RESUMEN
The effectiveness of radiotherapy depends on the sensitivities of 'normal' and cancer cells to the administered radiation dose. Increasing the radiosensitivity of cancers by inhibiting DNA damage repair is a goal of much current research, however success depends on avoiding concomitant sensitization of normal tissues inevitably irradiated during therapy. In this study we investigated the mechanisms of radiosensitization for DNA-PK and PARP inhibitors by examining the impacts on proliferating vs quiescent cell populations. Experiments were performed in BRCA1/2null and wild-type parental cancer models in vitro and in vivo. Overall AZD7648 has greater radiosensitizing activity relative to Olaparib, with BRCA2-deficient models showing the greatest sensitivity. However, DNA-PK inhibitor AZD7648 also produced greater toxicity in all irradiated mice. While both DNA-PK and PARP inhibition sensitizes wild type tumor cells to radiation, in BRCA1/2 deficient cells PARP inhibition by Olaparib had limited radiosensitization capacity. Quiescent cells are more radioresistant than proliferating cells, and these were also effectively sensitized by AZD7648 while Olaparib was unable to increase radiation-induced cell kill, even in BRCA1/2null cells. These findings underscore the distinct mechanisms of radiosensitization for DNA-PK and PARP inhibitors. While DNA-PK inhibitors are able to target both proliferating and non-proliferating tumor cells for greater overall anti-cancer benefit, their application is limited by exacerbation of normal tissue toxicities. Conversely, PARP inhibitors exhibit selective activity for proliferating cells, providing a mechanism for targeting activity to cancers, but due to poor activity in non-proliferating cells they have an overall reduced impact on tumor growth control. This study highlights the importance of creating a therapeutic ratio with DNA damage repair inhibition radiation sensitizing strategies.
Asunto(s)
Proteína BRCA1 , Proteína BRCA2 , Proteína Quinasa Activada por ADN , Ftalazinas , Piperazinas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Fármacos Sensibilizantes a Radiaciones , Ftalazinas/farmacología , Piperazinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Animales , Humanos , Proteína BRCA1/metabolismo , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/metabolismo , Ratones , Línea Celular Tumoral , Femenino , Proteína BRCA2/genética , Proliferación Celular/efectos de los fármacos , Tolerancia a Radiación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The oxygen depletion hypothesis has been proposed as a rationale to explain the observed phenomenon of FLASH-radiotherapy (FLASH-RT) sparing normal tissues while simultaneously maintaining tumor control. In this study we examined the distribution of DNA Damage Response (DDR) markers in irradiated 3D multicellular spheroids to explore the relationship between FLASH-RT protection and radiolytic-oxygen-consumption (ROC) in tissues. METHODS: Studies were performed using a Varian Truebeam linear accelerator delivering 10 MeV electrons with an average dose rate above 50 Gy/s. Irradiations were carried out on 3D spheroids maintained under a range of O2 and temperature conditions to control O2 consumption and create gradients representative of in vivo tissues. RESULTS: Staining for pDNA-PK (Ser2056) produced a linear radiation dose response whereas γH2AX (Ser139) showed saturation with increasing dose. Using the pDNA-PK staining, radiation response was then characterised for FLASH compared to standard-dose-rates as a function of depth into the spheroids. At 4 °C, chosen to minimize the development of metabolic oxygen gradients within the tissues, FLASH protection could be observed at all distances under oxygen conditions of 0.3-1 % O2. Whereas at 37 °C a FLASH-protective effect was limited to the outer cell layers of tissues, an effect only observed at 3 % O2. Modelling of changes in the pDNA-PK-based oxygen enhancement ratio (OER) yielded a tissue ROC g0-value estimate of 0.73 ± 0.25 µM/Gy with a km of 5.4 µM at FLASH dose rates. CONCLUSIONS: DNA damage response markers are sensitive to the effects of transient oxygen depletion during FLASH radiotherapy. Findings support the rationale that well-oxygenated tissues would benefit more from FLASH-dose-rate protection relative to poorly-oxygenated tissues.
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Daño del ADN , Esferoides Celulares , Daño del ADN/efectos de la radiación , Humanos , Esferoides Celulares/efectos de la radiación , Histonas/metabolismo , Histonas/análisis , Consumo de Oxígeno/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Tratamientos Conservadores del Órgano/métodosRESUMEN
Inhibitors of DNA-PK sensitize cancers to radiotherapy and DNA-damaging chemotherapies, with candidates in clinical trials. However, the degree to which DNA-PK inhibitors also sensitize normal tissues remains poorly characterized. In this study we compare tumor growth control and normal tissue sensitization following DNA-PK inhibitors in combination with radiation and etoposide. FaDu tumor xenografts implanted in mice were treated with 10 - 15Gy irradiation ± 3 - 100 mg/kg AZD7648. A dose-dependent increase in time to tumor volume doubling following AZD7648 was proportional to an increase in toxicity scores of the overlying skin. Similar effects were seen in the intestinal jejunum, tongue and FaDu tumor xenografts of mice assessed for proliferation rates at 3.5 days after treatment with etoposide or 5Gy whole body irradiation ± DNA-PK inhibitors AZD7648 or peposertib (M3814). Additional organs were examined for sensitivity to DNA-PK inhibitor activity in ATM-deficient mice, where DNA-PK activity is indicated by surrogate marker γH2AX. Inhibition was observed in heart, brain, pancreas, thymus, tongue and salivary glands of ATM-deficient mice treated with the DNA-PK inhibitors relative to radiation alone. Similar reductions are also seen in ATM-deficient FaDu tumor xenografts where both pDNA-PK and γH2AX staining could be performed. Conclusions: DNA-PK inhibitor-mediated sensitization to radiation and DNA-damaging chemotherapy is not limited to tumor tissues, but also extends to normal tissues sustaining DNA damage. These data are useful for interpretation of the sensitizing effects of DNA damage repair inhibitors, where a therapeutic index showing greater cell-killing effects on cancer cells is crucial for optimal clinical translation.
RESUMEN
Type II topoisomerase (Top2) poisoning therapy is used to treat a broad range of cancers via induction of double strand breaks (DSBs) in cells undergoing replication and transcription. Preventing the repair of DSBs via inhibition of DNA-PK, an inhibitor of non-homologous end-joining (NHEJ), increases cell kill with Top2 poisons and has led to the initiation of several clinical trials. To elucidate the cellular mechanisms leading to synergistic activity of dual DNA-PK/Top2 inhibition we looked at their effects in cycling versus non-cycling cells, in 3D spheroids and in xenograft models. Combined DNA-PK/Top2 inhibition was found to not only increase the cell kill in proliferating cells, the cell population that is typically most vulnerable to Top2 poisoning, but also in non-proliferative but transcriptionally active cells. This effect was observed in both cancer and normal tissue models, killing more cells than high concentrations of etoposide alone. The combination treatment delayed tumor growth in mice compared to Top2 poisoning alone, but also led to increased toxicity. These findings demonstrate sensitization of Top2ß-expressing, non-cycling cells to Top2 poisoning by DNA-PK inhibition. Expansion of the target cell population of Top2 poison treatment to include non-proliferating cells via combination with DNA damage repair inhibitors has implications for efficacy and toxicity of these combinations, including for inhibitors of DNA-PK currently in clinical trial.
Asunto(s)
Proteínas de Unión al ADN , Neoplasias , Humanos , Animales , Ratones , Proteínas de Unión al ADN/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Etopósido/farmacología , Isomerasas/genética , Proteína Quinasa Activada por ADN/genética , Neoplasias/tratamiento farmacológico , ADN , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1-/- mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with ß-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash1 in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy.
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Células Endoteliales/metabolismo , Pulmón/crecimiento & desarrollo , Óxido Nítrico/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Línea Celular , GMP Cíclico/metabolismo , Pérdida del Embrión/metabolismo , Pérdida del Embrión/patología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Endotelio/metabolismo , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Pulmón/ultraestructura , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo III/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Alveolos Pulmonares/patología , Proteínas Asociadas a Surfactante Pulmonar/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Treatment strategies involving immune-checkpoint blockade (ICB) have significantly improved survival for a subset of patients across a broad spectrum of advanced solid cancers. Despite this, considerable room for improving response rates remains. The tumor microenvironment (TME) is a hurdle to immune function, as the altered metabolism-related acidic microenvironment of solid tumors decreases immune activity. Here, we determined that expression of the hypoxia-induced, cell-surface pH regulatory enzyme carbonic anhydrase IX (CAIX) is associated with worse overall survival in a cohort of 449 patients with melanoma. We found that targeting CAIX with the small-molecule SLC-0111 reduced glycolytic metabolism of tumor cells and extracellular acidification, resulting in increased immune cell killing. SLC-0111 treatment in combination with immune-checkpoint inhibitors led to the sensitization of tumors to ICB, which led to an enhanced Th1 response, decreased tumor growth, and reduced metastasis. We identified that increased expression of CA9 is associated with a reduced Th1 response in metastatic melanoma and basal-like breast cancer TCGA cohorts. These data suggest that targeting CAIX in the TME in combination with ICB is a potential therapeutic strategy for enhancing response and survival in patients with hypoxic solid malignancies.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Anhidrasas Carbónicas/química , Hipoxia/fisiopatología , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Sulfonamidas/farmacología , Animales , Apoptosis , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Antígeno CTLA-4/antagonistas & inhibidores , Anhidrasas Carbónicas/metabolismo , Proliferación Celular , Quimioterapia Combinada , Inducción Enzimática , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/secundario , Melanoma/enzimología , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Pronóstico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Tasa de Supervivencia , Células Tumorales Cultivadas , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
PURPOSE: There is a critical need for non-invasive imaging biomarkers of tumor oxygenation to assist in patient stratification and development of hypoxia targeting therapies. Using a cycling gas challenge and independent component analysis (ICA), we sought to improve the sensitivity and speed of existing oxygen enhanced MRI (OE-MRI) techniques to detect changes in oxygenation with dynamically acquired T1 W signal intensity images (dOE-MRI). METHODS: Mice were implanted with SCCVII, HCT-116, BT-474, or SKOV3 tumors in the dorsal subcutaneous region and imaged at 7T. T1 W images were acquired during a respiratory challenge with alternating 2-minute periods of air and 100% oxygen for three cycles. Data were analyzed with ICA and oxygenation maps were generated and compared to corresponding histology sections stained for hypoxia (pimonidazole) and blood vessels (CD31). RESULTS: Cycling air-oxygen-air gas challenges were well tolerated and ICA permitted extraction of the oxygen-enhancing component in all imaged tumors from four different models. Comparison with synthetic response functions showed that dOE-MRI does not require any a-priori knowledge of the physiological response. The fraction of O2 -negative dOE-MRI voxels that correlate inversely with the ICA gas-cycling component correspond well with the histological hypoxic fraction in SCCVII tumors (r = 0.91, p = 0.0016) but did not correlate in HCT-116 tumors (r = 0.13, p = 0.81). CONCLUSIONS: Using ICA and adding a cycling gas challenge extends the sensitivity of OE-MRI and allows the oxygenation status of tumors to be assessed in as little as six minutes. These findings support further development of OE-MRI as a biomarker of tumor oxygenation.
Asunto(s)
Biomarcadores/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Neoplasias/diagnóstico por imagen , Oxígeno/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Femenino , Células HCT116 , Humanos , Ratones , Trasplante de Neoplasias , Nitroimidazoles/uso terapéutico , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Microambiente TumoralRESUMEN
Autophagy, a lysosome-mediated degradation and recycling process, functions in advanced malignancies to promote cancer cell survival and contribute to cancer progression and drug resistance. While various autophagy inhibition strategies are under investigation for cancer treatment, corresponding patient selection criteria for these autophagy inhibitors need to be developed. Due to its central roles in the autophagy process, the cysteine protease ATG4B is one of the autophagy proteins being pursued as a potential therapeutic target. In this study, we investigated the expression of ATG4B in breast cancer, a heterogeneous disease comprised of several molecular subtypes. We examined a panel of breast cancer cell lines, xenograft tumors, and breast cancer patient specimens for the protein expression of ATG4B, and found a positive association between HER2 and ATG4B protein expression. We showed that HER2-positive cells, but not HER2-negative breast cancer cells, require ATG4B to survive under stress. In HER2-positive cells, cytoprotective autophagy was dependent on ATG4B under both starvation and HER2 inhibition conditions. Combined knockdown of ATG4B and HER2 by siRNA resulted in a significant decrease in cell viability, and the combination of ATG4B knockdown with trastuzumab resulted in a greater reduction in cell viability compared to trastuzumab treatment alone, in both trastuzumab-sensitive and -resistant HER2 overexpressing breast cancer cells. Together these results demonstrate a novel association of ATG4B positive expression with HER2 positive breast cancers and indicate that this subtype is suitable for emerging ATG4B inhibition strategies.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Relacionadas con la Autofagia/biosíntesis , Autofagia/fisiología , Neoplasias de la Mama/metabolismo , Cisteína Endopeptidasas/biosíntesis , Resistencia a Antineoplásicos/efectos de los fármacos , Adulto , Anciano , Animales , Autofagia/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Femenino , Xenoinjertos , Humanos , Ratones , Persona de Mediana Edad , Receptor ErbB-2/biosíntesis , Trastuzumab/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Metronomic chemotherapy has shown promising activity against solid tumors and is believed to act in an antiangiogenic manner. The current study describes and quantifies the therapeutic efficacy, and mode of activity, of metronomic gemcitabine and a dedicated antiangiogenic agent (DC101) in patient-derived xenografts of pancreatic cancer. METHODS: Two primary human pancreatic cancer xenograft lines were dosed metronomically with gemcitabine or DC101 weekly. Changes in tumor growth, vascular function, and metabolism over time were measured with magnetic resonance imaging, positron emission tomography, and immunofluorescence microscopy to determine the anti-tumor effects of the respective treatments. RESULTS: Tumors treated with metronomic gemcitabine were 10-fold smaller than those in the control and DC101 groups. Metronomic gemcitabine, but not DC101, reduced the tumors' avidity for glucose, proliferation, and apoptosis. Metronomic gemcitabine-treated tumors had higher perfusion rates and uniformly distributed blood flow within the tumor, whereas perfusion rates in DC101-treated tumors were lower and confined to the periphery. DC101 treatment reduced the tumor's vascular density, but did not change their function. In contrast, metronomic gemcitabine increased vessel density, improved tumor perfusion transiently, and decreased hypoxia. CONCLUSION: The aggregate data suggest that metronomic gemcitabine treatment affects both tumor vasculature and tumor cells continuously, and the overall effect is to significantly slow tumor growth. The observed increase in tumor perfusion induced by metronomic gemcitabine may be used as a therapeutic window for the administration of a second drug or radiation therapy. Non-invasive imaging could be used to detect early changes in tumor physiology before reductions in tumor volume were evident.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Desoxicitidina/análogos & derivados , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Administración Metronómica , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Proliferación Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Humanos , Masculino , Ratones SCID , Microvasos/efectos de los fármacos , Microvasos/patología , Necrosis , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/patología , Perfusión , GemcitabinaRESUMEN
Macromolecular gadolinium (Gd)-based contrast agents are in development as blood pool markers for MRI. HPG-GdF is a 583 kDa hyperbranched polyglycerol doubly tagged with Gd and Alexa 647 nm dye, making it both MR and histologically visible. In this study we examined the location of HPG-GdF in whole-tumor xenograft sections matched to in vivo DCE-MR images of both HPG-GdF and Gadovist. Despite its large size, we have shown that HPG-GdF extravasates from some tumor vessels and accumulates over time, but does not distribute beyond a few cell diameters from vessels. Fractional plasma volume (fPV) and apparent permeability-surface area product (aPS) parameters were derived from the MR concentration-time curves of HPG-GdF. Non-viable necrotic tumor tissue was excluded from the analysis by applying a novel bolus arrival time (BAT) algorithm to all voxels. aPS derived from HPG-GdF was the only MR parameter to identify a difference in vascular function between HCT116 and HT29 colorectal tumors. This study is the first to relate low and high molecular weight contrast agents with matched whole-tumor histological sections. These detailed comparisons identified tumor regions that appear distinct from each other using the HPG-GdF biomarkers related to perfusion and vessel leakiness, while Gadovist-imaged parameter measures in the same regions were unable to detect variation in vascular function. We have established HPG-GdF as a biocompatible multi-modal high molecular weight contrast agent with application for examining vascular function in both MR and histological modalities.
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Medios de Contraste/administración & dosificación , Glicerol/administración & dosificación , Aumento de la Imagen/métodos , Imagen por Resonancia Magnética/métodos , Polímeros/administración & dosificación , Animales , Línea Celular Tumoral , Medios de Contraste/química , Gadolinio DTPA/administración & dosificación , Gadolinio DTPA/química , Glicerol/química , Humanos , Ratones , Neovascularización Patológica/diagnóstico por imagen , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/química , Polímeros/química , Radiografía , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Metástasis de la Neoplasia , Biosíntesis de Proteínas , Sarcoma/patología , Proteína 1 de Unión a la Caja Y/fisiología , Humanos , Invasividad Neoplásica , Sarcoma/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiologíaRESUMEN
PURPOSE: We aimed to assess the prognostic significance of follicular lymphoma-associated macrophages in the era of rituximab treatment and maintenance. EXPERIMENTAL DESIGN: We applied immunohistochemistry for CD68 and CD163 to two large tissue microarrays (TMA). The first TMA included samples from 186 patients from the BC Cancer Agency (BCCA) who had been treated with first-line systemic treatment including rituximab, cyclophosphamide, vincristine, and prednisone. The second contained 395 samples from PRIMA trial patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, and randomized to rituximab maintenance or observation. Macrophage infiltration was assessed using Aperio image analysis. Each of the two cohorts was randomly split into training/validation sets. RESULTS: An increased CD163-positive pixel count was predictive of adverse outcome in the BCCA dataset [5-year progression-free survival (PFS) 38% vs. 72%, respectively, P = 0.004 in the training cohort and 5-year PFS 29% vs. 61%, respectively, P = 0.004 in the validation cohort]. In the PRIMA trial, an increased CD163 pixel count was associated with favorable outcome (5-year PFS 60% vs. 44%, respectively, P = 0.011 in the training cohort and 5-year PFS 55% vs. 37%, respectively, P = 0.030 in the validation cohort). CONCLUSIONS: CD163-positive macrophages predict outcome in follicular lymphoma, but their prognostic impact is highly dependent on treatment received.
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Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Linfoma Folicular/tratamiento farmacológico , Linfoma Folicular/genética , Pronóstico , Receptores de Superficie Celular/biosíntesis , Rituximab/administración & dosificación , Anciano , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Ciclofosfamida/administración & dosificación , Supervivencia sin Enfermedad , Doxorrubicina/administración & dosificación , Femenino , Humanos , Linfoma Folicular/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Receptores de Superficie Celular/genética , Análisis de Matrices Tisulares , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Vincristina/administración & dosificaciónRESUMEN
Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.
Asunto(s)
Carcinogénesis/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Neoplasias/genética , Receptores Notch/genética , Acetilación , Animales , Carcinogénesis/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Histonas/metabolismo , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , FN-kappa B/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Receptores Notch/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Trasplante HeterólogoRESUMEN
BACKGROUND/AIM: Human papilloma virus-associated head and neck squamous cell carcinomas (HNSCC) represent a distinct subgroup of HNSCC characterized by a favorable prognosis and a distinct molecular biology. There is a range of unresolved questions regarding the different biology and clinical outcome of HPV-positive HNSCC. The purpose of the present project was to obtain insight into the biology of treatment responsiveness of HPV-related HNSCC. MATERIALS AND METHODS: Tumor xenografts were established from HPV-negative (FaDuDD,) and HPV-positive (UD2 and UMSCC47) HNSCC cell lines. Tumors were treated with 10 Gy or 20 Gy and the effect on the tumor microenvironment was studied at different time points after treatment. Cryosections were imaged for cell proliferation, hypoxia, vessel density and vessel perfusion. RESULTS: In the HPV-positive tumor models the levels of cell proliferation decreased significantly following irradiation. This was not seen in the HPV-negative model (FaDuDD). Furthermore, it was found that the tumor hypoxic fraction decreased over time after treatment in irradiated HPV-positive tumors and not in the HPV-negative tumors. CONCLUSION: The radiosensitivity previously observed in vitro could be applied in vivo in respect to a radiation-induced decrease in proliferating cells. A decreasing hypoxic fraction following irradiation in the HPV-positive tumors could explain the lack of benefit from hypoxic modifiers observed in patients.
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Carcinoma de Células Escamosas/radioterapia , Hipoxia de la Célula/efectos de la radiación , Proliferación Celular/efectos de la radiación , Neoplasias de Cabeza y Cuello/radioterapia , Infecciones por Papillomavirus/radioterapia , Animales , Apoptosis/efectos de la radiación , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Rayos gamma , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/virología , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Papillomaviridae/fisiología , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Tolerancia a Radiación , Células Tumorales CultivadasRESUMEN
The ability of a panel of camptothecin derivatives to access the tumor compartment was evaluated to determine the mechanisms by which the architecture of solid tumors may act to limit their activity. Microregional localization and activity of members of the camptothecin class of topoisomerase I targeting agents, including topotecan, irinotecan, and irinophore C, a lipid-based nanoparticulate formulation of irinotecan, were evaluated over time in HCT116 and HT29 colorectal tumor xenografts. Using native drug fluorescence, their distributions in tissue cryosections were related to the underlying tumor vasculature, tumor cell proliferation, and apoptosis. Topotecan exhibited a relatively uniform tumor distribution; in tissue 100 µm away from vessels, it reached 94% ± 5% of levels seen around blood vessels, whereas irinotecan and irinophore C were found to reach only 41% ± 10% and 5% ± 2%, respectively. Surprisingly, all three agents were able to initially inhibit proliferation uniformly throughout the tumors, and it was their rate of washout (topotecan > irinotecan > irinophore C) that correlated with activity. To explain this discrepancy, we looked at SN38, the active metabolite of irinotecan, and found it to penetrate tissue similarly to topotecan. Hence, the poor access to the tumor compartment of irinotecan and irinophore C could be offset by their systemic conversion to SN38. It was concluded that all three agents were effective at reaching tumor cells, and that despite the poor access to the extravascular compartment of irinophore C, its extended plasma exposure and systemic conversion to the diffusible metabolite SN38 enabled it to effectively target solid tumors.
Asunto(s)
Camptotecina/análogos & derivados , Camptotecina/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Animales , Camptotecina/farmacocinética , Proliferación Celular/efectos de los fármacos , Femenino , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Endogámicos NOD , Inhibidores de Topoisomerasa I/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels.
Asunto(s)
Melanoma Experimental/enzimología , Neovascularización Patológica/enzimología , Óxido Nítrico Sintasa de Tipo III/fisiología , Receptores Notch/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Línea Celular Tumoral , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Microvasos/efectos de los fármacos , Microvasos/patología , Trasplante de Neoplasias , Óxido Nítrico/metabolismo , Pericitos/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Hipoxia , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Citotoxinas/administración & dosificación , Femenino , Células HCT116 , Células HT29 , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/administración & dosificación , Tirapazamina , Resultado del Tratamiento , Triazinas/administración & dosificación , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The angiopoietins Ang1 (ANGPT1) and Ang2 (ANGPT2) are secreted factors that bind to the endothelial cell-specific receptor tyrosine kinase Tie2 (TEK) and regulate angiogenesis. Ang1 activates Tie2 to promote blood vessel maturation and stabilization. In contrast, Ang2, which is highly expressed by tumor endothelial cells, is thought to inhibit Tie2 activity and destabilize blood vessels, thereby facilitating VEGF-dependent vessel growth. Here, we show that the inhibition of tumor xenograft growth caused by an Ang2-specific antibody (REGN910) is reversed by systemic administration of the Tie2 agonist Ang1. These results indicate that Ang2 blockade inhibits tumor growth by decreasing Tie2 activity, showing that Ang2 is a Tie2 activator. REGN910 treatment of tumors resulted in increased expression of genes that are repressed by Tie2 activation, providing further evidence that REGN910 inhibits Tie2 signaling. Combination treatment with REGN910 plus the VEGF blocker aflibercept reduced tumor vascularity and tumor perfusion more dramatically than either single agent, resulting in more extensive tumor cell death and more potent inhibition of tumor growth. Challenging the prevailing model of Ang2 as a destabilizing factor, our findings indicate that Ang2 plays a protective role in tumor endothelial cells by activating Tie2, thereby limiting the antivascular effects of VEGF inhibition. Thus, blockade of Ang2 might enhance the clinical benefits currently provided by anti-VEGF agents. .
Asunto(s)
Angiopoyetina 2/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Experimentales/metabolismo , Receptor TIE-2/agonistas , Receptor TIE-2/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/metabolismo , Transducción de Señal/fisiología , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.