RESUMEN
BACKGROUND: Glioblastoma is one of the most lethal forms of cancer, with 5-year survival rates of only 6%. Glioblastoma-targeted therapeutics have been challenging to develop due to significant inter- and intra-tumoral heterogeneity. Telomerase reverse transcriptase gene (TERT) promoter mutations are the most common known clonal oncogenic mutations in glioblastoma. Telomerase is therefore considered to be a promising therapeutic target against this tumor. However, an important limitation of this strategy is that cell death does not occur immediately after telomerase ablation, but rather after several cell divisions required to reach critically short telomeres. We, therefore, hypothesize that telomerase inhibition would only be effective in glioblastomas with low tumor burden. METHODS: We used CRISPR interference to knock down TERT expression in TERT promoter-mutant glioblastoma cell lines and patient-derived models. We then measured viability using serial proliferation assays. We also assessed for features of telomere crisis by measuring telomere length and chromatin bridge formation. Finally, we used a doxycycline-inducible CRISPR interference system to knock down TERT expression in vivo early and late in tumor development. RESULTS: Upon TERT inactivation, glioblastoma cells lose their proliferative ability over time and exhibit telomere shortening and chromatin bridge formation. In vivo, survival is only prolonged when TERT knockdown is induced shortly after tumor implantation, but not when the tumor burden is high. CONCLUSIONS: Our results support the idea that telomerase inhibition would be most effective at treating glioblastomas with low tumor burden, for example in the adjuvant setting after surgical debulking and chemoradiation.
Asunto(s)
Glioblastoma , Telomerasa , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Telomerasa/genética , Telomerasa/metabolismo , Carga Tumoral , Mutación , Telómero/genética , Telómero/metabolismo , Telómero/patologíaRESUMEN
PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders.
Asunto(s)
PPAR gamma , Neoplasias de la Vejiga Urinaria , Humanos , PPAR gamma/agonistas , Agonismo Inverso de Drogas , Agonistas de PPAR-gamma , Regulación de la Expresión GénicaRESUMEN
The ligand-activated nuclear receptor peroxisome-proliferator-activated receptor-γ (PPARG or PPARγ) represents a potential target for a new generation of cancer therapeutics, especially in muscle-invasive luminal bladder cancer where PPARγ is a critical lineage driver. Here we disclose the discovery of a series of chloro-nitro-arene covalent inverse-agonists of PPARγ that exploit a benzoxazole core to improve interactions with corepressors NCOR1 and NCOR2. In vitro treatment of sensitive cell lines with these compounds results in the robust regulation of PPARγ target genes and antiproliferative effects. Despite their imperfect physicochemical properties, the compounds showed modest pharmacodynamic target regulation in vivo. Improvements to the in vitro potency and efficacy of BAY-4931 and BAY-0069 compared to those of previously described PPARγ inverse-agonists show that these compounds are novel tools for probing the in vitro biology of PPARγ inverse-agonism.
Asunto(s)
PPAR gamma , PPAR gamma/metabolismo , LigandosRESUMEN
Androgen receptor (AR) signaling is the central driver of prostate cancer across disease states. While androgen deprivation therapy (ADT) is effective in the initial treatment of prostate cancer, resistance to ADT or to next-generation androgen pathway inhibitors invariably arises, most commonly through the re-activation of the AR axis. Thus, orthogonal approaches to inhibit AR signaling in advanced prostate cancer are essential. Here, via genome-scale CRISPR-Cas9 screening, we identify protein arginine methyltransferase 1 (PRMT1) as a critical mediator of AR expression and signaling. PRMT1 regulates the recruitment of AR to genomic target sites and the inhibition of PRMT1 impairs AR binding at lineage-specific enhancers, leading to decreased expression of key oncogenes, including AR itself. In addition, AR-driven prostate cancer cells are uniquely susceptible to combined AR and PRMT1 inhibition. Our findings implicate PRMT1 as a key regulator of AR output and provide a preclinical framework for co-targeting of AR and PRMT1 in advanced prostate cancer.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Antagonistas de Andrógenos/farmacología , Antagonistas de Andrógenos/uso terapéutico , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
Both previous and additional genetic knockdown studies reported herein implicate G protein-coupled receptor kinase 6 (GRK6) as a critical kinase required for the survival of multiple myeloma (MM) cells. Therefore, we sought to develop a small molecule GRK6 inhibitor as an MM therapeutic. From a focused library of known kinase inhibitors, we identified two hits with moderate biochemical potencies against GRK6. From these hits, we developed potent (IC50 < 10 nM) analogues with selectivity against off-target kinases. Further optimization led to the discovery of an analogue (18) with an IC50 value of 6 nM against GRK6 and selectivity against a panel of 85 kinases. Compound 18 has potent cellular target engagement and antiproliferative activity against MM cells and is synergistic with bortezomib. In summary, we demonstrate that targeting GRK6 with small molecule inhibitors represents a promising approach for MM and identify 18 as a novel, potent, and selective GRK6 inhibitor.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Quinasas de Receptores Acoplados a Proteína-G/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Quinazolinas/síntesis química , Quinazolinas/química , Relación Estructura-ActividadRESUMEN
Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Evolución Molecular , Variación Genética/genética , Inestabilidad Genómica/genética , Transcripción Genética/genética , Neoplasias de la Mama/patología , Proliferación Celular , Forma de la Célula , Células Clonales/citología , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Variación Genética/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Humanos , Células MCF-7 , Reproducibilidad de los ResultadosRESUMEN
Functional redundancy shared by paralog genes may afford protection against genetic perturbations, but it can also result in genetic vulnerabilities due to mutual interdependency1-5. Here, we surveyed genome-scale short hairpin RNA and CRISPR screening data on hundreds of cancer cell lines and identified MAGOH and MAGOHB, core members of the splicing-dependent exon junction complex, as top-ranked paralog dependencies6-8. MAGOHB is the top gene dependency in cells with hemizygous MAGOH deletion, a pervasive genetic event that frequently occurs due to chromosome 1p loss. Inhibition of MAGOHB in a MAGOH-deleted context compromises viability by globally perturbing alternative splicing and RNA surveillance. Dependency on IPO13, an importin-ß receptor that mediates nuclear import of the MAGOH/B-Y14 heterodimer9, is highly correlated with dependency on both MAGOH and MAGOHB. Both MAGOHB and IPO13 represent dependencies in murine xenografts with hemizygous MAGOH deletion. Our results identify MAGOH and MAGOHB as reciprocal paralog dependencies across cancer types and suggest a rationale for targeting the MAGOHB-IPO13 axis in cancers with chromosome 1p deletion.
Asunto(s)
Cromosomas Humanos Par 1 , Neoplasias/genética , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Exones/genética , Femenino , Eliminación de Gen , Células HEK293 , Humanos , Carioferinas/genética , Ratones , Ratones Desnudos , Proteínas Nucleares/genética , Empalme del ARN/genética , ARN Interferente Pequeño/genéticaRESUMEN
The PPARG gene encoding the nuclear receptor PPARγ is activated in bladder cancer, either directly by gene amplification or mutation, or indirectly by mutation of the RXRA gene, which encodes the heterodimeric partner of PPARγ. Here, we show that activating alterations of PPARG or RXRA lead to a specific gene expression signature in bladder cancers. Reducing PPARG activity, whether by pharmacologic inhibition or genetic ablation, inhibited proliferation of PPARG-activated bladder cancer cells. Our results offer a preclinical proof of concept for PPARG as a candidate therapeutic target in bladder cancer. Cancer Res; 77(24); 6987-98. ©2017 AACR.
Asunto(s)
Terapia Molecular Dirigida , PPAR gamma/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/terapia , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Amplificación de Genes/fisiología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Análisis por Micromatrices , Mutación/fisiología , Transcriptoma/fisiologíaRESUMEN
Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting ß-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Tanquirasas/antagonistas & inhibidores , Administración Oral , Disponibilidad Biológica , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Tanquirasas/metabolismoRESUMEN
Tankyrase (TNKS) is a poly-ADP-ribosylating protein (PARP) whose activity suppresses cellular axin protein levels and elevates ß-catenin concentrations, resulting in increased oncogene expression. The inhibition of tankyrase (TNKS1 and 2) may reduce the levels of ß-catenin-mediated transcription and inhibit tumorigenesis. Compound 1 is a previously described moderately potent tankyrase inhibitor that suffers from poor pharmacokinetic properties. Herein, we describe the utilization of structure-based design and molecular modeling toward novel, potent, and selective tankyrase inhibitors with improved pharmacokinetic properties (39, 40).
Asunto(s)
Bencimidazoles/síntesis química , Oxazolidinonas/síntesis química , Tanquirasas/antagonistas & inhibidores , Administración Oral , Animales , Bencimidazoles/farmacocinética , Bencimidazoles/farmacología , Sitios de Unión , Disponibilidad Biológica , Técnicas In Vitro , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Oxazolidinonas/farmacocinética , Oxazolidinonas/farmacología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Manipulation of gene expression is an invaluable tool to study gene function in vitro and in vivo. The application of small inhibitory RNAs to knock down gene expression provides a relatively simple, elegant, but transient approach to study gene function in many cell types as well as in whole animals. Short hairpin structures (shRNAs) are a logical advance as they can be expressed continuously and are hence suitable for stable gene knockdown. Drug-inducible systems have now been developed; however, application of the technology has been hampered by persistent problems with low or transient expression, leakiness or poor inducibility of the short hairpin, and lack of reversibility. We have developed a robust, versatile, single lentiviral vector tool that delivers tightly regulated, fully reversible, doxycycline-responsive knockdown of target genes (FOXP3 and MYB), using single short hairpin RNAs. To demonstrate the capabilities of the vector we targeted FOXP3 because it plays a critical role in the development and function of regulatory T cells. We also targeted MYB because of its essential role in hematopoiesis and implication in breast cancer progression. The versatility of this vector is hence demonstrated by knockdown of distinct genes in two biologically separate systems.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Vectores Genéticos , Lentivirus/genética , ARN Interferente Pequeño/metabolismo , Animales , Doxiciclina/metabolismo , Factores de Transcripción Forkhead/genética , Expresión Génica , Marcación de Gen , Células HEK293 , Humanos , Lentivirus/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-myb/genética , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) selectively induces apoptosis in transformed cells. Normal cells and certain tumor cells can evade Apo2L/TRAIL induced cell death, but the determinants of Apo2L/TRAIL sensitivity are poorly understood. To better understand the factors that contribute to Apo2L/TRAIL resistance, we characterized two colon carcinoma lines with pronounced differences in Apo2L/TRAIL sensitivity. Colo205 cells are highly sensitive to Apo2L/TRAIL whereas Colo320 cells are unresponsive. Components of the DISC (death inducing signaling complex) could be immunoprecipitated from both cell lines in response to Apo2L/TRAIL. Sensitizing agents including a proteasome inhibitor conferred Apo2L/TRAIL sensitivity in Colo320 cells, indicating that the apoptotic machinery was intact and functional. We specifically suppressed the expression of Bcl-2, FLIP or XIAP in Colo320 cells. Downregulation of either FLIP or XIAP but not Bcl-2 restored sensitivity of Colo320 cells to Apo2L/TRAIL. Moreover, stable knockdown of XIAP expression in Colo320 subcutaneous tumors resulted in suppression of tumor growth and sensitivity to Apo2L/TRAIL in vivo. Our results indicate that only a specific subset of anti-apoptotic proteins can confer resistance to Apo2L/TRAIL in Colo320 cells. Elucidation of the factors that contribute to Apo2L/TRAIL resistance in tumor cells may provide insight into combination therapies with Apo2L/TRAIL in a clinical setting.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Carcinoma/genética , Neoplasias del Colon/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Carcinoma/patología , Neoplasias del Colon/patología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones SCID , Receptores de Muerte Celular/metabolismo , Células Tumorales Cultivadas , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously reported that interleukin (IL)-4 treatment of nonobese diabetic (NOD) mice elevates intrapancreatic CCL4 expression and protects from type 1 diabetes. Here, we show that antibody neutralization of CCL4 abrogates the ability of T-cells from IL-4-treated NOD mice to transfer protection against type 1 diabetes. Intradermal delivery of CCL4 via a plasmid vector stabilized by incorporation of the Epstein-Barr virus EBNA1/oriP episomal maintenance replicon (pHERO8100-CCL4) to NOD mice beginning at later stages of disease progression protects against type 1 diabetes. This protection was associated with a Th2-like response in the spleen and pancreas; decreased recruitment of activated CD8(+) T-cells to islets, accompanied by diminished CCR5 expression on CD8(+) T-cells; and regulatory T-cell activity in the draining pancreatic lymph nodes. Thus, inflammatory responses that target islet beta-cells are suppressed by CCL4, which implicates the use of CCL4 therapeutically to prevent type 1 diabetes.
Asunto(s)
Quimiocinas CC/metabolismo , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/prevención & control , Células Secretoras de Insulina/patología , Envejecimiento , Animales , Quimiocina CCL4 , Quimiocinas CC/genética , Diabetes Mellitus Tipo 1/metabolismo , Terapia Genética , Inflamación/prevención & control , Interleucina-4/inmunología , Interleucina-4/farmacología , Trasplante de Islotes Pancreáticos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplanteRESUMEN
Successful attainment of tumor-specific gene expression was achieved in nasopharyngeal carcinoma (NPC) by exploiting the exclusive presence of the Epstein-Barr virus (EBV) genome in the cancer cells. In the current study, we have utilized an EBV-dependent transcriptional targeting strategy to construct a novel conditionally replicating adenovirus, adv.oriP.E1A. After treatment with adv.oriP.E1A, we observed extensive cell death in the EBV-positive NPC cell line C666-1. In contrast, no cytotoxicity was observed in a panel of other human EBV-negative cell lines, including fibroblasts from the nasopharynx. In vitro adenoviral replication was confirmed by the time-dependent increase in the expression of adenoviral capsid fiber protein and adenoviral DNA after C666-1 cells were infected with adv.oriP.E1A. Tumor formation was inhibited for more than 100 days after ex vivo infection of C666-1 cells with adv.oriP.E1A. Combination of local tumor radiation and adv.oriP.E1A caused complete disappearance of established tumors for at least 2 weeks in two distinct EBV-positive NPC xenograft models. Safety of this treatment was determined through the systemic delivery of adv.oriP.E1A in vivo, whereby minimal temporary perturbation of liver function was observed. We have successfully established a conditionally replicating adenovirus for EBV-positive NPC, which is both safe and efficacious, indicating a strategy that may be therapeutically applicable.
Asunto(s)
Adenoviridae/genética , Carcinoma/terapia , Terapia Genética/métodos , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/terapia , Replicación Viral/genética , Proteínas E1A de Adenovirus/genética , Animales , Línea Celular Tumoral , Efecto Citopatogénico Viral , Replicación del ADN , Humanos , Riñón/patología , Hígado/patología , Ratones , Ratones SCID , Bazo/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Stimulation of T cells through their antigen receptors (TCRs) causes a transient increase in the intracellular concentration of cyclic AMP (cAMP). However, sustained high levels of cAMP inhibit T-cell responses, suggesting that TCR signaling is coordinated with the activation of cyclic nucleotide phosphodiesterases (PDEs). The molecular basis of such a pathway is unknown. Here we show that TCR-dependent signaling activates PDE4B2 and that this enhances interleukin-2 production. Such an effect requires the regulatory N terminus of PDE4B2 and correlates with partitioning within lipid rafts, early targeting of this PDE to the immunological synapse, and subsequent accumulation in the antipodal pole of the T cell as activation proceeds.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunología , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Compartimento Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Activación Enzimática , Humanos , Técnicas In Vitro , Interleucina-2/biosíntesis , Células Jurkat , Activación de Linfocitos , Microdominios de Membrana/enzimología , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Transducción de SeñalRESUMEN
We have characterized a splice variant (isoform) of the human CD28 T cell costimulatory receptor. The nucleotide sequence of this CD28 isoform was identical to that of CD28 in the signal peptide, the transmembrane domain, and the cytoplasmic tail, but it was missing a large segment of the extracellular ligand-binding domain, which is encoded by the second exon. This isoform (CD28i), whose message level exceeded 25% of CD28, was a transmembrane homodimer. CD28i was found noncovalently associated with CD28 and was tyrosine-phosphorylated/PI3-kinase-complexed following the crosslinking of CD28, and the CD28 costimulatory signal was enhanced in T cells expressing CD28i. These data demonstrate that CD28i, via noncovalent association with CD28, plays a role as a costimulatory signal amplifier in human T cells.
Asunto(s)
Empalme Alternativo/inmunología , Antígenos CD28/genética , Antígenos CD28/fisiología , Transducción de Señal , Linfocitos T/inmunología , Secuencia de Bases , Sitios de Unión , Antígenos CD28/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Eliminación de Secuencia , Activación TranscripcionalRESUMEN
The unique feature of human nasopharyngeal carcinoma (NPC) is its almost universal association with the EBV, which is expressed in a latent form exclusively in cancer cells, and not in the surrounding tissues. We have exploited this differential by constructing a novel replication-deficient adenovirus vector (ad5.oriP) in which transgene expression is under the transcriptional regulation of the family of repeats domain of the origin of replication (oriP) of EBV. When EBNA1, one of the latent gene products of EBV, binds to the family of repeats sequence, this activates transcription of downstream genes. Vector constructs were made using the beta-galactosidase and luciferase reporter genes (ad5oriP.betagal and ad5oriP.luc) or the p53 tumor suppressor gene (ad5oriP.p53). 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining demonstrated extensive expression only in EBV-positive NPC cells, specifically in response to the presence of EBNA1. The relative difference in expression between EBV-positive and -negative cell lines is approximately 1000-fold. This selective expression was corroborated in EBV-positive and -negative tumor models, along with an absence of transgene expression in the host liver. Significant cytotoxicity was achieved using the adv.oriP.p53 therapeutic gene only in EBV-positive NPC cells, which was enhanced with the addition of ionizing radiation. Cytotoxicity was mediated primarily by induction of apoptosis. These results demonstrate that the oriP sequence can achieve high levels of gene expression targeted specifically to EBV-positive NPC cells in the context of the adv vector. This has now provided the tumor-specific expression system from which additional interventions can be evaluated in future treatment strategies for patients with nasopharyngeal cancers.