RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.
Asunto(s)
Carcinoma Ductal Pancreático , Viroterapia Oncolítica , Virus Oncolíticos , Neoplasias Pancreáticas , Humanos , Proteínas Nucleares/genética , Epigénesis Genética , Virus Oncolíticos/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patología , Viroterapia Oncolítica/métodos , Adenoviridae/genética , Microambiente Tumoral , Proteínas que Contienen Bromodominio , Proteínas de Ciclo Celular/metabolismoRESUMEN
Lung cancer is one of the most commonly diagnosed cancer and despite therapeutic advances, mortality remains high. The long period of clinical latency associated with lung cancer provides an ideal window of opportunity to administer vaccines to at-risk individuals that can prevent tumor progression and initiate long-term anti-tumor immune surveillance. Here we describe a personalized vaccination regime that could be applied for both therapeutic and prophylactic prevention of lung cancer, based on the derivation of lung cancer cells from induced pluripotent stem cells. Stem cells from healthy mice were modified to express Cre-dependent KRASG12D and Trp53R172H prior to differentiation to lung progenitor cells. Subsequent viral delivery of Cre caused activation of exogenous driver mutations, resulting in transformation and development of lung cancer cells. iPSC-derived lung cancer cells were highly antigenically related to lung cancer cells induced in LSL-KRASG12D/+; Trp53R172H/+ transgenic mice and were antigenically unrelated to original pluripotent stem cells or pancreatic cancer cells derived using the same technological platform. For vaccination, induced lung cancer cells were infected with oncolytic Adenovirus or Vaccinia virus, to act as vaccine adjuvants, prior to delivery of vaccines sequentially to a murine inducible transgenic model of lung cancer. Application of this Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime primed tumor-specific T cell responses that significantly prolonged survival in both subcutaneous post-vaccine challenge models and induced transgenic models of lung cancer, demonstrating that stem cell-derived prophylactic vaccines may be a feasible intervention for treatment or prevention of lung cancer development in at-risk individuals.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Neoplasias Pulmonares/terapia , Animales , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/administración & dosificación , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos/genética , Inmunización , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/prevención & control , Masculino , Ratones , Ratones Transgénicos , Virus Oncolíticos/genética , Sobrevida , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transducción Genética , Resultado del Tratamiento , Carga TumoralRESUMEN
PURPOSE: Pancreatic cancer remains one of the most lethal cancers, and late detection renders most tumors refractory to conventional therapies. Development of cancer prophylaxis may be the most realistic option for improving mortality associated with this disease. Here, we develop a novel individualized prophylactic and therapeutic vaccination regimen using induced pluripotent stem cells (iPSC), gene editing, and tumor-targeted replicating oncolytic viruses. EXPERIMENTAL DESIGN: We created a Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCs from healthy cells were induced to pancreatic tumor cells using in situ gene editing via stable provision of KRas G12D and p53 R172H tumor driver mutations. These cells were preinfected with oncolytic Adenovirus (AdV) as prime or Vaccinia virus (VV) as boost, to improve vaccine immunogenicity, prior to delivery of vaccines in a sequential regime to young KPC transgenic mice, genetically programmed to develop pancreatic cancer, to prevent and delay disease development. RESULTS: Tumor cells preinfected with oncolytic AdV as prime or VV as boost were the best regime to induce tumor-specific immunity. iPSC-derived tumor cells were highly related in antigen repertoire to pancreatic cancer cells of KPC transgenic mice, suggesting that an individual's stem cells can provide an antigenically matched whole tumor cell vaccine. The VIReST vaccination primed tumor-specific T-cell responses, resulting in delayed disease emergence and progression and significantly prolonged survival of KPC transgenic mice. Importantly, this regime was well-tolerated and nontoxic. CONCLUSIONS: These results provide both proof of concept and a robust technology platform for the development of personalized prophylactic cancer vaccines to prevent pancreatic malignancies in at-risk individuals.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Células Madre Pluripotentes Inducidas/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Experimentales/prevención & control , Viroterapia Oncolítica , Virus Oncolíticos/inmunología , Neoplasias Pancreáticas/prevención & control , Animales , Vacunas contra el Cáncer/inmunología , Chlorocebus aethiops , Progresión de la Enfermedad , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Tasa de Supervivencia , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics.
Asunto(s)
Adenocarcinoma , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Neoplasias Pancreáticas , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Adenoviridae/genética , Camptotecina/análogos & derivados , Camptotecina/farmacología , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Regulación hacia Abajo , Genes Virales , Humanos , Irinotecán , Proteína Homóloga de MRE11/biosíntesis , GemcitabinaRESUMEN
The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (~90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.
RESUMEN
PURPOSE: Noninvasive biomarkers for early detection of pancreatic ductal adenocarcinoma (PDAC) are currently not available. Here, we aimed to identify a set of urine proteins able to distinguish patients with early-stage PDAC from healthy individuals. EXPERIMENTAL DESIGN: Proteomes of 18 urine samples from healthy controls, chronic pancreatitis, and patients with PDAC (six/group) were assayed using GeLC/MS/MS analysis. The selected biomarkers were subsequently validated with ELISA assays using multiple logistic regression applied to a training dataset in a multicenter cohort comprising 488 urine samples. RESULTS: LYVE-1, REG1A, and TFF1 were selected as candidate biomarkers. When comparing PDAC (n = 192) with healthy (n = 87) urine specimens, the resulting areas under the receiver-operating characteristic curves (AUC) of the panel were 0.89 [95% confidence interval (CI), 0.84-0.94] in the training (70% of the data) and 0.92 (95% CI, 0.86-0.98) in the validation (30% of the data) datasets. When comparing PDAC stage I-II (n = 71) with healthy urine specimens, the panel achieved AUCs of 0.90 (95% CI, 0.84-0.96) and 0.93 (95% CI, 0.84-1.00) in the training and validation datasets, respectively. In PDAC stage I-II and healthy samples with matching plasma CA19.9, the panel achieved a higher AUC of 0.97 (95% CI, 0.94-0.99) than CA19.9 (AUC = 0.88; 95% CI, 0.81-0.95, P = 0.005). Adding plasma CA19.9 to the panel increased the AUC from 0.97 (95% CI, 0.94-0.99) to 0.99 (95% CI, 0.97-1.00, P = 0.04), but did not improve the comparison of stage I-IIA PDAC (n = 17) with healthy urine. CONCLUSIONS: We have established a novel, three-protein biomarker panel that is able to detect patients with early-stage pancreatic cancer in urine specimens.
Asunto(s)
Adenocarcinoma/orina , Biomarcadores de Tumor/orina , Detección Precoz del Cáncer , Litostatina/orina , Neoplasias Pancreáticas/orina , Proteínas Supresoras de Tumor/orina , Proteínas de Transporte Vesicular/orina , Adenocarcinoma/genética , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Carbohidratos Asociados a Tumores/orina , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteoma/genética , Espectrometría de Masas en Tándem , Factor Trefoil-1RESUMEN
Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions.
Asunto(s)
Sistemas CRISPR-Cas , Genoma Viral , Biología Molecular/métodos , Recombinación Genética , Virus Vaccinia/genética , Tecnología Farmacéutica/métodosRESUMEN
Oncolytic viruses (OVs) have the ability to selectively replicate in and lyse cancer cells. Angiogenesis is an essential requirement for tumor growth. Like OVs, the therapeutic effect of many angiogenesis inhibitors has been limited, leading to the development of more effective approaches to combine antiangiogenic therapy with OVs. Angiogenesis can be targeted either directly by OV infection of vascular endothelial cells, or by arming OVs with antiangiogenic transgenes, which are subsequently expressed locally in the tumor microenvironment. In this review, we describe the development and targeting of OVs, the role of angiogenesis in cancer, and the progress made in arming viruses with antiangiogenic transgenes. Future developments required to optimize this approach are addressed.
RESUMEN
Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922-947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922-947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics.
Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Antineoplásicos/uso terapéutico , Mitoxantrona/uso terapéutico , Taxoides/uso terapéutico , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Docetaxel , Citometría de Flujo , Humanos , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Mitoxantrona/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Taxoides/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AdΔΔ is an oncolytic adenoviral mutant that has been engineered to selectively target tumors with deregulated cell cycle and apoptosis pathways. AdΔΔ potentiates apoptotic cell death induced by drugs, including mitoxantrone and docetaxel, which are commonly used to treat prostate cancer. Here, we demonstrate that AdΔΔ can also interact synergistically with dietary phytochemicals known to have anti-cancer activities, without incurring the toxic side effects of chemodrugs. Curcumin, genistein, epigallocatechin-gallate, equol, and resveratrol efficiently killed both androgen-receptor positive (22Rv1) and negative cell lines (PC-3, DU145) in combination with adenoviral mutants. Synergistic cell killing was demonstrated with wild-type virus (Ad5) and AdΔΔ in combination with equol and resveratrol. EC(50) values for both phytochemicals and viruses were reduced three- to eightfold in all three combination-treated cell lines. The most potent efficacy was achieved in the cytotoxic drug- and virus-insensitive PC-3 cells, both in vitro and in vivo, while cell killing in normal bronchial epithelial cells was not enhanced. Although equol and resveratrol induced only low levels of apoptosis when administered alone, in combination with wild-type virus or AdΔΔ, the level of apoptotic cell death was significantly increased in PC-3 and DU145 cells. In vivo studies using suboptimal doses of AdΔΔ and equol or resveratrol, showed reduced tumor growth without toxicity to normal tissue. These findings identify novel functions for AdΔΔ and phytochemicals in promoting cancer cell killing and apoptosis, suggesting the use of these natural nontoxic compounds might be a feasible and currently unexploited anti-cancer strategy.
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Adenoviridae , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Suplementos Dietéticos , Equol/farmacología , Mutación , Virus Oncolíticos , Neoplasias de la Próstata/terapia , Estilbenos/farmacología , Animales , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fitoestrógenos/farmacología , Neoplasias de la Próstata/patología , Resveratrol , Trasplante HeterólogoRESUMEN
Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.
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Angiostatinas/genética , Carcinoma de Células Escamosas/terapia , Endostatinas/genética , Vectores Genéticos/genética , Neoplasias de Cabeza y Cuello/terapia , Virus Oncolíticos/genética , Virus Vaccinia/genética , Adenoviridae/genética , Angiostatinas/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Efecto Citopatogénico Viral , Endostatinas/metabolismo , Femenino , Regulación Viral de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Viroterapia Oncolítica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello , Vacunas Virales , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND AIMS: Metastasis accounts for the poor outcome of patients with pancreatic cancer. We recently discovered PRSS3 to be over-expressed in metastatic human pancreatic cancer cells. This study aimed to elucidate the role of PRSS3 in the growth and metastasis of human pancreatic cancer. METHODS: PRSS3 expression in human pancreatic cancer cell lines was detected by qPCR and immunoblotting. The effect of PRSS3 on cancer cell proliferation, migration and invasion in vitro, tumour growth and metastasis in vivo were investigated by manipulation of PRSS3 expression in human pancreatic cancer cell lines. VEGF expression was detected by ELISA, and the pathway through which PRSS3 regulates VEGF expression was investigated. The therapeutic effect of targeting this pathway on metastasis was assessed in vivo. Immunohistochemistry was employed to detect PRSS3 expression in human pancreatic cancer tissues. RESULTS: PRSS3 was over-expressed in the metastatic PaTu8988s cell line, but not in the non-metastatic PaTu8988t cell line. Over-expression of PRSS3 promoted pancreatic cancer cell proliferation as well as invasion in vitro, and tumour progression and metastasis in vivo. Stepwise investigations demonstrated that PRSS3 upregulates VEGF expression via the PAR1-mediated ERK pathway. ERK inhibitor significantly delayed the progression of metastases of pancreatic cancer and prolonged the survival of animals bearing metastatic pancreatic cancer (p<0.05). 40.54% of human pancreatic cancers (n=74) were positive for PRSS3 protein. A significant correlation was observed between PRSS3 expression and metastasis (p<0.01). Multivariate Cox regression analysis indicated that patients with PRSS3 expression in their tumours had a shorter survival time compared to those without PRSS3 expression (p<0.05). CONCLUSION: PRSS3 plays an important role in the progression, metastasis and prognosis of human pancreatic cancer. Targeting the PRSS3 signalling pathway may be an effective and feasible approach for treatment of this lethal cancer.
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Proteínas de Neoplasias/fisiología , Neoplasias Pancreáticas/patología , Tripsina/fisiología , Adulto , Anciano , Animales , Butadienos/uso terapéutico , Proliferación Celular , Progresión de la Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Nitrilos/uso terapéutico , Neoplasias Pancreáticas/enzimología , Pronóstico , Análisis de Supervivencia , Tripsina/farmacología , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Prostate cancer is significantly more common in Western men than in Asian men, but the basis for this difference remains unknown. Because genomic studies of Asian prostate cancer are very limited, we used a genome-wide approach to reveal the genomic alterations in Chinese prostate cancers. We found a significant reduction in the frequency of certain somatic genomic changes that are commonly found in Western prostate cancers, including the 21q22.2-22.3 deletion, which involves the TMPRSS2:ERG fusion gene, and 10q deletion, which causes PTEN inactivation. Array results were confirmed by PCR-based molecular copy-number counting in selected samples. The different frequencies of these genomic changes were further evaluated by fluorescent in situ hybridization and immunohistochemistry analyses of tissue microarray samples. These alterations might be key genetic changes underlying the regional/ethnic difference in clinical incidence and might be induced by specific environmental and/or genetic risk factors that Western men are exposed to. Our findings suggest that tumors arise in Western and Chinese populations by alternative pathogenetic mechanisms.
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Pueblo Asiatico/genética , Neoplasias de la Próstata/etnología , Neoplasias de la Próstata/genética , Población Blanca/genética , China , Reordenamiento Génico , Genoma Humano , Humanos , Masculino , Proteínas de Fusión Oncogénica/genética , Fosfohidrolasa PTEN/genética , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/patología , Transactivadores/genética , Regulador Transcripcional ERG , Reino UnidoRESUMEN
PURPOSE: Replication-selective oncolytic adenoviruses are a promising class of tumor-targeting agents with proven safety in hundreds of patients. However, clinical responses have been limited and viral mutants with higher potency are needed. Here, we report on the generation of a novel set of mutants with improved efficacy in prostate and pancreatic carcinoma models. Currently, no curative treatments are available for late-stage metastatic prostate or rapidly progressing pancreatic cancers. EXPERIMENTAL DESIGN: Adenovirus type 5 mutants were created with deletions in the E1ACR2 region for tumor selectivity and/or the E1B19K gene for attenuated replication in vivo; all constructs retain the E3 genes intact. Cell-killing efficacy, replication, and cytotoxicity in combination with chemotherapeutics were investigated in normal cells (PrEC and NHBE), seven carcinoma cell lines, and human (PC3 and DU145) and murine (TRAMPC, CMT-64, and CMT-93) tumor models in vivo. RESULTS: The double-deleted AdDeltaDelta (DeltaE1ACR2 and DeltaE1B19K) mutant had high cell-killing activity in prostate, pancreatic, and lung carcinomas. Replication was similar to wild-type in all tumor cells and was attenuated in normal cells to levels less than the single-deleted AdDeltaCR2 mutant. AdDeltaDelta combined with the chemotherapeutics docetaxel and mitoxantrone resulted in synergistically enhanced cell killing and greatly improved antitumor efficacy in prostate xenografts in vivo. In murine immunocompetent in vivo models efficacy was greater for mutants with the E3B genes intact even in the absence of viral replication, indicating attenuated macrophage-dependent clearance. CONCLUSIONS: These data suggest that the novel oncolytic mutant AdDeltaDelta is a promising candidate for targeting of solid tumors specifically in combination with chemotherapeutics.
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Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Carcinoma/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Neoplasias Pancreáticas/terapia , Adenoviridae/genética , Animales , Línea Celular Tumoral , Eliminación de Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Virus Oncolíticos/fisiología , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/fisiología , Especificidad por Sustrato , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lung cancer is the commonest cancer killer. Small cell lung cancer (SCLC) is initially chemosensitive, but rapidly relapses in a chemoresistant form with an overall survival of <5%. Consequently, novel therapies are urgently required and will likely arise from an improved understanding of the disease biology. Our previous work showed that fibroblast growth factor-2 induces proliferation and chemoresistance in SCLC cells. Here, we show that the selective fibroblast growth factor receptor (FGFR) inhibitor PD173074 blocks H-510 and H-69 SCLC proliferation and clonogenic growth in a dose-dependent fashion and prevents FGF-2-induced chemoresistance. These effects correlate with the inhibition of both FGFR1 and FGFR2 transphosphorylation. We then determined the efficacy of daily oral administration of PD173074 for 28 days in two human SCLC models. In the H-510 xenograft, tumor growth was impaired similar to that seen with single-agent cisplatin administration, increasing median survival compared with control sham-treated animals. Crucially, the effect of cisplatin was significantly potentiated by coadministration of PD173074. More dramatically, in H-69 xenografts, PD173074 induced complete responses lasting >6 months in 50% of mice. These effects were not a consequence of disrupted tumor vasculature but instead correlated with increased apoptosis (caspase 3 and cytokeratin 18 cleavage) in excised tumors. Moreover, in vivo imaging with 3'-deoxy-3'-[(18)F]fluorothymidine-positron emission tomography ([(18)F]FLT-PET) showed decreased intratumoral proliferation in live animals treated with the compound at 7 to 14 days. Our results suggest that clinical trials of FGFR inhibitors should be undertaken in patients with SCLC and that [(18)F]FLT-PET imaging could provide early in vivo evidence of response.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/fisiología , Neoplasias Pulmonares/tratamiento farmacológico , Pirimidinas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Sinergismo Farmacológico , Factor 2 de Crecimiento de Fibroblastos/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ratones , Tomografía de Emisión de Positrones , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The Na/I symporter (hNIS) promotes concentration of iodine in cells. In cancer gene therapy, this transgene has potential as a reporter gene for molecular imaging of viral biodistribution and as a therapeutic protein promoting (131)I-mediated radiotherapy. Here, we combined the imaging and therapeutic potential of hNIS in an oncolytic adenoviruses targeting colorectal cancer cells. EXPERIMENTAL DESIGN: We generated an adenovirus (AdIP2) encoding hNIS and capable of selective replication in colorectal carcinoma cells. The selectivity of this virus was verified in vitro and in vivo. Its spread in tumors was monitored in vivo using single-photon emission computed tomography/CT imaging upon (99m)TcO(4)(-) injection and confirmed by immunohistochemistry. Metabolic radiotherapy was done through injection of therapeutic doses of (131)I(-). RESULTS: We showed in vitro and in vivo the selectivity of AdIP2 and that hNIS expression is restricted to the target cells. Imaging and immunohistochemical data showed that viral spread is limited and that the point of maximal hNIS expression is reached 48 hours after a single intratumoral injection. Administration of a single therapeutic dose of (131)I at this time point led to a dramatic reduction in tumor size not observed in hNIS-negative viruses. CONCLUSIONS: This report showed for the first time that the combination of the imaging and therapeutic potentials of hNIS can be applied to oncolytic adenoviruses in experimental models of cancer.
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Adenoviridae/genética , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/terapia , Genes Reporteros , Terapia Genética/métodos , Radioisótopos de Yodo/uso terapéutico , Simportadores/genética , Proteínas Wnt/metabolismo , Animales , Línea Celular Tumoral , Diagnóstico por Imagen , Marcación de Gen , Vectores Genéticos , Ratones , Ratones Desnudos , Cintigrafía , Simportadores/metabolismo , TransgenesRESUMEN
The changes in cancer cell surface molecules and intracellular signaling pathways during tumorigenesis make delivery of adenovirus-based cancer therapies inefficient. Here we have identified carcinoembryonic antigen- related cell adhesion molecule 6 (CEACAM6) as a cellular protein that restricts the ability of adenoviral vectors to infect cancer cells. We have demonstrated that CEACAM6 can antagonize the Src signaling pathway, downregulate cancer cell cytoskeleton proteins, and block adenovirus trafficking to the nucleus of human pancreatic cancer cells. Similar to CEACAM6 overexpression, treatment with a Src-selective inhibitor significantly reduced adenovirus replication in these cancer cells and normal human epithelial cells. In a mouse xenograft tumor model, siRNA-mediated knockdown of CEACAM6 also significantly enhanced the antitumor effect of an oncolytic adenovirus. We propose that CEACAM6-associated signaling pathways could be potential targets for the development of biomarkers to predict the response of patients to adenovirus-based therapies, as well as for the development of more potent adenovirus-based therapeutics.
Asunto(s)
Adenoviridae/fisiología , Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Adenoviridae/ultraestructura , Antígenos CD/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Neoplasias Colorrectales/genética , Citoesqueleto/metabolismo , Regulación hacia Abajo , Proteínas Ligadas a GPI , Regulación Neoplásica de la Expresión Génica , Humanos , Microscopía Electrónica de Transmisión , Neoplasias Pancreáticas/genética , ARN Interferente Pequeño/genética , Especificidad por Sustrato , Familia-src Quinasas/metabolismoRESUMEN
PURPOSE: Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal. We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. EXPERIMENTAL DESIGN: Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdDeltaE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. RESULTS: The DeltaE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdDeltaE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. CONCLUSIONS: Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways. These findings imply that less toxic doses than currently practiced in the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants.
Asunto(s)
Adenocarcinoma/patología , Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Apoptosis/efectos de los fármacos , Desoxicitidina/análogos & derivados , Eliminación de Gen , Mutación/genética , Virus Oncolíticos/fisiología , Neoplasias Pancreáticas/patología , Adenocarcinoma/genética , Proteínas E1B de Adenovirus/metabolismo , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Terapia Combinada , Desoxicitidina/uso terapéutico , Sinergismo Farmacológico , Citometría de Flujo , Terapia Genética , Vectores Genéticos , Humanos , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias Pancreáticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S/efectos de los fármacos , Fase S/fisiología , Replicación Viral , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Angiogenesis is essential for development and tissue repair, and is controlled by a balance of inhibitors and promoters. Overactive angiogenesis promotes tumor progression and other chronic disorders, including diabetic retinopathy and rheumatoid arthritis. The discovery of angiogenesis inhibitors has resulted in a promising therapeutic approach to these diseases. However, the benefits of anti-angiogenic drugs have been modest, stimulating interest in developing more effective approaches by combining anti-angiogenic therapy with other therapeutics. Oncolytic virotherapies are attractive therapeutics for cancer, but virotherapy alone has had similar problems to anti-angiogenic therapy, with few examples of clinical efficacy. This review summarizes the progress of the emerging field of combinations of anti-angiogenic therapy and virotherapy, and highlights future challenges in experimental and translational research that need to be addressed in order for these therapeutics to advance into the clinic.