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1.
Immunity ; 41(5): 843-52, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25517616

RESUMO

Ionizing radiation-mediated tumor regression depends on type I interferon (IFN) and the adaptive immune response, but several pathways control I IFN induction. Here, we demonstrate that adaptor protein STING, but not MyD88, is required for type I IFN-dependent antitumor effects of radiation. In dendritic cells (DCs), STING was required for IFN-? induction in response to irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) mediated sensing of irradiated-tumor cells in DCs. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-? treatment rescued the cross-priming by cGAS or STING-deficient DCs. Accordingly, activation of STING by a second messenger cGAMP administration enhanced antitumor immunity induced by radiation. Thus radiation-mediated antitumor immunity in immunogenic tumors requires a functional cytosolic DNA-sensing pathway and suggests that cGAMP treatment might provide a new strategy to improve radiotherapy.


Assuntos
DNA/imunologia , Proteínas de Membrana/genética , Neoplasias/radioterapia , Nucleotidiltransferases/imunologia , Imunidade Adaptativa , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Antineoplásicos/farmacologia , Células Cultivadas , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Interferon beta/biossíntese , Interferon beta/imunologia , Interferon beta/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Neoplasias/imunologia , Nucleotídeos Cíclicos/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Radiação Ionizante , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Transdução de Sinais/imunologia , Xantonas/farmacologia
2.
Mol Cell ; 44(5): 785-96, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22152481

RESUMO

The functional significance of the signaling pathway induced by O(6)-methylguanine (O(6)-MeG) lesions is poorly understood. Here, we identify the p50 subunit of NF-κB as a central target in the response to O(6)-MeG and demonstrate that p50 is required for S(N)1-methylator-induced cytotoxicity. In response to S(N)1-methylation, p50 facilitates the inhibition of NF-κB-regulated antiapoptotic gene expression. Inhibition of NF-κB activity is noted to be an S phase-specific phenomenon that requires the formation of O(6)-MeG:T mismatches. Chk1 associates with p50 following S(N)1-methylation, and phosphorylation of p50 by Chk1 results in the inhibition of NF-κB DNA binding. Expression of an unphosphorylatable p50 mutant blocks inhibition of NF-κB-regulated antiapoptotic gene expression and attenuates S(N)1-methylator-induced cytotoxicity. While O(6)-MeG:T-induced, p50-dependent signaling is not sufficient to induce cell death, this pathway sensitizes cells to the cytotoxic effects of DNA breaks.


Assuntos
Dano ao DNA , Metilação de DNA , Subunidade p50 de NF-kappa B/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Subunidade p50 de NF-kappa B/deficiência
3.
Proc Natl Acad Sci U S A ; 111(4): E484-91, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24434553

RESUMO

An siRNA screen targeting 89 IFN stimulated genes in 14 different cancer cell lines pointed to the RIG-I (retinoic acid inducible gene I)-like receptor Laboratory of Genetics and Physiology 2 (LGP2) as playing a key role in conferring tumor cell survival following cytotoxic stress induced by ionizing radiation (IR). Studies on the role of LGP2 revealed the following: (i) Depletion of LGP2 in three cancer cell lines resulted in a significant increase in cell death following IR, (ii) ectopic expression of LGP2 in cells increased resistance to IR, and (iii) IR enhanced LGP2 expression in three cell lines tested. Studies designed to define the mechanism by which LGP2 acts point to its role in regulation of IFNß. Specifically (i) suppression of LGP2 leads to enhanced IFNß, (ii) cytotoxic effects following IR correlated with expression of IFNß inasmuch as inhibition of IFNß by neutralizing antibody conferred resistance to cell death, and (iii) mouse embryonic fibroblasts from IFN receptor 1 knockout mice are radioresistant compared with wild-type mouse embryonic fibroblasts. The role of LGP2 in cancer may be inferred from cumulative data showing elevated levels of LGP2 in cancer cells are associated with more adverse clinical outcomes. Our results indicate that cytotoxic stress exemplified by IR induces IFNß and enhances the expression of LGP2. Enhanced expression of LGP2 suppresses the IFN stimulated genes associated with cytotoxic stress by turning off the expression of IFNß.


Assuntos
Sobrevivência Celular/fisiologia , RNA Helicases DEAD-box/fisiologia , Neoplasias Experimentais/patologia , RNA Helicases/fisiologia , Radiação Ionizante , Animais , Apoptose , Neoplasias Encefálicas/patologia , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Glioblastoma/patologia , Humanos , Interferon Tipo I/biossíntese , Camundongos , Camundongos Knockout , Neoplasias Experimentais/metabolismo , RNA Helicases/metabolismo , Células Tumorais Cultivadas
4.
J Immunol ; 190(11): 5874-81, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23630355

RESUMO

Local failures following radiation therapy are multifactorial, and the contributions of the tumor and the host are complex. Current models of tumor equilibrium suggest that a balance exists between cell birth and cell death due to insufficient angiogenesis, immune effects, or intrinsic cellular factors. We investigated whether host immune responses contribute to radiation-induced tumor equilibrium in animal models. We report an essential role for immune cells and their cytokines in suppressing tumor cell regrowth in two experimental animal model systems. Depletion of T cells or neutralization of IFN-γ reversed radiation-induced equilibrium, leading to tumor regrowth. We also demonstrate that PD-L1 blockade augments T cell responses, leading to rejection of tumors in radiation-induced equilibrium. We identify an active interplay between tumor cells and immune cells that occurs in radiation-induced tumor equilibrium and suggest a potential role for disruption of the PD-L1/PD-1 axis in increasing local tumor control.


Assuntos
Citotoxicidade Imunológica , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Antígeno B7-H1 , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Humanos , Imunoterapia , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos , Metástase Neoplásica , Neoplasias/patologia , Neoplasias/cirurgia , Receptor de Morte Celular Programada 1 , Radiocirurgia , Carga Tumoral/imunologia , Carga Tumoral/efeitos da radiação
5.
Mol Ther ; 20(5): 1046-55, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22334019

RESUMO

Radiotherapy offers an effective treatment for advanced cancer but local and distant failures remain a significant challenge. Here, we treated melanoma and pancreatic carcinoma in syngeneic mice with ionizing radiation (IR) combined with the poly(ADP-ribose) polymerase inhibitor (PARPi) veliparib to inhibit DNA repair and promote accelerated senescence. Based on prior work implicating cytotoxic T lymphocytes (CTLs) as key mediators of radiation effects, we discovered that senescent tumor cells induced by radiation and veliparib express immunostimulatory cytokines to activate CTLs that mediate an effective antitumor response. When these senescent tumor cells were injected into tumor-bearing mice, an antitumor CTL response was induced which potentiated the effects of radiation, resulting in elimination of established tumors. Applied to human cancers, radiation-inducible immunotherapy may enhance radiotherapy responses to prevent local recurrence and distant metastasis.


Assuntos
Benzimidazóis/farmacologia , Vacinas Anticâncer/uso terapêutico , Imunoterapia/métodos , Melanoma Experimental/terapia , Neoplasias Pancreáticas/terapia , Radiossensibilizantes/farmacologia , Animais , Vacinas Anticâncer/imunologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Terapia Combinada , Citocinas/biossíntese , Citocinas/imunologia , Citotoxicidade Imunológica , Feminino , Humanos , Ativação Linfocitária , Melanoma Experimental/imunologia , Melanoma Experimental/mortalidade , Camundongos , Transplante de Neoplasias , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/mortalidade , Inibidores de Poli(ADP-Ribose) Polimerases , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas
6.
Mol Ther ; 18(5): 912-20, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20197756

RESUMO

Ad.Egr-TNF is a radioinducible adenovector currently in phase 3 trials for inoperable pancreatic cancer. The combination of Ad.Egr-TNF and ionizing radiation (IR) contributes to local tumor control through the production of tumor necrosis factor-alpha (TNFalpha) in the tumor microenvironment. Moreover, clinical and preclinical studies with Ad.Egr-TNF/IR have suggested that this local approach suppresses the growth of distant metastatic disease; however, the mechanisms responsible for this effect remain unclear. These studies have been performed in wild-type (WT) and TNFR1,2(-/-) mice to assess the role of TNFalpha-induced signaling in the suppression of draining lymph node (DLN) metastases. The results demonstrate that production of TNFalpha in the tumor microenvironment induces expression of interferon (IFNbeta). In turn, IFNbeta stimulates the production of chemokines that recruit CD8(+) T cells to the tumor. The results further demonstrate that activation of tumor antigen-specific CD8(+) CTLs contributes to local antitumor activity and suppression of DLN metastases. These findings support a model in which treatment of tumors with Ad.Egr-TNF and IR is mediated by local and distant immune-mediated antitumor effects that suppress the development of metastases.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Metástase Neoplásica/prevenção & controle , Metástase Neoplásica/terapia , Radiação Ionizante , Fator de Necrose Tumoral alfa/metabolismo , Adenoviridae/genética , Animais , Proliferação de Células , Vetores Genéticos/genética , Humanos , Interferon beta/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Metástase Neoplásica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genética
7.
Int J Radiat Biol ; 85(5): 421-31, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19437244

RESUMO

PURPOSE: To determine the mechanisms of Signal Transducer and Activator of Transcription 1 (Stat1)-associated radioresistance developed by nu61 tumour selected in vivo by fractionated irradiation of the parental radiosensitive tumour SCC61. MATERIALS AND METHODS: Radioresistence of nu61 and SCC61 in vitro was measured by clonogenic assay. Apoptotic response of nu61 and SCC61 cells to genotoxic stress was examined using caspase-based apoptotic assays. Co-cultivation of carboxyfluorescein diacetate, succinimidyl ester (CFDE-SE)-labeled nu61 with un-labeled SCC61 was performed at 1:1 ratio. Production of interleukin-6, interleukin-8 and soluble receptor of interleukin 6 (IL6, IL8 and sIL6R) was measured using Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Radioresistant nu61 was also resistant to interferon-gamma (IFNgamma) and the death ligands of tumour necrosis factor alpha receptor (TNFR) family when compared to SCC61. This combined resistance is due to an impaired apoptotic response in nu61. Relative to SCC61, nu61 produced more IL6, IL8 and sIL6R. Using Stat1 knock-downs we demonstrated that IL6 and IL8 production is Stat1-dependent. Treatment with neutralising antibodies to IL6 and IL8, but not to either cytokine alone sensitised nu61 to genotoxic stress induced apoptosis. CONCLUSION: Nu61, which over-expresses Stat1 pathway, is deficient in apoptotic response to ionising radiation and cytotoxic ligands. This resistance to apoptosis is associated with Stat1-dependent production of IL6 and IL8 and suppression of caspases 8, 9 and 3.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Interleucinas/metabolismo , Neoplasias/patologia , Tolerância a Radiação , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Animais , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Técnicas de Cocultura , Citocinas/toxicidade , Citotoxinas/toxicidade , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Interferon gama/farmacologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Neoplasias/genética , Radiação Ionizante , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação
8.
Cancer Res ; 67(14): 6889-98, 2007 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-17638900

RESUMO

The alkylating agent temozolomide, commonly used in the treatment of malignant glioma, causes cellular cytotoxicity by forming O(6)-methylguanine adducts. In this report, we investigated whether temozolomide alters the activity of the transcription factor nuclear factor-kappaB (NF-kappaB). Temozolomide inhibits basal and tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB transcriptional activity without altering phosphorylation or degradation of inhibitor of kappaB-alpha. Inhibition of NF-kappaB is secondary to attenuation of p65 DNA binding, not nuclear translocation. Inhibition of DNA binding is shown both in vitro, with gel shift studies and DNA binding assays, and in vivo at kappaB sites. Consistent with inhibition of NF-kappaB activity, temozolomide reduces basal and TNFalpha-induced kappaB-dependent gene expression. Temozolomide also inhibits NF-kappaB activated by inducers other than TNFalpha, including lipopolysaccharide, doxorubicin, and phorbol 12-myristate 13-acetate. The inhibitory action of temozolomide on NF-kappaB is observed to be maximal following pretreatment of cells with temozolomide for 16 h and is also seen with the S(N)1-type methylating agent methylnitrosourea. The ability of temozolomide to form O(6)-methylguanine adducts is important for inhibition of NF-kappaB as is the presence of a functioning mismatch repair system. Activation of NF-kappaB with TNFalpha before administration of temozolomide reduces the cytotoxicity of temozolomide, whereas 16-h pretreatment with temozolomide resensitizes cells to killing. This work shows a mechanism whereby O(6)-methylguanine adducts formed by temozolomide lead to inhibition of NF-kappaB activity and illustrates a link between mismatch repair processing of alkylator-induced DNA damage and cell death.


Assuntos
Dacarbazina/análogos & derivados , Guanina/análogos & derivados , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Dano ao DNA , Dacarbazina/farmacologia , Guanina/química , Humanos , Luciferases/metabolismo , Microscopia de Fluorescência , Ligação Proteica , Frações Subcelulares/metabolismo , Temozolomida , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Fator de Transcrição RelA/antagonistas & inibidores
9.
Cancer Res ; 67(19): 9214-20, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17909027

RESUMO

Elsewhere, we reported that multiple serial in vivo passage of a squamous cell carcinoma cells (SCC61) concurrent with ionizing radiation (IR) treatment resulted in the selection of radioresistant tumor (nu61) that overexpresses the signal transducer and activator of transcription 1 (Stat1)/IFN-dependent pathway. Here, we report that (a) the Stat1 pathway is induced by IR, (b) constitutive overexpression of Stat1 is linked with failure to transmit a cytotoxic signal by radiation or IFNs, (c) selection of parental cell line SCC61 against IFN-alpha and IFN-gamma leads to the same IR- and IFN-resistant phenotype as was obtained by IR selection, and (d) suppression of Stat1 by short hairpin RNA renders the IR-resistant nu61 cells radiosensitive to IR. We propose a model that transient induction of Stat1 by IFN, IR, or other stress signals activates cytotoxic genes and cytotoxic response. Constitutive overexpression of Stat1 on the other hand leads to the suppression of the cytotoxic response and induces prosurvival genes that, at high levels of Stat1, render the cells resistant to IR or other inducers of cell death.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Humanos , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Interferente Pequeno/genética , Tolerância a Radiação , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT1/biossíntese , Fator de Transcrição STAT1/genética , Transplante Heterólogo
10.
J Clin Invest ; 110(3): 403-10, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12163460

RESUMO

Ionizing radiation (IR) and radical oxygen intermediates (ROIs) activate the early growth response-1 (Egr1) promoter through specific cis-acting sequences termed CArG elements. Ad.Egr.TNF.11D, a replication-deficient adenoviral vector containing CArG elements cloned upstream of the cDNA for human recombinant TNF-alpha was used to treat human esophageal adenocarcinoma and rat colon adenocarcinoma cells in culture and as xenografts in athymic nude mice. Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating ROIs. The present studies demonstrate induction of TNF-alpha production in tumor cells and xenografts treated with the combination of Ad.Egr.TNF.11D and cisplatin. The results show that the Egr1 promoter is induced by cisplatin and that this induction is mediated in part through the CArG elements. These studies also demonstrate an enhanced antitumor response without an increase in toxicity following treatment with Ad.Egr.TNF.11D and cisplatin, compared with either agent alone. Chemo-inducible cancer gene therapy thus provides a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents.


Assuntos
Cisplatino/farmacologia , Proteínas Imediatamente Precoces , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Adenocarcinoma/terapia , Adenoviridae/genética , Adenoviridae/fisiologia , Animais , Neoplasias do Colo , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce , Neoplasias Esofágicas/terapia , Feminino , Terapia Genética , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Regiões Promotoras Genéticas , Ratos , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/biossíntese
11.
Int J Oncol ; 31(6): 1519-28, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17982679

RESUMO

TNFalpha was initially described as inducing necrotic death in tumors in vivo, and more recently as a cytokine that mediates cytoprotection and inflammation. The anti-tumor effects of TNFalpha are poorly characterized because TNFalpha-induced death of human tumor cells has largely been studied in the presence of agents that block transcription or protein synthesis. Also, most reports in model cell systems describe apoptosis within relatively early time points as the principal mode of cell death induced by TNFalpha. We investigated the cytotoxic effects of 10 ng/ml TNFalpha on human tumor cells of different histological types without concomitant exposure to these inhibitors. Eleven of 21 human tumor cell lines underwent TNFalpha-induced cell death which ranged from 41% to complete loss of viability. Only one cell line demonstrated caspase-dependent apoptosis within 24 h. Nine cell lines underwent death between 48 h and 21 days. Seven of these lines underwent caspase-3 independent death consistent with necrosis. One tumor line exhibited characteristics of senescence following TNFalpha exposure. Nine of 9 cell lines activated NF-kappaB following TNFalpha exposure by 24 h. In all cell lines studied, with the exception of the epidermoid carcinoma cell line that underwent early apoptosis, expression of one or more NF-kappaB target genes was demonstrated at 24-96 h. BMS-345541, a specific IKK inhibitor, increased TNFalpha killing in TNFalpha resistant tumor cell lines by increasing apoptosis, suggesting that inhibition of NF-kappaB may be an effective strategy to enhance the tumoricidal effects of TNFalpha.


Assuntos
Apoptose/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , NF-kappa B/fisiologia , Necrose , Neoplasias/patologia , Quinoxalinas/farmacologia
12.
Cancer Res ; 65(20): 9479-84, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16230412

RESUMO

Ionizing radiation potentiates the oncolytic activity of attenuated herpes simplex viruses in tumors exposed to irradiation at specific time intervals by inducing higher virus yields. Cell culture studies have shown that an attenuated virus lacking the viral gamma(1)34.5 genes underproduces late proteins whose synthesis depends on sustained synthesis of viral DNA. Here we report that ionizing radiation enhances gene expression from late viral promoters in transduced cells in the absence of other viral gene products. Consistent with this result, we show that in tumors infected with the attenuated virus, ionizing radiation increases 13.6-fold above baseline the gene expression from a late viral promoter as early as 2 hours after virus infection, an interval too short to account for viral DNA synthesis. The radiation-dependent up-regulation of late viral genes is mediated by the p38 pathway, inasmuch as the enhancement is abolished by p38 inhibitors or a p38 dominant-negative construct. The p38 pathway is not essential for wild-type virus gene expression. The results suggest that ionizing radiation up-regulates late promoters active in the course of viral DNA synthesis and provide a rationale for use of radiation to up-regulate cytotoxic genes introduced into tumor cells by viral vectors for cytoreductive therapy.


Assuntos
Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/efeitos da radiação , Neoplasias/terapia , Neoplasias/virologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Chlorocebus aethiops , DNA Viral/biossíntese , DNA Viral/genética , Ativação Enzimática , Regulação Viral da Expressão Gênica/efeitos da radiação , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/virologia , Fosforilação , Regiões Promotoras Genéticas/efeitos da radiação , Coelhos , Regulação para Cima/efeitos da radiação , Células Vero , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
13.
Cancer Res ; 65(8): 3146-54, 2005 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15833844

RESUMO

We did expressional profiling on 24 paired samples of normal esophageal epithelium, Barrett's metaplasia, and esophageal adenocarcinomas. Matching tissue samples representing the three different histologic types were obtained from each patient undergoing esophagectomy for adenocarcinoma. Our analysis compared the molecular changes accompanying the transformation of normal squamous epithelium with Barrett's esophagus and adenocarcinoma in individual patients rather than in a random cohort. We tested the hypothesis that expressional profiling may reveal gene sets that can be used as molecular markers of progression from normal esophageal epithelium to Barrett's esophagus and adenocarcinoma. Expressional profiling was done using U133A GeneChip (Affymetrix), which represent approximately two thirds of the human genome. The final selection of 214 genes permitted the discrimination of differential gene expression of normal esophageal squamous epithelium, Barrett's esophagus, and adenocarcinoma using two-dimensional hierarchical clustering of selected genes. These data indicate that transformation of Barrett's esophagus to adenocarcinoma is associated with suppression of the genes involved in epidermal differentiation, including genes in 1q21 loci and corresponding to the epidermal differentiation complex. Correlation analysis of genes concordantly expressed in Barrett's esophagus and adenocarcinoma revealed 21 genes that represent potential genetic markers of disease progression and pharmacologic targets for treatment intervention. PCR analysis of genes selected based on DNA array experiments revealed that estimation of the ratios of GATA6 to SPRR3 allows discrimination among normal esophageal epithelium, Barrett's dysplasia, and adenocarcinoma.


Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Transformação Celular Neoplásica/genética , Neoplasias Esofágicas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Diferenciação Celular/genética , Proteínas Ricas em Prolina do Estrato Córneo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Progressão da Doença , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Fator de Transcrição GATA6 , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Família Multigênica , Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
14.
Oncotarget ; 7(18): 26496-515, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27034163

RESUMO

Emerging evidence indicates that ionizing radiation (IR) and chemotherapy activate Type I interferon (IFN) signaling in tumor and host cells. However, the mechanism of induction is poorly understood. We identified a novel radioprotective role for the DEXH box RNA helicase LGP2 (DHX58) through its suppression of IR-induced cytotoxic IFN-beta [1]. LGP2 inhibits activation of the RIG-I-like receptor (RLR) pathway upon binding of viral RNA to the cytoplasmic sensors RIG-I (DDX58) and MDA5 (IFIH1) and subsequent IFN signaling via the mitochondrial adaptor protein MAVS (IPS1). Here we show that MAVS is necessary for IFN-beta induction and interferon-stimulated gene expression in the response to IR. Suppression of MAVS conferred radioresistance in normal and cancer cells. Germline deletion of RIG-I, but not MDA5, protected mice from death following total body irradiation, while deletion of LGP2 accelerated the death of irradiated animals. In human tumors depletion of RIG-I conferred resistance to IR and different classes of chemotherapy drugs. Mechanistically, IR stimulated the binding of cytoplasmic RIG-I with small endogenous non-coding RNAs (sncRNAs), which triggered IFN-beta activity. We demonstrate that the small nuclear RNAs U1 and U2 translocate to the cytoplasm after IR treatment, thus stimulating the formation of RIG-I: RNA complexes and initiating downstream signaling events. Taken together, these findings suggest that the physiologic responses to radio-/chemo-therapy converge on an antiviral program in recruitment of the RLR pathway by a sncRNA-dependent activation of RIG-I which commences cytotoxic IFN signaling. Importantly, activation of interferon genes by radiation or chemotherapy is associated with a favorable outcome in patients undergoing treatment for cancer. To our knowledge, this is the first demonstration of a cell-intrinsic response to clinically relevant genotoxic treatments mediated by an RNA-dependent mechanism.


Assuntos
Proteína DEAD-box 58/efeitos dos fármacos , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/efeitos da radiação , Interferon beta/biossíntese , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/terapia , Pequeno RNA não Traduzido/efeitos dos fármacos , Pequeno RNA não Traduzido/metabolismo , Pequeno RNA não Traduzido/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação
15.
Oncotarget ; 6(6): 3540-52, 2015 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-25686838

RESUMO

Oligometastasis is a clinically distinct subset of metastasis characterized by a limited number of metastases potentially curable with localized therapies. We analyzed pathways targeted by microRNAs over-expressed in clinical oligometastasis samples and identified suppression of cellular adhesion, invasion, and motility pathways in association with the oligometastatic phenotype. We identified miR-127-5p, miR-544a, and miR-655-3p encoded in the 14q32 microRNA cluster as co-regulators of multiple metastatic pathways through repression of shared target genes. These microRNAs suppressed cellular adhesion and invasion and inhibited metastasis development in an animal model of breast cancer lung colonization. Target genes, including TGFBR2 and ROCK2, were key mediators of these effects. Understanding the role of microRNAs expressed in oligometastases may lead to improved identification of and interventions for patients with curable metastatic disease, as well as an improved understanding of the molecular basis of this unique clinical entity.


Assuntos
Cromossomos Humanos Par 14 , MicroRNAs/genética , Neoplasias/genética , Neoplasias/patologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Fenótipo
16.
FEBS Lett ; 565(1-3): 167-70, 2004 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-15135073

RESUMO

We demonstrate that human umbilical vein endothelial cells (HUVEC) grown in co-culture (CC) with U87 glioblastoma cells transfected with green fluorescent protein (GFP-U87) exhibit resistance to radiation-mediated apoptosis. cDNA macroarray analysis reveals increases in the accumulation of RNAs for HUVEC genes encoding cell adhesion molecules, growth factor-related proteins, and cell cycle regulatory/DNA repair proteins. An increase in protein expression of integrin alphav, integrin beta1, MAPK(p42), Rad51, DNA-PK(CS), and ataxia telangiectasia gene (ATM) was detected in HUVEC grown in CC with GFP-U87 cells compared with HUVEC grown in mono-culture. Treatment with anti-VEGF antibody decreases the expression of integrin alphav, integrin beta1, DNA-PK(CS) and ATM with a corresponding increase in ionizing radiation (IR)-induced apoptosis. These data support the concept that endothelial cells growing in the tumor microenvironment may develop resistance to cytotoxic therapies due to the up-regulation by tumor cells of endothelial cells genes associated with survival.


Assuntos
Apoptose , Células Endoteliais/patologia , Glioblastoma/patologia , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Adesão Celular , Ciclo Celular , Proteínas de Ciclo Celular , Divisão Celular , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Reparo do DNA , DNA Complementar/metabolismo , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/efeitos da radiação , Endotélio Vascular/citologia , Glioblastoma/radioterapia , Proteínas de Fluorescência Verde , Humanos , Raios Infravermelhos , Integrina alfaV/biossíntese , Integrina alfaVbeta3/metabolismo , Integrina beta1/biossíntese , Proteínas Luminescentes/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/metabolismo , Rad51 Recombinase , Radioterapia , Recombinação Genética , Transcrição Gênica , Transfecção , Proteínas Supressoras de Tumor , Veias Umbilicais/citologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
J Clin Invest ; 124(2): 687-95, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24382348

RESUMO

High-dose ionizing irradiation (IR) results in direct tumor cell death and augments tumor-specific immunity, which enhances tumor control both locally and distantly. Unfortunately, local relapses often occur following IR treatment, indicating that IR-induced responses are inadequate to maintain antitumor immunity. Therapeutic blockade of the T cell negative regulator programmed death-ligand 1 (PD-L1, also called B7-H1) can enhance T cell effector function when PD-L1 is expressed in chronically inflamed tissues and tumors. Here, we demonstrate that PD-L1 was upregulated in the tumor microenvironment after IR. Administration of anti-PD-L1 enhanced the efficacy of IR through a cytotoxic T cell-dependent mechanism. Concomitant with IR-mediated tumor regression, we observed that IR and anti-PD-L1 synergistically reduced the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which suppress T cells and alter the tumor immune microenvironment. Furthermore, activation of cytotoxic T cells with combination therapy mediated the reduction of MDSCs in tumors through the cytotoxic actions of TNF. Our data provide evidence for a close interaction between IR, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and radiotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Experimentais/imunologia , Microambiente Tumoral , Animais , Apoptose , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Linhagem Celular Tumoral , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Células Mieloides/citologia , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/radioterapia , Radiação Ionizante , Fatores de Tempo
18.
Sci Transl Med ; 6(229): 229ra42, 2014 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-24670686

RESUMO

Mutagenesis is a hallmark of malignancy, and many oncologic treatments function by generating additional DNA damage. Therefore, DNA damage repair is centrally important in both carcinogenesis and cancer treatment. Homologous recombination (HR) and nonhomologous end joining are alternative pathways of double-strand DNA break repair. We developed a method to quantify the efficiency of DNA repair pathways in the context of cancer therapy. The recombination proficiency score (RPS) is based on the expression levels for four genes involved in DNA repair pathway preference (Rif1, PARI, RAD51, and Ku80), such that high expression of these genes yields a low RPS. Carcinoma cells with low RPS exhibit HR suppression and frequent DNA copy number alterations, which are characteristic of error-prone repair processes that arise in HR-deficient backgrounds. The RPS system was clinically validated in patients with breast or non-small cell lung carcinomas (NSCLCs). Tumors with low RPS were associated with greater mutagenesis, adverse clinical features, and inferior patient survival rates, suggesting that HR suppression contributes to the genomic instability that fuels malignant progression. This adverse prognosis associated with low RPS was diminished if NSCLC patients received adjuvant chemotherapy, suggesting that HR suppression and associated sensitivity to platinum-based drugs counteract the adverse prognosis associated with low RPS. Therefore, RPS may help oncologists select which therapies will be effective for individual patients, thereby enabling more personalized care.


Assuntos
Antineoplásicos/uso terapêutico , Reparo do DNA/genética , Tratamento Farmacológico , Regulação Neoplásica da Expressão Gênica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Prognóstico , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética
19.
Oncol Rep ; 27(5): 1625-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22294050

RESUMO

Signaling pathways that activate mTOR (mammalian target of rapamycin) are altered in many human cancers and these alterations are associated with prognosis and treatment response. mTOR inhibition can restore sensitivity to DNA damaging agents such as cisplatin. The rapamycin derivative everolimus exhibits antitumor activity and is approved for patients with renal cell cancer. Clinically, everolimus has also been evaluated in patients with advanced non-small cell lung cancer (NSCLC) that were refractory to chemotherapy and epidermal growth factor receptor tyrosine kinase inhibitors. We tested the effects of combined treatment with everolimus (RAD001) and fractionated radiation using a xenograft model of human NSCLC (A549 cells). In growth studies, mean tumor volume was reduced in the everolimus plus 30 Gy cohort with significant tumor growth suppression compared to 30 Gy alone (p=0015), or everolimus alone (p<0.001, ANOVA). everolimus (20 nM) significantly reduced protein levels of the mTOR downstream effector p70-S6K compared with radiation and vehicle (p=0.05, ANOVA) and significantly suppressed phospho-p70-S6K levels compared with all other treatments (p<0.001, ANOVA). We also evaluated everolimus and radiation effects on gene expression in A549 cells. Everolimus ± 5 Gy suppressed endothelin 1 and lactate dehydrogenase expression and increased VEGFA, p21, hypoxia-inducible factor-1α and SLC2A1 (facilitated glucose transporter 1). mTOR mRNA levels were unaffected while TNF-α levels were increased with everolimus + 5 Gy compared to either treatment alone. These findings suggest that everolimus increases the antitumor activity of radiation. Clinical trials combining everolimus with fractionated radiation in patients with NSCLC are warranted.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Radiossensibilizantes/uso terapêutico , Sirolimo/análogos & derivados , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Linhagem Celular Tumoral , Terapia Combinada , Everolimo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Radiação Ionizante , Radiossensibilizantes/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
20.
PLoS One ; 7(10): e46104, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056240

RESUMO

BACKGROUND: Vascular endothelial cells contribute to the pathogenesis of numerous human diseases by actively regulating the stromal inflammatory response; however, little is known regarding the role of endothelial inflammation in the growth of human tumors and its influence on the prognosis of human cancers. METHODS: Using an experimental model of tumor necrosis factor-alpha (TNF-α)-mediated inflammation, we characterized inflammatory gene expression in immunopurified tumor-associated endothelial cells. These genes formed the basis of a multivariate molecular predictor of overall survival that was trained and validated in four types of human cancer. RESULTS: We report that expression of experimentally derived tumor endothelial genes distinguished pathologic tissue specimens from normal controls in several human diseases associated with chronic inflammation. We trained these genes in human cancer datasets and defined a six-gene inflammatory signature that predicted significantly reduced overall survival in breast cancer, colon cancer, lung cancer, and glioma. This endothelial-derived signature predicted outcome independently of, but cooperatively with, standard clinical and pathological prognostic factors. Consistent with these findings, conditioned culture media from human endothelial cells stimulated by pro-inflammatory cytokines accelerated the growth of human colon and breast tumors in immunodeficient mice as compared with conditioned media from untreated endothelial cells. CONCLUSIONS: This study provides the first prognostic cancer gene signature derived from an experimental model of tumor-associated endothelial inflammation. These findings support the notion that activation of inflammatory pathways in non-malignant tumor-infiltrating endothelial cells contributes to tumor growth and progression in multiple human cancers. Importantly, these results identify endothelial-derived factors that could serve as potential targets for therapy in diverse human cancers.


Assuntos
Endotélio Vascular/metabolismo , Inflamação/genética , Neoplasias/genética , Neovascularização Patológica/genética , Adulto , Idoso , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/patologia , Feminino , Perfilação da Expressão Gênica , Glioma/genética , Glioma/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/patologia , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Análise Multivariada , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Necrose Tumoral alfa/farmacologia
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