RESUMO
BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.
Assuntos
Receptores de Antígenos Quiméricos , Anticorpos de Cadeia Única , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Linfócitos T , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/terapia , Microambiente TumoralRESUMO
INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.
Assuntos
Leptina/metabolismo , Cirrose Hepática/metabolismo , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Osteopontina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Deleção de Genes , Hepatócitos/metabolismo , Hepatócitos/patologia , Leptina/genética , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Osteopontina/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).
Assuntos
Fator IX/genética , Terapia Genética , Vetores Genéticos/administração & dosagem , Hemofilia B/terapia , Adulto , Alanina Transaminase/sangue , Dependovirus/genética , Fator IX/metabolismo , Seguimentos , Expressão Gênica , Terapia Genética/efeitos adversos , Hemofilia B/sangue , Hemofilia B/genética , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Transgenes , Adulto JovemAssuntos
Antineoplásicos Imunológicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/imunologia , Linfócitos T/imunologia , Adenina/análogos & derivados , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Recidiva Local de Neoplasia/imunologia , Piperidinas , Células Tumorais CultivadasRESUMO
We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.
Assuntos
Dependovirus , Deficiência do Fator VII/terapia , Fator VII/uso terapêutico , Terapia Genética/métodos , Vetores Genéticos , Hemorragia/prevenção & controle , Assistência Perinatal , Animais , Animais Recém-Nascidos , Códon , Dependovirus/genética , Fator VII/análise , Fator VII/biossíntese , Fator VII/genética , Deficiência do Fator VII/sangue , Deficiência do Fator VII/genética , Deficiência do Fator VII/fisiopatologia , Feminino , Terapias Fetais/efeitos adversos , Expressão Gênica , Terapia Genética/efeitos adversos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Hemorragia/etiologia , Células Hep G2 , Humanos , Injeções Intravenosas , Macaca mulatta , Masculino , Camundongos , Gravidez , Caracteres Sexuais , Análise de SobrevidaRESUMO
BACKGROUND: Our understanding of the mechanism regulating pancreatic cancer metastatic phenotype is limited. We analyzed the role of RHOA and PRKCZ in the motility attitude of two subclones of the pancreatic adenocarcinoma cell line SUIT-2 (S2), with different in vivo metastatic potential in nude mice: S2-m with a low metastatic potential and highly metastatic S2-CP9 using RHOA and PRKCZ cell-permeable inhibitory peptides. METHODS: Adhesion assays, cell permeable peptides, RHOA activity assay, western blotting RESULTS: When used in combination cell-permeable inhibitory peptides partially inhibited cell adhesion by about 50% in clone S2-CP9. In clone S2-m, the effect was limited to 15% inhibition. In a wound healing assay, S2-CP9 was sensitive only to treatment with the combination of both RHOA and PRKCZ inhibitory peptides. Conversely, S2-m was unable to migrate toward both ends of the wound in basal conditions. Migration of cells through a membrane with 8 mum pores was completely abolished in both clones by individual treatment with RHOA and PRKCZ inhibitory peptides. CONCLUSION: Herein, we demonstrate a critical role for RHOA and PRKCZ in the regulation of different aspects of cell motility of pancreatic adenocarcinoma and demonstrate the need to inhibit both pathways to obtain a functionally relevant effect in most assays. These results indicate that RHOA and PRKCZ, and their downstream effectors, can represent important pharmacological targets that could potentially control the highly metastatic attitude of PDAC.
Assuntos
Movimento Celular , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/secundário , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Proteína Quinase C/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Clonais , Camundongos , Camundongos Nus , Peptídeos/farmacologia , Proteína Quinase C/genética , Cicatrização/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
AIMS: To (i) determine whether methylarginine-specific antibodies can be employed for standard immunohistochemical analysis of paraffin-embedded tissues, (ii) analyse methylarginine expression in normal and neoplastic tissues and (iii) correlate methylarginine expression with that of protein arginine methyltransferase (PRMT1), the predominant cellular arginine methyltransferase. METHODS AND RESULTS: Immunohistochemistry of normal and cancer tissues was performed utilizing three commercial polyclonal antibodies: anti-methylarginine-specific antibody (anti-mRG) raised against a methylarginine peptide, Control antibody (anti-RG), a control antiserum raised against a corresponding arginine peptide without any methylated residues and anti-PRMT1. Nuclear and/or cytoplasmic methylarginine expression was detected in all keratinized and non-keratinized epithelia. A preliminary survey of a series of thyroid, pancreatic, colonic and gastric cancers identified a different pattern of methylarginine expression in comparison with normal tissue. A correlation between methylarginine staining and PRMT1 expression was found in all normal and cancer tissues analysed. CONCLUSION: Methylarginine-specific antibodies are capable of recognizing methylarginine proteins (MeRP) in paraffin-embedded tissues. Methylarginine proteins are expressed widely and show differences in subcellular localization in various organs and neoplastic conditions. The efficient detection of methylproteins by standard immunohistochemistry provides a new tool to investigate the role of methylarginine proteins (MeRP) in biological processes including carcinogenesis.
Assuntos
Arginina/imunologia , Arginina/metabolismo , Imuno-Histoquímica , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Anticorpos/imunologia , Especificidade de Anticorpos , Metilação , Neoplasias/patologia , Inclusão em Parafina , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas/metabolismoRESUMO
Aimed at developing accurate, reliable and cost-saving analytical techniques for drugs screening we evaluated the potential of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) as a quantitative pre-diagnostic approach for the rapid identification of IR signatures of drugs targeting specific molecular pathways causing Chronic Myeloid Leukemia (CML). To obtain reproducible FTIR absorbance spectra at the necessary spatial resolution we optimized sample preparation and acquisition parameters on a single channel Mercury-Cadmium-Telluride (MCT) detector in the spectral interval of frequencies from 4000 to 800 cm(-1). Single K562 cells were illuminated by Synchrotron Radiation (SR) and a number of ~15 K562 cells spread in monolayer were illuminated by a conventional IR source (Globar), respectively. Combining IR spectral data with the results of complementary biochemical investigations carried out in samples by different analytical methods we identified and cross-validated IR signatures of drugs targeting the oncogenic protein BCR/ABL and its associated abnormal tyrosine kinase activity in K562 cell line. Unsupervised pattern recognition performed by Hierarchical Cluster Analysis (HCA) clustered the spectra of single K562 cells in two distinct groups roughly corresponding to living and to apoptotic cells, respectively. The corresponding IR spectral profiles were assumed to represent drug-resistant and drug-sensitive cells. Significant variations with increasing percentages of apoptotic cells were observed after the treatment of K562 cells with drugs that directly or indirectly target BCR/ABL. In conclusion, we suggest that microFTIR associated with multivariate data analysis may be useful to assess drug compounds in ex vivo cancer cell models and possibly peripheral blast cells from CML patients.
Assuntos
Antineoplásicos/química , Microscopia/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Antineoplásicos/uso terapêutico , Apoptose , Análise por Conglomerados , Resistencia a Medicamentos Antineoplásicos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Microscopia/instrumentação , Proteínas/química , Reprodutibilidade dos Testes , Espectroscopia de Infravermelho com Transformada de Fourier/instrumentaçãoRESUMO
Anti-inflammatory properties of azithromycin (AZM) have been proposed as possible mechanisms of clinical beneficial effects in patients with cystic fibrosis (CF). Altered glutathione (GSH) transport in cystic fibrosis transmembrane regulator protein (CFTR)-deficient cells leads to the occurrence of oxidative stress that finally induces glutathione S-transferase (GST) activity. The present investigation was aimed to verify the effects of AZM on GST activity and expression in CF airway cells in vitro and in vivo. AZM exposure significantly decreased GSTT1 and GSTM1 mRNA and protein expression in IB3-1, restoring the levels to those observed in non-CF C38 cells, which also express lower levels of gamma-glutamyltransferase (GGT) activity than IB3-1. In another CF cell line, 2CFSMEo-, AZM produced 45% reduction in GSTT1 and GSTM1 mRNA levels. AZM reduced GST activity by approximately 25% and 40% in IB3-1 and 2CFSMEo- cells, respectively. GSTP1 was similarly expressed in all CF and non-CF cells and was unaffected by AZM. The anti-inflammatory cytokine IL-10 down-modulated GST activity at similar levels, supporting a link between GST inhibition and anti-inflammatory properties of AZM. In bronchoalveolar lavage fluid of CF mice homozygous for the F508 del mutation, GSTM1 protein levels were undetectable after AZM treatment. The association between increased GST expression and activity, together with its reversal by AZM treatment in vitro and in vivo, suggest novel antioxidant properties for this drug. The issue whether decreased GST activity may directly concur to anti-inflammatory properties of AZM or is rather a marker of the oxidative status of CF cells will require additional studies.
Assuntos
Anti-Inflamatórios/farmacologia , Azitromicina/farmacologia , Fibrose Cística , Células Epiteliais , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Mucosa Respiratória , Animais , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Feminino , Glutationa Transferase/genética , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Isoenzimas/genética , Camundongos , Camundongos Transgênicos , Oxirredução , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/enzimologia , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We have developed a humanized bi-specific T-cell engager (BiTE) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), a cell surface antigen present on a range of malignancies and cancer-initiating cells. Focusing initially on pancreatic cancer, we demonstrated that our ROR1 BiTE results in T cell mediated and antigen-specific cytotoxicity against ROR1-expressing pancreatic cancer cell lines in vitro at exceedingly low concentrations (0.1 ng/mL) and low effector to target ratios. Our BiTE prevented engraftment of pancreatic tumor xenografts in murine models and reduced the size of established subcutaneous tumors by at least 3-fold. To validate its wider therapeutic potential, we next demonstrated significant cytotoxicity against ovarian cancer in an in vitro and in vivo setting and T-cell-mediated killing of a range of histologically distinct solid tumor cell lines. Overall, our ROR1 BiTE represents a promising immunotherapy approach, because of its ability to target a broad range of malignancies, many with significant unmet therapeutic needs.
RESUMO
The purpose of this study is to identify novel proteins released by cancer cells that are involved in extracellular matrix (ECM) remodeling using small-volume samples and automated technology. We applied multidimensional protein identification technology (MudPIT), which incorporates two-dimensional capillary chromatography coupled to tandem mass spectrometry to small quantities of serum-free supernatants of resting or phorbol ester-activated Suit-2 pancreatic cancer cells. Selected markers were validated in additional pancreatic cancer cell lines, primary cancers, and xenografted cancer cells. MudPIT analysis of 10 microl of supernatants identified 46 proteins, 21 of which are classified as secreted, and 10 have never been associated with pancreatic cancer. These include CSPG2/versican, Mac25/angiomodulin, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, cyclophilin B, beta2 microglobulin, and ICA69. Evidence that cancer cells release these proteins in vivo was obtained for CSPG2/versican and Mac25/angiomodulin by immunohistochemistry on both primary pancreatic cancers and in a model consisting of Suit-2 cells embedded in an amorphous matrix and implanted in athymic mice. MudPIT allowed efficient and rapid identification of proteins released by cancer cells, including molecules previously undescribed in the type of cancer analyzed. Our finding that pancreatic cancer cells secrete a series of proteoglycans, including versican, perlecan, syndecan 1 and 4, challenges the common view that fibroblasts of tumor stroma are the sole source of these molecules.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Neoplasias/análise , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/química , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/análise , Cromatografia , Humanos , Imuno-Histoquímica , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Espectrometria de Massas , Proteínas do Tecido Nervoso/análise , Neoplasias Pancreáticas/química , Software , VersicanasRESUMO
We report the correction of hyperglycemia of STZ induced diabetic mice using one intravenous systemic administration of a single stranded serotype 8 pseudotyped adeno-associated virus (ssAAV2/8) vector encoding the human proinsulin gene under a constitutive liver specific promoter. In vivo dose titration experiments were carried out and we identified an optimal range that achieved maintenance of euglycaemia or a mild diabetic condition for at least 9 months and ongoing to beyond 1 year for some animals, accompanied by human C-peptide secretion and weight gain. Further DNA codon optimization of the insulin gene construct resulted in approximately 3-10 times more human C-peptide secreted in the blood of codon optimized treated animals thereby reducing the number of vector particles required to achieve the same extent of reduction in blood glucose levels as the non-codon optimized vector. The constitutive secretion of insulin achieved with a single administration of the vector could be of therapeutic value for some diabetic patients.
Assuntos
Vetores Genéticos/administração & dosagem , Hiperglicemia/terapia , Insulina/genética , Animais , Peptídeo C/metabolismo , Códon , Dependovirus/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/terapia , Humanos , Hiperglicemia/genética , Fígado/metabolismo , Camundongos Endogâmicos NOD , Pâncreas/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genéticaRESUMO
A novel selectively targeting gene delivery approach has been developed for advanced hepatocellular carcinoma (HCC), a leading cause of cancer mortality whose prognosis remains poor. We combine the strong liver tropism of serotype-8 capsid-pseudotyped adeno-associated viral vectors (AAV8) with a liver-specific promoter (HLP) and microRNA-122a (miR-122a)-mediated posttranscriptional regulation. Systemic administration of our AAV8 construct resulted in preferential transduction of the liver and encouragingly of HCC at heterotopic sites, a finding that could be exploited to target disseminated disease. Tumor selectivity was enhanced by inclusion of miR-122a-binding sequences (ssAAV8-HLP-TK-122aT4) in the expression cassette, resulting in abrogation of transgene expression in normal murine liver but not in HCC. Systemic administration of our tumor-selective vector encoding herpes simplex virus-thymidine kinase (TK) suicide gene resulted in a sevenfold reduction in HCC growth in a syngeneic murine model without toxicity. In summary, we have developed a systemically deliverable gene transfer approach that enables high-level expression of therapeutic genes in HCC but not normal tissues, thus improving the prospects of safe and effective treatment for advanced HCC.
Assuntos
Carcinoma Hepatocelular/terapia , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/farmacocinética , Neoplasias Hepáticas/terapia , MicroRNAs/genética , Timidina Quinase/genética , Proteínas Virais/genética , Animais , Capsídeo/química , Capsídeo/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Dependovirus/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Engenharia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Humanos , Fígado/patologia , Fígado/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Simplexvirus/química , Simplexvirus/enzimologia , Timidina Quinase/metabolismo , Timidina Quinase/farmacocinética , Distribuição Tecidual , Transplante Heterotópico , Proteínas Virais/metabolismo , Proteínas Virais/farmacocinéticaRESUMO
Chronic myelogenous leukemia (CML) is the most common myeloproliferative disease. Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene and a myeloid cell marker expressed by CD34(+) cells. Downregulation of PTPRG increases colony formation in the PTPRG-positive megakaryocytic cell lines MEG-01 and LAMA-84 but has no effect in the PTPRG-negative cell lines K562 and KYO-1. Its overexpression has an oncosuppressive effect in all these cell lines and is associated with myeloid differentiation and inhibition of BCR/ABL-dependent signaling. The intracellular domain of PTPRG directly interacts with BCR/ABL and CRKL, but not with signal transducers and activators of transcription 5. PTPRG is downregulated at the mRNA and protein levels in leukocytes of CML patients in both peripheral blood and bone marrow, including CD34(+) cells, and is reexpressed following molecular remission of disease. Reexpression was associated with a loss of methylation of a CpG island of PTPRG promoter occurring in 55% of the patients analyzed. In K562 cell line, the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced PTPRG expression and caused an inhibition of colony formation, partially reverted by downregulation of PTPRG expression. These findings establish, for the first time, PTPRG as a tumor suppressor gene involved in the pathogenesis of CML, suggesting its use as a potential diagnostic and therapeutic target.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Proliferação de Células , Regulação para Baixo , Feminino , Citometria de Fluxo , Imunofluorescência , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: We investigated the global gene expression in a large panel of pancreatic endocrine tumors (PETs) aimed at identifying new potential targets for therapy and biomarkers to predict patient outcome. PATIENTS AND METHODS: Using a custom microarray, we analyzed 72 primary PETs, seven matched metastases, and 10 normal pancreatic samples. Relevant differentially expressed genes were validated by either quantitative real-time polymerase chain reaction or immunohistochemistry on tissue microarrays. RESULTS: Our data showed that: tuberous sclerosis 2 (TSC2) and phosphatase and tensin homolog (PTEN) were downregulated in most of the primary tumors, and their low expression was significantly associated with shorter disease-free and overall survival; somatostatin receptor 2 (SSTR2) was absent or very low in insulinomas compared with nonfunctioning tumors; and expression of fibroblast growth factor 13 (FGF13) gene was significantly associated with the occurrence of liver metastasis and shorter disease-free survival. TSC2 and PTEN are two key inhibitors of the Akt/mammalian target of rapamycin (mTOR) pathway and the specific inhibition of mTOR with rapamycin or RAD001 inhibited cell proliferation of PET cell lines. CONCLUSION: Our results strongly support a role for PI3K/Akt/mTOR pathway in PET, which ties in with the fact that mTOR inhibitors have reached phase III trials in neuroendocrine tumors. The finding of differential SSTR expression raises the potential for SSTR expression to be evaluated as a marker of response to somatostatin analogs. Finally, we identified FGF13 as a new prognostic marker that predicted poorer outcome in patients who were clinically considered free from disease.
Assuntos
Carcinoma de Células das Ilhotas Pancreáticas/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Adolescente , Adulto , Idoso , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Transdução de Sinais , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
Protein Tyrosine Phosphatase gamma (PTPgamma) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPgamma is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPgamma mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPgamma specific antibody that recognizes the protein by flow cytometry. PTPgamma expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPgamma was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34(+) haematopoietic precursors express high PTPgamma level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPgamma as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation.