RESUMO
TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the ß cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Linfoma/metabolismo , Linfoma/patologia , Proteína Supressora de Tumor p53/deficiência , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/uso terapêutico , Linfoma/tratamento farmacológico , Linfoma/genética , Masculino , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Receptores da Calcitonina/metabolismo , Timo/metabolismo , Timo/patologia , Transativadores/genética , Transativadores/metabolismo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: The receptor for the cardiac hormone atrial natriuretic peptide (ANP), natriuretic peptide receptor A (NPRA), is expressed in cancer cells, and natriuretic peptides have been implicated in cancers. However, the direct role of NPRA signaling in prostate cancer remains unclear. RESULTS: NPRA expression was examined by western blotting, RT-PCR and immunohistochemistry. NPRA was downregulated by transfection of siRNA, shRNA and NPRA inhibitor (iNPRA). Antitumor efficacy of iNPRA was tested in mice using a TRAMP-C1 xenograft. Here, we demonstrated that NPRA is abundantly expressed on tumorigenic mouse and human prostate cells, but not in nontumorigenic prostate epithelial cells. NPRA expression showed positive correlation with clinical staging in a human PCa tissue microarray. Down-regulation of NPRA by siNPRA or iNPRA induced apoptosis in PCa cells. The mechanism of iNPRA-induced anti-PCa effects was linked to NPRA-induced expression of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine over-expressed in PCa and significantly reduced by siNPRA. Prostate tumor cells implanted in mice deficient in atrial natriuretic peptide receptor A (NPRA-KO) failed to grow, and treatment of TRAMP-C1 xenografts with iNPRA reduced tumor burden and MIF expression. Using the TRAMP spontaneous PCa model, we found that NPRA expression correlated with MIF expression during PCa progression. CONCLUSIONS: Collectively, these results suggest that NPRA promotes PCa development in part by regulating MIF. Our findings also suggest that NPRA is a potential prognostic marker and a target for PCa therapy.
Assuntos
Neoplasias da Próstata/fisiopatologia , Receptores do Fator Natriurético Atrial/metabolismo , Animais , Apoptose , Linhagem Celular , Progressão da Doença , Regulação para Baixo , Humanos , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/genética , Peptídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Coelhos , Ratos , Receptores do Fator Natriurético Atrial/antagonistas & inibidores , Receptores do Fator Natriurético Atrial/genética , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Natriuretic peptide receptor A (NPRA), the receptor for the cardiac hormone atrial natriuretic peptide (ANP), is expressed abundantly on cancer cells and disruption of ANP-NPRA signaling inhibits tumor burden and metastasis. Since antagonists of NPRA signaling have not provided reproducible results, we reasoned that a synthetic neutralizing antibody to ANP, which has high selectivity and affinity for ANP, could be used to regulate ANP levels and attenuate NPRA signaling. In this study, we prepared molecularly imprinted polymer nanoparticles (MIPNPs) for ANP using a short peptide of ANP as the template and determined their binding affinity and selectivity. The MIPNPs were prepared by precipitation polymerization using NH2-SLRRSS-CONH2, which is a short peptide from ANP as template, methacrylic acid (MAA) and N-isopropylacrylamide (NIPAm) as functional monomers, bis-acrylamide (BIS) as crosslinker. The average diameter of MIPNPs and non-imprinted nanoparticles (NIPNPs) in water is 215.8 ±4.6 nm and 197.7±3.1 nm respectively. The binding isotherm analysis showed that MIPNPs have a much higher binding affinity for template peptide and ANP than NIPNPs. Scatchard analysis gave an equilibrium dissociation constant, Kd of 7.3 µM with a binding capacity 106.7 µmol/g for template peptide and Kd of 7.9 µM with a binding capacity of 36.0 µmol/g for ANP. Measurements of binding kinetics revealed that MIPNPs reach protein adsorption equilibrium in 30 min. MIPNPs found to have high specificity for ANP with little affinity for BSA or scrambled ANP peptide. MIPNPs also recognized and adsorbed ANP in cell culture media spiked with ANP and human plasma. Taken together, these results indicate that MIPNPs have high affinity and selectivity for ANP and can be used as a synthetic antibody for modulating ANP-NPRA signaling in cancers.
RESUMO
A subset of hepatocellular carcinoma (HCC) overexpresses the chromosome 19 miRNA cluster (C19MC) and is associated with an undifferentiated phenotype marked by overexpression of cancer testis antigens (CTAs) including anti-apoptotic melanoma-A antigens (MAGEAs). However, the regulation of C19MC miRNA and MAGEA expression in HCCs are not understood. Here we show that, C19MC overexpression is tightly linked to a sub-set of HCCs with transcription-incompetent p53. Using next-generation and Sanger sequencing we found that, p53 in Hep3B cells is impaired by TP53-FXR2 fusion, and that overexpression of the C19MC miRNA-520G in Hep3B cells promotes the expression of MAGEA-3, 6 and 12 mRNAs. Furthermore, overexpression of p53-R175H and p53-R273H mutants promote miR-520G and MAGEA RNA expression and cellular transformation. Moreover, IFN-γ co-operates with miR-520G to promote MAGEA expression. On the other hand, metals such as nickel and zinc promote miR-526B but not miR-520G, to result in the suppression of MAGEA mRNA expression, and evoke cell death through mitochondrial membrane depolarization. Therefore our study demonstrates that a MAGEA-promoting network involving miR-520G, p53-defects and IFN-γ that govern cellular transformation and cell survival pathways, but MAGEA expression and survival are counteracted by nickel and zinc combination.
Assuntos
Antígenos de Neoplasias , Carcinoma Hepatocelular , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 19 , Humanos , Interferon gama/metabolismo , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/metabolismo , Mutação , Fusão Oncogênica , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Testículo/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
MYO18B has been proposed to contribute to the progression of hepatocellular carcinoma (HCC). However, the signals that govern MYO18B transcription are not known. Here we show that, a network of C19MC miRNA-520G, IFN-γ, CEBPB and p53 transcriptional-defects promote MYO18B mRNA expression in HCCs. IFN-γ by itself suppresses MYO18B transcription, but promotes it when miRNA-520G is stably overexpressed. Similarly, CEBPB-liver-enriched activator protein (LAP) isoform overexpression suppresses MYO18B transcription but promotes transcription when the cells are treated with IFN-γ. Furthermore, miR-520G together with mutant-p53 promotes MYO18B transcription. Conversely, bFGF suppresses MYO18B mRNA irrespective of CEBPB, miR-520G overexpression or IFN-γ treatment. Finally high MYO18B expression reflects poor prognosis while high MYL5 or MYO1B expression reflects better survival of HCC patients. Thus, we identified a network of positive and negative regulators of MYO18B mRNA expression which reflects the survival of HCC patients.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Carcinoma Hepatocelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/biossíntese , Regulação Neoplásica da Expressão Gênica , Interferon gama/biossíntese , Neoplasias Hepáticas/metabolismo , MicroRNAs/biossíntese , Miosinas/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Interferon gama/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/genética , Miosinas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
TP53 is highly mutated in human cancers, thus targeting this tumor suppressor pathway is highly desirable and will impact many cancer patients. (1,2) Therapeutic strategies to reactivate the p53-pathway have been challenging, (3,4) and no effective treatment exists. (5) We utilized the p53-family members, p63 and p73, which are not frequently mutated in cancer, to treat p53-defective cancers. The N-terminal splice variants of p63 and p73 are denoted as the TA and ΔN isoforms. We recently demonstrated that deletion of either ΔNp63 or ΔNp73 in p53-deficient mouse tumors results in tumor regression mediated by metabolic programming. Using this strategy, we identified pramlintide, a synthetic analog of amylin, as an effective treatment for p53 deficient and mutant tumors. Here, we show the utility of using pramlintide, as a potential cancer preventive option for p53-deficient tumors in mouse models. Additionally, we found that in vivo inhibition of both ΔNp63 and ΔNp73 in combination accelerates tumor regression and increases survival of p53-deficient mice. We report that inhibition of both ΔNp63 and ΔNp73 in combination results in upregulation of 3 key metabolic regulators, IAPP, GLS2, and TIGAR resulting in an increase in apoptosis and tumor regression in ΔNp63/ΔNp73/p53 deficient thymic lymphomas. These data highlight the value of generating inhibitors that will simultaneously target ΔNp63 and ΔNp73 to treat cancer patients with alterations in p53.
Assuntos
Anticarcinógenos/farmacologia , Proteínas de Ligação a DNA/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Linfoma/tratamento farmacológico , Proteínas Nucleares/genética , Fosfoproteínas/genética , Neoplasias do Timo/tratamento farmacológico , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Proteínas de Ligação a DNA/deficiência , Regulação Neoplásica da Expressão Gênica , Glicólise/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/agonistas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Linfoma/genética , Linfoma/mortalidade , Linfoma/patologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Fosfoproteínas/deficiência , Monoéster Fosfórico Hidrolases , Proteínas/agonistas , Proteínas/genética , Proteínas/metabolismo , Transdução de Sinais , Análise de Sobrevida , Neoplasias do Timo/genética , Neoplasias do Timo/mortalidade , Neoplasias do Timo/patologia , Transativadores/deficiência , Transaminases/genética , Transaminases/metabolismo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/deficiência , Proteínas Supressoras de Tumor/deficiênciaRESUMO
ΔNp63 is an oncogenic member of the p53 family and acts to inhibit the tumor-suppressive activities of the p53 family. By performing a chemical library screen, we identified histone deacetylase inhibitors (HDACi) as agents reducing ΔNp63 protein stability through the E3 ubiquitin ligase, Fbw7. ΔNp63 inhibition decreases the levels of its transcriptional target, DGCR8, and the maturation of let-7d and miR-128, which we found to be critical for HDACi function in vitro and in vivo. Our work identified Fbw7 as a predictive marker for HDACi response in squamous cell carcinomas and lymphomas, and unveiled let-7d and miR-128 as specific targets to bypass tumor resistance to HDACi treatment.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Inibidores de Histona Desacetilases/administração & dosagem , Linfoma/tratamento farmacológico , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Animais , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Linfoma/genética , Camundongos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2) are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s) as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid) was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs) and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC) model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162) viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections.
Assuntos
Antivirais , Infecções por HIV/virologia , HIV-1/genética , RNA Interferente Pequeno/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Animais , Antivirais/administração & dosagem , Antivirais/química , Linhagem Celular , Modelos Animais de Doenças , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Genes Virais , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Vetores Genéticos/genética , Infecções por HIV/prevenção & controle , Humanos , Macaca mulatta , Camundongos , Mucosa/virologia , Nanocompostos/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Coelhos , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Técnicas de Cultura de Tecidos , Transfecção , Replicação ViralRESUMO
Gene therapy is a promising therapeutic approach for treating disease, but the efficient delivery of genes to desired locations with minimal side effects remains a challenge. In addition to gene therapy, it is also highly desirable to provide sensitive imaging information in patients for disease diagnosis, screening and post-therapy monitoring. Here, we report on the development of dual-purpose chitosan and polyethyleneimine (PEI) coated magnetic micelles (CP-mag-micelles) that can deliver nucleic acid-based therapeutic agents and also provide magnetic resonance imaging (MRI). These 'theranostic' CP-mag-micelles are composed of monodisperse hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) loaded into the cores of micelles that are self-assembled from a block copolymer of poly (D, L-lactide) (PLA) and monomethoxy polyethylene glycol (mPEG). For efficient loading and protection of the nucleic acids the micelles were coated with cationic polymers, such as chitosan and PEI. The morphology and size distribution of the CP-mag-micelles were characterized and their potential for use as an MRI-probe was tested using an MRI scanner. The T(2) relaxivity of mag-micelles was similar to CP-mag-micelles confirming that coating with cationic polymers did not alter magnetism. Nanoparticles coated with chitosan:PEI at a weight ratio of 5:5 showed higher transfection efficiency in HEK293, 3T3 and PC3 cells than with weight ratios of 3:7 or 7:3. CP-mag-micelles are biocompatible, can be delivered to various organs and are safe. A single injection of CP-mag-micelles carrying reporter plasmids in vivo expressed genes for at least one week. Collectively, our results demonstrate that a structural reinforcement of SPIONs loaded in the core of an mPEG-PLA micelle coated with cationic polymers provides efficient DNA delivery and enhanced MRI potential, and affords a promising candidate for theranostics in the future.