RESUMEN
Spinal muscular atrophy is an autosomal recessive disease resulting in motor neuron degeneration and progressive life-limiting motor deficits when untreated. Onasemnogene abeparvovec is an adeno-associated virus serotype 9-based gene therapy that improves survival, motor function, and motor milestone achievement in symptomatic and presymptomatic patients. Although the adeno-associated virus genome is maintained as an episome, theoretical risk of tumorigenicity persists should genomic insertion occur. We present the case of a 16-month-old male with spinal muscular atrophy who was diagnosed with an epithelioid neoplasm of the spinal cord approximately 14 months after receiving onasemnogene abeparvovec. In situ hybridization analysis detected an onasemnogene abeparvovec nucleic acid signal broadly distributed in many but not all tumor cells. Integration site analysis on patient formalin-fixed, paraffin-embedded tumor samples failed to detect high-confidence integration sites of onasemnogene abeparvovec. The finding was considered inconclusive because of limited remaining tissue/DNA input. The improved life expectancy resulting from innovative spinal muscular atrophy therapies, including onasemnogene abeparvovec, has created an opportunity to analyze the long-term adverse events and durability of these therapies as well as identify potential disease associations that were previously unrecognized because of the premature death of these patients.
RESUMEN
BACKGROUND: Engineered tissues and cell therapies based on human induced pluripotent stem cells (iPSCs) represent a promising approach for novel medicines. However, iPSC-derived cells and tissues may contain residual undifferentiated iPSCs that could lead to teratoma formation after implantation into patients. As a consequence, highly sensitive and specific methods for detecting residual undifferentiated iPSCs are indispensable for safety evaluations of iPSC-based therapies. The present study provides an approach for identifying potential marker genes for iPSC impurities in iPSC-derived cells using RNA sequencing data from iPSCs and various differentiated cell types. METHODS: Identifying iPSC marker genes for each cell type individually provided a larger and more specific set of potential marker genes than considering all cell types in the analysis. Thus, the authors focused on identifying markers for iPSC impurities in iPSC-derived cardiomyocytes (iCMs) and validated the selected genes by reverse transcription quantitative polymerase chain reaction. The sensitivity of the candidate genes was determined by spiking different amounts of iPSCs into iCMs and their performance was compared with the previously suggested marker lin-28 homolog A (LIN28A). RESULTS: Embryonic stem cell-related gene (ESRG), long intergenic non-protein coding RNA 678 (LINC00678), CaM kinase-like vesicle-associated (CAMKV), indoleamine 2,3-dioxygenase 1 (IDO1), chondromodulin (CNMD), LINE1-type transposase domain containing 1 (L1DT1), LIN28A, lymphocyte-specific protein tyrosine kinase (LCK), vertebrae development-associated (VRTN) and zinc finger and SCAN domain containing 10 (ZSCAN10) detected contaminant iPSCs among iCMs with a limit of detection that ranged from 0.001% to 0.1% depending on the gene and iCM batch used. CONCLUSIONS: Using the example of iCMs, the authors provide a strategy for identifying a set of highly specific and sensitive markers that can be used for quality assessment of iPSC-derived products.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Diferenciación Celular/genética , Células Madre EmbrionariasRESUMEN
Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.
Asunto(s)
Eritropoyetina/metabolismo , Telocitos/patología , Factores de Transcripción/metabolismo , Anemia/etiología , Anemia/patología , Animales , Hipoxia de la Célula , Línea Celular , Eritropoyetina/genética , Retroalimentación Fisiológica , Riñón/citología , Riñón/patología , Ratones , Ratones Transgénicos , Cultivo Primario de Células , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/patología , Telocitos/metabolismoRESUMEN
Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.
Asunto(s)
Proteínas Portadoras/fisiología , Regulación del Desarrollo de la Expresión Génica , Melanocitos/citología , Proteínas de Microfilamentos/fisiología , Animales , Proteínas Portadoras/genética , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Humanos , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Pigmentación , Piel/patología , Factores de TiempoRESUMEN
The presence of residual undifferentiated pluripotent stem cells (PSCs) in PSC-derived cell therapy products (CTPs) is a major safety issue for their clinical application, due to the potential risk of PSC-derived tumor formation. An international multidisciplinary multisite study to evaluate a droplet digital PCR (ddPCR) approach to detect residual undifferentiated PSCs in PSC-derived CTPs was conducted as part of the Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee. To evaluate the use of ddPCR in quantifying residual iPSCs in a cell sample, different quantities of induced pluripotent stem cells (iPSCs) were spiked into a background of iPSC-derived cardiomyocytes (CMs) to mimic different concentrations of residual iPSCs. A one step reverse transcription ddPCR (RT-ddPCR) was performed to measure mRNA levels of several iPSC-specific markers and to evaluate the assay performance (precision, sensitivity, and specificity) between and within laboratories. The RT-ddPCR assay variability was initially assessed by measuring the same RNA samples across all participating facilities. Subsequently, each facility independently conducted the entire process, incorporating the spiking step, to discern the parameters influencing potential variability. Our results show that a RT-ddPCR assay targeting ESRG, LINC00678, and LIN28A genes offers a highly sensitive and robust detection of impurities of iPSC-derived CMs and that the main contribution to variability between laboratories is the iPSC-spiking procedure, and not the RT-ddPCR. The RT-ddPCR assay would be generally applicable for tumorigenicity evaluation of PSC-derived CTPs with appropriate marker genes suitable for each CTP.
RESUMEN
Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.
Asunto(s)
Terapia Genética , Terapia Genética/efectos adversos , Terapia Genética/métodos , Humanos , Factores de Riesgo , Animales , Vectores Genéticos/efectos adversos , Consenso , Neoplasias/terapia , Neoplasias/genética , Medición de RiesgoRESUMEN
It has been reported previously that both Cdk1 and Cdk2 phosphorylate Chk1 in a cell-cycle dependent manner. Cdk-mediated phosphorylation is required for efficient activation of Chk1 and checkpoint proficiency in response to DNA damage. Here, we demonstrate that Cdk-mediated phosphorylation is also required for replication stress induced Chk1 activation and S/M checkpoint proficiency. Re-introduction of Chk1 mutant (S286A/S301A) into Chk1 deficient cells is capable of restraining mitosis in cells with completely unreplicated DNA, but the mitotic delay at later stage of the cell cycle is largely impaired. The mutation strongly attenuates aphidicolin induced Chk1 activation without altering the S-phase dependent Chk1 activation. These data indicate that Cdk-mediated phosphorytion is required for efficient Chk1 activation and multiple checkpoint proficiency.
Asunto(s)
Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Quinasas/metabolismo , División Celular , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Replicación del ADN , Activación Enzimática , Fase G2 , Humanos , Mitosis , Mutación , Fosforilación , Proteínas Quinasas/genética , Fase SRESUMEN
Off-target editing is one of the main safety concerns for the use of CRISPR-Cas9 genome editing in gene therapy. These unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of gene therapy products indispensable. In this study, we established two in vitro transformation assays, the soft agar colony-forming assay (SACF) and the growth in low attachment assay (GILA) as alternative methods for tumorigenicity evaluation of genome-edited cells. Using a CRISPR-Cas9-based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as a valid positive control in GILA and SACF. Next, we measured the limit of detection for both assays and proved that SACF is more sensitive than GILA (0.8% versus 3.1% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls and by testing the suitability of another cell line (THLE-2). Moreover, in contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12 deletion, demonstrating the relevance of GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to preclinical safety assessment of gene therapy products.
RESUMEN
Aurora/Ipl1-related kinases are a conserved family of proteins that are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumours and have been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Previous studies of our group have shown that Aurora B expression is restricted to specific germinal cells. In this study, we have evaluated by immunohistochemical analysis Aurora B expression in post-puberal testicular germ cell tumours (22 seminomas, 2 teratomas, 15 embryonal carcinomas, 5 mixed germinal tumours with a prominent yolk sac tumour component and 1 choriocarcinoma). The Aurora B protein expression was detected in all intratubular germ cell tumours, seminomas and embryonal carcinomas analysed but not in teratomas and yolk sac carcinomas. The immunohistochemical data were further confirmed by Western blot analysis. In addition, the kinase Aurora B was vigorously expressed in GC-1 cells line derived from murine spermatogonia. The block of Aurora B function induced by a pharmacological inhibitor significantly reduced the growth of GC-1 cells suggesting that Aurora B is a potential therapeutic target.
Asunto(s)
Neoplasias de Células Germinales y Embrionarias/enzimología , Neoplasias de Células Germinales y Embrionarias/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Pubertad/metabolismo , Neoplasias Testiculares/enzimología , Neoplasias Testiculares/patología , Adulto , Aurora Quinasa B , Aurora Quinasas , Western Blotting , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: Anaplastic thyroid carcinoma is a prime target for innovative therapy because it represents one of the most lethal human neoplasms and is refractory to conventional treatments such as chemotherapy and radiotherapy. We have evaluated a novel therapeutic approach based on the oncolytic replication-selective adenovirus dl922-947. EXPERIMENTAL DESIGN: The antitumor efficacies of the E1ADeltaCR2 (dl922-947) and DeltaE1B55K (dl1520) mutants were compared in human thyroid anaplastic carcinoma cells in culture and in xenografts in vivo. To enhance the effects of dl922-947, anaplastic thyroid carcinoma tumor xenografts were treated with dl922-947 in combination with bevacizumab. RESULTS: We showed that the efficacy of dl922-947 exceeded that of dl1520 in all tested anaplastic thyroid carcinoma cells in vitro and in vivo. Furthermore, bevacizumab in combination with dl922-947 significantly reduced tumor growth compared with single treatments alone. Bevacizumab treatment significantly improved viral distribution in neoplastic tissues. CONCLUSIONS: Our data showed that dl922-947 had a higher oncolytic activity compared with dl1520 in anaplastic thyroid carcinoma cell lines and might represent a better option for virotherapy of anaplastic thyroid carcinoma. Moreover, bevacizumab increased the oncolytic effects of dl922-947 by enhancing viral distribution in tumors. The results described herein encourage the use of the dl922-947 virus in combination with bevacizumab.
Asunto(s)
Adenoviridae/efectos de los fármacos , Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Carcinoma/terapia , Viroterapia Oncolítica , Neoplasias de la Tiroides/terapia , Adenoviridae/genética , Infecciones por Adenoviridae/patología , Infecciones por Adenoviridae/terapia , Infecciones por Adenoviridae/virología , Proteínas E1A de Adenovirus/deficiencia , Animales , Anticuerpos Monoclonales Humanizados , Bevacizumab , Carcinoma/patología , Carcinoma/virología , Células Cultivadas , Terapia Combinada , Sinergismo Farmacológico , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/virología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Replicación Viral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The phosphorylation of histone H2AX in Serine 139 (gamma-H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma-H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma-H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma-H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma-H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma-H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma-H2AX staining allows the detection of tissue-specific effects in situ. Moreover, since gamma-H2AX staining can be performed on formalin-fixed and paraffin-embedded tissue sections generated during repeated-dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals. Environ. Mol. Mutagen. 60:4-16, 2019. © 2018 Wiley Periodicals, Inc.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de los fármacos , Doxorrubicina/toxicidad , Metanosulfonato de Etilo/toxicidad , Etilnitrosourea/toxicidad , Histonas/metabolismo , Pruebas de Mutagenicidad/métodos , Fosfoproteínas/metabolismo , Animales , Reparación del ADN/genética , Técnica del Anticuerpo Fluorescente/métodos , Procesamiento de Imagen Asistido por Computador , Masculino , Fosforilación , Ratas , Ratas WistarRESUMEN
Anaplastic thyroid carcinoma (ATC) is one of the most aggressive solid tumors and shows morphological features of a highly malignant, undifferentiated neoplasm. Patients with ATC have a poor prognosis with a mean survival time of 2-6 months; surgery, radiotherapy, and chemotherapy do not improve survival. Gene therapy approaches and oncolytic viruses have been tested for the treatment of ATC. To enhance the antineoplastic effects of the oncolytic adenovirus dl1520 (Onyx-015), we treated ATC cells with lovastatin (3-hydroxy-methylglutaryl-CoA reductase inhibitor), a drug used for the treatment of hypercholesterolemia, which has previously been reported to exert growth-inhibitory and apoptotic activity on ATC cells. Lovastatin treatment significantly increased the effects of dl1520 against ATC cells. The replication of dl1520 in ATC cells was enhanced by lovastatin treatment, and a significant increase of the expression of the early gene E1A 13 S and the late gene Penton was observed in lovastatin-treated cells. Furthermore, lovastatin treatment significantly enhanced the effects of dl1520 against ATC tumor xenografts. Lovastatin treatment could be exploited to increase the efficacy of oncolytic adenoviruses, and further studies are warranted to confirm the feasibility of the approach in ATC patients.
Asunto(s)
Adenoviridae/efectos de los fármacos , Antineoplásicos/uso terapéutico , Carcinoma/terapia , Lovastatina/farmacología , Lovastatina/uso terapéutico , Viroterapia Oncolítica , Neoplasias de la Tiroides/terapia , Replicación Viral/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Carcinoma/tratamiento farmacológico , Células Cultivadas , Terapia Combinada , Femenino , Humanos , Ratones , Ratones Desnudos , Virus Oncolíticos/efectos de los fármacos , Vacunas Virales , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ped/pea-15 is a cytosolic protein performing a broad antiapoptotic function. We show that, upon DMBA/TPA-induced skin carcinogenesis, transgenic mice overexpressing ped/pea-15 (Tg(ped/pea-15)) display early development of papillomas and a four-fold increase in papilloma number compared to the nontransgenic littermates (P<0.001). The malignant conversion frequency was 24% for the Tg(ped/pea-15) mice and only 5% in controls (P<0.01). The isolated application of TPA, but not that of DMBA, was sufficient to reversibly upregulate ped/pea-15 in both untransformed skin and cultured keratinocytes. ped/pea-15 protein levels were also increased in DMBA/TPA-induced papillomas of both Tg(ped/pea-15) and control mice. Isolated TPA applications induced Caspase-3 activation and apoptosis in nontransformed mouse epidermal tissues. The induction of both Caspase-3 and apoptosis by TPA were four-fold inhibited in the skin of the Tg(ped/pea-15) compared to the nontransgenic mice, accompanied by a similarly sized reduction in TPA-induced JNK and p38 stimulation and by constitutive induction of cytoplasmic ERK activity in the transgenics. ped/pea-15 expression was stably increased in cell lines from DMBA/TPA-induced skin papillomas and carcinomas, paralleled by protection from TPA apoptosis. In the A5 spindle carcinoma cell line, antisense inhibition of ped/pea-15 expression simultaneously rescued sensitivity to TPA-induced Caspase-3 function and apoptosis. The antisense also reduced A5 cell ability to grow in semisolid media by 65% (P<0.001) and increased by three-fold tumor latency time (P<0.01). Thus, the expression levels of ped/pea-15 control Caspase-3 function and epidermal cell apoptosis in vivo and determine susceptibility to skin tumor development.
Asunto(s)
Apoptosis , Transformación Celular Neoplásica , Cocarcinogénesis , Fosfoproteínas/fisiología , Neoplasias Cutáneas , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Carcinógenos/toxicidad , Caspasa 3 , Caspasas/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Células Cultivadas , ADN sin Sentido/farmacología , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Papiloma/inducido químicamente , Papiloma/genética , Papiloma/patología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Sarcoma/inducido químicamente , Sarcoma/genética , Sarcoma/patología , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol/toxicidad , TransfecciónRESUMEN
Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFß Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through Braf(V600E) or Kras(G12D) knockin) and TGFß signalling ablation (through Tgfbr1 deletion) in LGR5(+ve) stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5(+ve) cells also results in cSCC development. These findings indicate that LGR5(+ve) stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFß signalling, are driving events of skin tumorigenesis.
Asunto(s)
Antineoplásicos/efectos adversos , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/genética , Melanoma/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Animales , Biopsia , Carcinogénesis/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Análisis Mutacional de ADN/métodos , Femenino , Humanos , Indoles/efectos adversos , Masculino , Ratones , Ratones Endogámicos , Mutación , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/patología , Células Madre , Sulfonamidas/efectos adversos , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/genética , Vemurafenib , Secuenciación del ExomaRESUMEN
Alterations in chromosome number (aneuploidy) are common in human neoplasias. Loss of mitotic regulation is believed to induce aneuploidy in cancer cells and act as a driving force during the malignant progression. The serine/theronine protein kinases of aurora family genes play a critical role in the regulation of key cell cycle processes. Aurora B mediates chromosome segregation by ensuring orientation of sister chromatids and overexpression of Aurora B in diploid human cells NHDF (normal human diploid fibroblast) induces multinuclearity. We analyzed Aurora B expression in human thyroid carcinomas. Cell lines originating from different histotypes showed an increase in Aurora B expression. Immunohistochemical analysis of archive samples showed a high expression of Aurora B in anaplastic thyroid carcinomas; conversely, Aurora B expression was not detectable in normal thyroid tissue. Real-time PCR analysis confirmed a strong expression of Aurora B in anaplastic thyroid carcinomas. The block of Aurora B expression induced by RNA interference or by using an inhibitor of Aurora kinase activity significantly reduced the growth of thyroid anaplastic carcinoma cells.
Asunto(s)
Proteínas Serina-Treonina Quinasas/genética , Neoplasias de la Tiroides/genética , Animales , Aurora Quinasa B , Aurora Quinasas , Secuencia de Bases , Carcinoma/enzimología , Carcinoma/genética , Carcinoma/patología , División Celular , Línea Celular Tumoral , Cartilla de ADN , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/patología , Trasplante HeterólogoRESUMEN
PARP inhibitors are mostly effective as anticancer drugs in association with DNA damaging agents. We have previously shown that the oncolytic adenovirus dl922-947 induces extensive DNA damage, therefore we hypothesized a synergistic antitumoral effect of the PARP inhibitor olaparib in association with dl922-947. Anaplastic thyroid carcinoma was chosen as model since it is a particularly aggressive tumor and, because of its localized growth, it is suitable for intratumoral treatment with oncolytic viruses. Here, we show that dl922-947 infection induces PARP activation, and we confirm in vitro and in vivo that PARP inhibition increases dl922-947 replication and oncolytic activity. In vitro, the combination with olaparib exacerbates the appearance of cell death markers, such as Annexin V positivity, caspase 3 cleavage, cytochrome C release and propidium iodide permeability. In vivo, we also observed a better viral distribution upon PARP inhibition. Changes in CD31 levels suggest a direct effect of olaparib on tumor vascularization and on the viral distribution within the tumor mass. The observation that PARP inhibition enhances the effects of dl922-947 is highly promising not only for the treatment of anaplastic thyroid carcinoma but, in general, for the treatment of other tumors that could benefit from the use of oncolytic viruses.
Asunto(s)
Adenoviridae , Viroterapia Oncolítica/métodos , Virus Oncolíticos , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Carcinoma Anaplásico de Tiroides/terapia , Neoplasias de la Tiroides/terapia , Animales , Línea Celular Tumoral , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Carcinoma Anaplásico de Tiroides/enzimología , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/patologíaRESUMEN
ONYX-015 is a genetically modified adenovirus with a deletion of the E1B early gene and therefore is designed to replicate preferentially in p53-mutated cells causing their death. We previously demonstrated that the ONYX-015 virus kills anaplastic thyroid carcinoma (ATC) cells and enhances the antineoplastic effects of doxorubicin and paclitaxel. Here we report that ONYX-015 increased the cytopathic effect of radiotherapy in three ATC cell lines. In fact, ONYX-015 and radiation induced a significant cytopathic effect on ATC cells. DNA fragmentation analysis showed that ATC ONYX-015-treated cells were very sensitive to radiation-induced apoptosis. In addition, low doses of ONYX-015 associated with a single radiation dose of 10 Gy delayed the growth of a xenograft tumor induced by ARO cells in athymic mice. Our results suggest that the combination of ONYX-015 and radiotherapy should be considered for experimental trials in patients with anaplastic thyroid carcinoma.
Asunto(s)
Carcinoma/radioterapia , Neoplasias de la Tiroides/radioterapia , Vacunas Virales/farmacología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Muerte Celular/efectos de la radiación , Terapia Combinada , Fragmentación del ADN/efectos de la radiación , Ratones , Ratones Desnudos , Tolerancia a Radiación , Células Tumorales Cultivadas/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
dl922-947 is an oncolytic adenovirus potentially suitable for the treatment of aggressive localized tumors, such as anaplastic thyroid carcinoma (ATC). In this study, we have analyzed the effects of dl922-947 in combination with ionizing radiations, testing different schedules of administration and observing synergistic effects only when ATC cells were irradiated 24 h prior to viral infection. Cells undergoing combined treatment exhibited a marked increase in cell death and viral replication, suggesting that irradiation blocks cells in a more permissive state for viral life cycle. We also show that dl922-947 triggers a DNA damage response, characterized by mobilization of the MRN complex (composed by Mre11-Rad50-Nbs1), accumulation of γH2AX, and activation of the checkpoint kinases ataxia telangiectasia mutated (ATM) and Chk1. Based on these observations, we speculate that the DNA damage response acts as a cellular protective mechanism to hinder viral infection and replication. To confirm this hypothesis, we demonstrate that the ATM inhibitor KU55933 increased the oncolytic activity of dl922-947 and its replication. Finally, we validate the potential therapeutic use of this approach by showing in vivo that the combined treatment slows tumor xenograft growth more potently than either irradiation or infection alone.
Asunto(s)
Adenoviridae/fisiología , Viroterapia Oncolítica , Virus Oncolíticos/fisiología , Radiación Ionizante , Neoplasias de la Tiroides/terapia , Animales , Muerte Celular , Línea Celular Tumoral , Terapia Combinada , Daño del ADN , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides/patología , Carga Tumoral , Replicación ViralRESUMEN
Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-IâLC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.
Asunto(s)
Adenoviridae/genética , Adyuvantes Inmunológicos/farmacología , Autofagia/efectos de los fármacos , Vectores Genéticos/uso terapéutico , Glioma/terapia , Virus Oncolíticos/genética , Adenina/análogos & derivados , Adenina/farmacología , Adenoviridae/inmunología , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/inmunología , Animales , Línea Celular Tumoral , Cloroquina/farmacología , Técnica del Anticuerpo Fluorescente , Vectores Genéticos/genética , Humanos , Ratones , Ratones Desnudos , Virus Oncolíticos/inmunología , Reacción en Cadena de la Polimerasa , Transducción de SeñalRESUMEN
Novel therapeutic approaches are required for the treatment of anaplastic thyroid carcinoma (ATC), an incurable disease resistant to current available therapies. Aurora B is an important mitotic kinase involved in chromosome segregation and cytokinesis. It is overexpressed in many cancers including ATC and represents a potential target for chemotherapy. The effects of AZD1152, a specific Aurora B kinase inhibitor, have been evaluated against ATC, showing G(2)/M accumulation, polyploidy and subsequent cell death by mitotic catastrophe upon drug treatment. Only three administrations of AZD1152 significantly reduced the growth of ATC tumour xenogratfs. Oncolytic viruses in association with other forms of treatment have proven highly promising in preclinical and clinical reports. The oncolytic adenovirus dl922-947 is active against ATC cells, and we have evaluated the effects of the association between AZD1152 and dl922-947. In cells treated with virus and drug, we report additive/synergistic killing effects. Interestingly, the phosphorylation of histone H3 (Ser10), the main Aurora B substrate, is inhibited by dl922-947 in a dose-dependent manner, and completely abolished in association with AZD1152. The combined treatment significantly inhibited the growth of ATC tumour xenografts with respect to single treatments. Our data demonstrate that the Aurora B inhibitor AZD1152, alone or in combination with oncolytic virus dl922-947, could represent a novel therapeutic option for the treatment of ATC.