erlotinib and radiation was not synergistic. CONCLUSIONS/SIGNIFICANCE: EGFR inhibitors have shown clinical benefit when used in combination with conventional cytotoxic therapy. Our studies show that targeting tumor cells with EGFR inhibitors may modulate the TME via vascular normalization to increase response to chemotherapy and radiotherapy. These studies suggest ways to assess the response of tumors to EGFR inhibition using non-invasive imaging of the TME.
Assuntos
Receptores ErbB/antagonistas & inibidores , Neoplasias Experimentais/irrigação sanguínea , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cloridrato de Erlotinib , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/radioterapia , Oxigênio/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes. METHODOLOGY AND RESULTS: In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation. CONCLUSION: These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo.
Assuntos
Sistema de Sinalização das MAP Quinases , Fator Estimulador de Colônias de Macrófagos/fisiologia , Neovascularização Fisiológica , Fator de Transcrição Sp1/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Humanos , Camundongos , Monócitos , Fator de Transcrição Sp1/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Glioblastomas are malignant brain tumors that are rarely curable, even with aggressive therapy (surgery, chemotherapy, and radiation). Glioblastomas frequently display loss of PTEN and/or epidermal growth factor receptor activation, both of which activate the PI3K pathway. This pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha expression. We examined the effects of two human immunodeficiency virus protease inhibitors, nelfinavir and amprenavir, which inhibit Akt signaling, on VEGF and HIF-1alpha expression and on angiogenesis. Nelfinavir decreased VEGF mRNA expression and VEGF secretion under normoxia. Downregulation of P-Akt decreased VEGF secretion in a manner similar to that of nelfinavir, but the combination of the two had no greater effect, consistent with the idea that nelfinavir decreases VEGF through the PI3K/Akt pathway. Nelfinavir also decreased the hypoxic induction of VEGF and the hypoxic induction of HIF-1alpha, which regulates VEGF promoter. The effect of nelfinavir on HIF-1alpha was most likely mediated by decreased protein translation. Nelfinavir's effect on VEGF expression had the functional consequence of decreasing angiogenesis in in vivo Matrigel plug assays. Similar effects on VEGF and HIF-1alpha expression were seen with a different protease inhibitor, amprenavir. Our results support further research into these protease inhibitors for use in future clinical trials for patients with glioblastoma multiformes.