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1.
J Pathol ; 249(3): 332-342, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31259422

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvß6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of ß6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10-8 ). In two separate cohorts, we showed that over 80% of PDACs expressed αvß6 protein and that paired metastases retained αvß6 expression. In vitro, integrin αvß6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvß6-positive human PDAC xenografts and transgenic mice bearing αvß6-positive PDAC with the αvß6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p < 0.0001) and increased survival (log-rank test, p < 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase-3) and suppression of the pro-tumourigenic microenvironment (suppression of TGFß signalling, fewer αSMA-positive myofibroblasts, decreased blood vessel density). These data show that αvß6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Integrinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Antígenos de Neoplasias/genética , Antineoplásicos Imunológicos/farmacologia , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/secundário , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Fosfatase 6 de Especificidade Dupla/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes ras , Humanos , Integrases/genética , Integrinas/antagonistas & inibidores , Integrinas/genética , Itália , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Carga Tumoral , Microambiente Tumoral , Reino Unido , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Mol Ther ; 25(1): 259-273, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28129120

RESUMO

Expression of the αvß6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvß6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αß) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvß6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvß6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvß6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin.


Assuntos
Antígenos de Neoplasias/imunologia , Engenharia Celular , Integrinas/antagonistas & inibidores , Integrinas/imunologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Antígenos de Neoplasias/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Imunoterapia Adotiva , Integrinas/genética , Camundongos , Camundongos SCID , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biochem Soc Trans ; 44(2): 349-55, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27068939

RESUMO

Immunotherapy of cancer using chimeric antigen receptor (CAR) T-cells is a rapidly expanding field. CARs are fusion molecules that couple the binding of a tumour-associated cell surface target to the delivery of a tailored T-cell activating signal. Re-infusion of such genetically engineered T-cells to patients with haematological disease has demonstrated unprecedented response rates in Phase I clinical trials. However, such successes have not yet been observed using CAR T-cells against solid malignancies and this is, in part, due to a lack of safe tumour-specific targets. The αvß6 integrin is strongly up-regulated in multiple solid tumours including those derived from colon, lung, breast, cervix, ovaries/fallopian tube, pancreas and head and neck. It is associated with poorer prognosis in several cancers and exerts pro-tumorigenic activities including promotion of tumour growth, migration and invasion. By contrast, physiologic expression of αvß6 is largely restricted to wound healing. These attributes render this epithelial-specific integrin a highly attractive candidate for targeting using immunotherapeutic strategies such as CAR T-cell adoptive immunotherapy. This mini-review will discuss the role and expression of αvß6 in cancer, as well as its potential as a therapeutic target.


Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia , Integrinas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/fisiologia , Biomarcadores Tumorais/imunologia , Humanos , Imunoterapia/efeitos adversos , Integrinas/química , Integrinas/fisiologia , Neoplasias/imunologia , Neoplasias/terapia , Prognóstico
5.
Sci Rep ; 6: 24006, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-27035095

RESUMO

Although squamous cell carcinomas (SqCCs) of the lungs, head and neck, oesophagus, and cervix account for up to 30% of cancer deaths, the mechanisms that regulate disease progression remain incompletely understood. Here, we use gene transduction and human tumor xenograft assays to establish that the tumour suppressor Cell adhesion molecule 1 (CADM1) inhibits SqCC proliferation and invasion, processes fundamental to disease progression. We determine that the extracellular domain of CADM1 mediates these effects by forming a complex with HER2 and integrin α6ß4 at the cell surface that disrupts downstream STAT3 activity. We subsequently show that treating CADM1 null tumours with the JAK/STAT inhibitor ruxolitinib mimics CADM1 gene restoration in preventing SqCC growth and metastases. Overall, this study identifies a novel mechanism by which CADM1 prevents SqCC progression and suggests that screening tumours for loss of CADM1 expression will help identify those patients most likely to benefit from JAK/STAT targeted chemotherapies.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Moléculas de Adesão Celular/metabolismo , Imunoglobulinas/metabolismo , Neoplasias Pulmonares/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Carcinoma de Células Escamosas/patologia , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Imunoglobulinas/genética , Integrina alfa6beta4/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Nitrilas , Pirazóis/química , Pirimidinas , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
6.
J Natl Cancer Inst ; 106(8)2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24974129

RESUMO

BACKGROUND: Integrin αvß6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvß6 has yet to be elucidated in breast cancer. METHODS: Protein expression of integrin subunit beta6 (ß6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of ß6 in breast cell lines, the role of αvß6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student's t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni's Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided. RESULTS: High expression of either the mRNA or protein for the integrin subunit ß6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of ß6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts. CONCLUSIONS: Targeting αvß6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígenos de Neoplasias/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Terapia de Alvo Molecular , Receptor ErbB-2/metabolismo , Animais , Antígenos de Neoplasias/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Integrinas/genética , Estimativa de Kaplan-Meier , Camundongos , Camundongos SCID , Terapia de Alvo Molecular/métodos , Receptor ErbB-2/genética , Trastuzumab , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Eur Respir J ; 44(2): 513-22, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24435005

RESUMO

Chronic respiratory diseases, including pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and lung cancer, are the second leading cause of death among Europeans. Despite this, there have been only a few therapeutic advances in these conditions over the past 20 years. In this review we provide evidence that targeting the epidermal growth factor receptor (EGFR) signalling pathway may represent a novel therapeutic panacea for treating chronic lung disease. Using evidence from human patient samples, transgenic animal models, and cell and molecular biology studies we highlight the roles of this signalling pathway in lung development, homeostasis, repair, and disease ontogeny. We identify mechanisms underlying lung EGFR pathway regulation and suggest how targeting these mechanisms using new and existing therapies has the potential to improve future lung cancer, COPD and pulmonary fibrosis patient outcomes.


Assuntos
Receptores ErbB/metabolismo , Pneumopatias/fisiopatologia , Animais , Doença Crônica , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Humanos , Inflamação , Pulmão/fisiologia , Neoplasias Pulmonares/metabolismo , Camundongos , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fibrose Pulmonar/fisiopatologia , Transdução de Sinais , Resultado do Tratamento
8.
Hum Gene Ther ; 23(9): 960-79, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22708837

RESUMO

Achieving high-efficiency tumor targeting after systemic delivery is a considerable challenge facing oncolytic gene therapists. Efficient retargeting should be combined with efforts to improve in vivo safety, reduce hepatotoxicity, minimize off-target interactions, and improve antitumoral potency and efficacy. We previously described the successful retargeting of adenovirus serotype 5 (Ad5) to α(v)ß(6), an integrin that is highly overexpressed in numerous human carcinomas. In this study, we have further modified this construct by introducing mutations that ablate coxsackievirus-adenovirus receptor (CAR) binding and putative interactions with factor IX (FIX)/C4b-binding protein (C4BP). We have found that the resulting vector, Ad5-477dlTAYT(A20), displays a desirable in vivo safety profile. This vector does not agglutinate human erythrocytes, fails to cause thrombocytopenia after intravenous delivery, has limited induction of proinflammatory cytokines, and results in low-level toxicity (aspartate aminotransferase/alanine aminotransferase) when compared with Ad5-EGFP(WT). Furthermore, it has reduced accumulation in Kupffer cells (1 hr) and limited hepatocyte transduction at later time points (24 and 96 hr). The parental vector, Ad5-EGFP(A20), also displayed many of these desirable properties. As a result of the improved safety profile of both A20-modified vectors, we escalated the dose from 2×10(10) to 4×10(10) viral particles in an antitumoral efficacy study. We observed improvements in reducing percent tumor growth at early time points (96 hr) when compared with Ad5-EGFP(WT), although increasing the dose did not affect the therapeutic outcome beneficially. On completion of the experiment, we detected increased E1A staining in the tumors of all A20-treated groups and we determined that E1A expression was localized largely within α(v)ß(6)(+) tumor cells. However, in spite of apparently efficient tumor transduction, this did not result in enhanced antitumoral efficacy as the virus failed to disseminate effectively throughout the tumor mass, presumably due to physical intratumoral restrictions. This highlights a remaining challenge that needs to be overcome before such vectors can be developed for future cancer gene therapy applications.


Assuntos
Adenoviridae , Antígenos de Neoplasias/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Integrinas/metabolismo , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Tropismo Viral , Proteínas E1A de Adenovirus/biossíntese , Animais , Antígenos de Neoplasias/genética , Sítios de Ligação , Células CHO , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Cricetinae , Cricetulus , Feminino , Regulação Viral da Expressão Gênica/genética , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Integrinas/genética , Células de Kupffer/metabolismo , Células de Kupffer/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/virologia , Transdução Genética
9.
Breast Cancer Res Treat ; 125(1): 43-53, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20224929

RESUMO

Nicastrin is an essential component of the gamma secretase (GS) enzyme complex, required for its synthesis and recognition of substrates for proteolytic cleavage. The purpose of this study was to investigate whether nicastrin has prognostic value or potential as a therapeutic target in breast cancer (BC). The suitability of nicastrin as a target in BC was assessed using BC tissue microarrays (TMAs) (n = 1050), and its biological role in vitro was evaluated in BC cell lines following gene silencing. Nicastrin blocking antibodies were developed and evaluated for their suitability as potential clinical therapeutics. TMA and cell line analysis confirmed that nicastrin expression was upregulated in BC compared to normal breast cells. In TMA patient samples, high nicastrin expression was observed in 47.5% of cases and correlated with ERα expression, patient age, and tumor grade. In pre-defined subset analysis, high nicastrin expression predicted for worse BC specific survival in the ERα -ve cohort. In vitro gene silencing of nicastrin resulted in disruption of the GS complex and a decrease in notch1 cleavage. This was sufficient to increase E-cadherin expression and its co-localization with p120 catenin at cell-cell junctions in MCF7 cells. Nicastrin silencing in invasive MDA-MB-231 cells resulted in loss of vimentin expression and a marked reduction in both cell motility and invasion; which was concomitant with the de novo formation of cell-cell junctions characterized by the colocalization of p120 catenin and F-actin. These data indicate that nicastrin can function to maintain epithelial to mesenchymal transition during BC progression. Anti-nicastrin polyclonal and monoclonal antibodies were able to decrease notch1 and vimentin expression and reduced the invasive capacity of BC cells in vitro. This supports our hypothesis that a nicastrin blocking antibody could be used to limit metastatic dissemination in invasive BC.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Glicoproteínas de Membrana/metabolismo , Actinas/metabolismo , Fatores Etários , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Caderinas/metabolismo , Cateninas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Junções Intercelulares/metabolismo , Estimativa de Kaplan-Meier , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Interferência de RNA , Fatores de Tempo , Análise Serial de Tecidos , Vimentina/metabolismo , delta Catenina
10.
Chem Commun (Camb) ; 46(40): 7533-5, 2010 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-20838674

RESUMO

We report the first example of peptide-protein heteronuclear two-dimensional (2D) saturation transfer difference nuclear magnetic resonance (STD NMR). This method, resulting in dramatically reduced overlap, was applied to the interaction of the integrin αvß6 with a known peptide ligand and highlights novel contact points between the substrate and target protein.


Assuntos
Antígenos de Neoplasias/metabolismo , Vírus da Febre Aftosa/metabolismo , Integrinas/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Sítios de Ligação , Vírus da Febre Aftosa/química , Humanos , Integrinas/química , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Receptores Virais/química
11.
J Virol ; 83(13): 6416-28, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19369326

RESUMO

A key impediment to successful cancer therapy with adenoviral vectors is the inefficient transduction of malignant tissue in vivo. Compounding this problem is the lack of cancer-specific targets, coupled with a shortage of corresponding high-efficiency ligands, permitting selective retargeting. The epithelial cell-specific integrin alphavbeta6 represents an attractive target for directed therapy since it is generally not expressed on normal epithelium but is upregulated in numerous carcinomas, where it plays a role in tumor progression. We previously have characterized a high-affinity, alphavbeta6-selective peptide (A20FMDV2) derived from VP1 of foot-and-mouth disease virus. We generated recombinant adenovirus type 5 (Ad5) fiber knob, incorporating A20FMDV2 in the HI loop, for which we validated the selectivity of binding and functional inhibition of alphavbeta6. The corresponding alphavbeta6-retargeted virus Ad5-EGFP(A20) exhibited up to 50-fold increases in coxsackievirus- and-adenovirus-receptor-independent transduction and up to 480-fold-increased cytotoxicity on a panel of alphavbeta6-positive human carcinoma lines compared with Ad5-EGFP(WT). Using an alphavbeta6-positive (DX3-beta6) xenograft model, we observed a approximately 2-fold enhancement in tumor uptake over Ad5-EGFP(WT) following systemic delivery. Furthermore, approximately 5-fold-fewer Ad5-EGFP(A20) genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. Warfarin pretreatment, to deplete coagulation factors, did not improve tumor uptake significantly with either virus but did significantly reduce liver sequestration and hepatic toxicity. The ability of Ad5-EGFP(A20) to improve delivery to alphavbeta6, combined with its reduced hepatic tropism and toxicity, highlights its potential as a prototype virus for future clinical investigation.


Assuntos
Adenoviridae/genética , Antígenos de Neoplasias/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Integrinas/metabolismo , Terapia Viral Oncolítica , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Feminino , Genoma Viral , Humanos , Camundongos , Camundongos Nus , Ligação Proteica , Receptores Virais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética
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