RESUMEN
Targeted inhibition of oncogenic miRNA-21 has been proposed to treat glioblastoma by rescuing tumor suppressors, PTEN and PDCD4. However, systemic delivery of anti-miR-21 sequences requires a robust and efficient delivery platform to successfully inhibit this druggable target. Three-way-junction (3WJ)-based RNA nanoparticles (RNP), artificially derived from pRNA of bacteriophage phi29 DNA packaging motor, was recently shown to target glioblastoma. Here, we report that multi-valent folate (FA)-conjugated 3WJ RNP constructed to harbor anti-miR-21 LNA sequences (FA-3WJ-LNA-miR21) specifically targeted and delivered anti-miR-21 LNA and knocked down miR-21 expression in glioblastoma cells in vitro and in vivo with favorable biodistribution. Systemically injected FA-3WJ-LNA-miR21 RNP efficiently rescued PTEN and PDCD4, resulting in glioblastoma cell apoptosis and tumor growth regression. Overall survival rate was also significantly improved by FA-3WJ-LNA-miR21 RNP. These results are indicative of the clinical benefit of FA-3WJ RNP-based gene therapy for the successful targeted therapy of developing and even recurring glioblastoma.
Asunto(s)
Antagomirs/farmacología , Neoplasias Encefálicas/terapia , Regulación Neoplásica de la Expresión Génica , Glioblastoma/terapia , MicroARNs/antagonistas & inhibidores , Nanopartículas/administración & dosificación , Animales , Antagomirs/química , Antagomirs/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Portadores de Fármacos , Femenino , Receptores de Folato Anclados a GPI/genética , Receptores de Folato Anclados a GPI/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Glioblastoma/genética , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Nanopartículas/química , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Malignant gliomas (glioblastomas; GBMs) are extremely aggressive and have a median survival of approximately 15 months. Current treatment modalities, which include surgical resection, radiation and chemotherapy, have done little to prolong the lives of GBM patients. Chondroitin sulfate proteoglycans (CSPG) are critical for cell-cell and cell-extracellular matrix (ECM) interactions and are implicated in glioma growth and invasion. Chondroitinase (Chase) ABC is a bacterial enzyme that cleaves chondroitin sulfate disaccharide chains from CSPGs in the tumor ECM. Wild-type Chase ABC has limited stability and/or activity in mammalian cells; therefore, we created a mutant humanized version (Chase M) with enhanced function in mammalian cells. METHODS: We hypothesized that disruption of cell-cell and cell-ECM interactions by ChaseM and temozolomide (TMZ) will enhance the chemotherapeutic availability and sensitivity of glioma cells. RESULTS: Utilizing primary patient-derived neurospheres, we found that ChaseM decreases glioma neurosphere aggregation in vitro. Furthermore, an oncolytic HSV-1 virus expressing secreted ChaseM (OV-ChaseM) enhanced viral spread and glioma cell killing compared to OV-Control, in vitro. OV-ChaseM plus TMZ combinatorial treatment resulted in a significant synergistic enhancement of glioma cell killing accompanied by an increase in apoptotic cell death. Intracellular flow cytometric analysis revealed a significant reduction in the phosphorylation of the pro-survival AKT protein following OV-ChaseM plus TMZ treatment. Lastly, in nude mice bearing intracranial GBM30 glioma xenografts, intratumoral OV-ChaseM plus TMZ (10 mg/kg by oral gavage) combination therapy resulted in a significant (p < 0.02) enhancement of survival compared to each individual treatment alone. CONCLUSIONS: These data reveal that OV-ChaseM enhances glioma cell viral susceptibility and sensitivity to TMZ.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Condroitina ABC Liasa/genética , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Glioblastoma/genética , Alelos , Sustitución de Aminoácidos , Animales , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Chlorocebus aethiops , Condroitina ABC Liasa/metabolismo , Dacarbazina/farmacología , Modelos Animales de Enfermedad , Activación Enzimática , Expresión Génica , Vectores Genéticos/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Herpesvirus Humano 1/genética , Humanos , Ratones , Mutación , Viroterapia Oncolítica , Temozolomida , Transducción Genética , Resultado del Tratamiento , Carga Tumoral , Células Tumorales Cultivadas , Células Vero , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 µM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 µM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells.
Asunto(s)
Janus Quinasa 1/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Células Cultivadas , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Interferón-alfa/farmacología , Interleucina-2/farmacología , Células K562 , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones Endogámicos BALB C , Niacinamida/análogos & derivados , Niacinamida/farmacología , Compuestos de Fenilurea/farmacología , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sorafenib , Neoplasias de la Tiroides/sangre , Neoplasias de la Tiroides/tratamiento farmacológico , Quinasas raf/antagonistas & inhibidores , Quinasas raf/metabolismoRESUMEN
The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 µg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.
Asunto(s)
Adenocarcinoma/terapia , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias del Colon/terapia , Interferón gamma/biosíntesis , Interleucina-12/uso terapéutico , Células Asesinas Naturales/inmunología , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Anticuerpos Monoclonales Humanizados , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Pruebas Inmunológicas de Citotoxicidad , Femenino , Interferón gamma/fisiología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos BALB C , Distribución Aleatoria , Receptor ErbB-2/inmunología , Trastuzumab , Regulación hacia Arriba/inmunologíaRESUMEN
Interferon-alpha (IFN-α) is an immunomodulatory cytokine that is used clinically for the treatment of melanoma in the adjuvant setting. The cellular actions of IFN-α are regulated by the suppressors of cytokine signaling (SOCS) family of proteins. We hypothesized that the anti-tumor activity of exogenous IFN-α would be enhanced in SOCS1-deficient mice. SOCS1-deficient (SOCS1(-/-)) or control (SOCS1(+/+)) mice on an IFN-γ(-/-) C57BL/6 background bearing intraperitoneal (i.p.) JB/MS murine melanoma cells were treated for 30 days with i.p. injections of IFN-A/D or PBS (vehicle). Log-rank Kaplan-Meier survival curves were used to evaluate survival. Tumor-bearing control SOCS1(+/+) mice receiving IFN-A/D had significantly enhanced survival versus PBS-treated mice (P = 0.0048). The anti-tumor effects of IFN-A/D therapy were significantly enhanced in tumor-bearing SOCS1(-/-) mice; 75% of these mice survived tumor challenge, whereas PBS-treated SOCS1(-/-) mice all died at 13-16 days (P = 0.00038). Antibody (Ab) depletion of CD8(+) T cells abrogated the anti-tumor effects of IFN-A/D in SOCS1(-/-) mice as compared with mice receiving a control antibody (P = 0.0021). CD4(+) T-cell depletion from SOCS1(-/-) mice also inhibited the effects of IFN-A/D (P = 0.0003). IFN-A/D did not alter expression of CD80 or CD86 on splenocytes of SOCS1(+/+) or SOCS1(-/-) mice, or the proportion of T regulatory cells or myeloid-derived suppressor cells in SOCS1(+/+) or SOCS1(-/-) mice. An analysis of T-cell function did reveal increased proliferation of SOCS1-deficient splenocytes at baseline and in response to mitogenic stimuli. These data suggest that modulation of SOCS1 function in T-cell subsets could enhance the anti-tumor effects of IFN-α in the setting of melanoma.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón-alfa/farmacología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Proteínas Supresoras de la Señalización de Citocinas/deficiencia , Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Humanos , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Linfocitos T/metabolismoRESUMEN
Because most patients with multiple myeloma (MM) develop resistance to current regimens, novel approaches are needed. Genetically modified, replication-competent oncolytic viruses exhibit high tropism for tumor cells regardless of cancer stage and prior treatment. Receptors of oncolytic herpes simplex virus 1 (oHSV-1), NECTIN-1, and HVEM are expressed on MM cells, prompting us to investigate the use of oHSV-1 against MM. Using oHSV-1-expressing GFP, we found a dose-dependent increase in the GFP+ signal in MM cell lines and primary MM cells. Whereas NECTIN-1 expression is variable among MM cells, we discovered that HVEM is ubiquitously and highly expressed on all samples tested. Expression of HVEM was consistently higher on CD138+/CD38+ plasma cells than in non-plasma cells. HVEM blocking demonstrated the requirement of this receptor for infection. However, we observed that, although oHSV-1 could efficiently infect and kill all MM cell lines tested, no viral replication occurred. Instead, we identified that oHSV-1 induced MM cell apoptosis via caspase-3 cleavage. We further noted that oHSV-1 yielded a significant decrease in tumor volume in two mouse xenograft models. Therefore, oHSV-1 warrants exploration as a novel potentially effective treatment option in MM, and HVEM should be investigated as a possible therapeutic target.
RESUMEN
PURPOSE: To examine the effect of oncolytic herpes simplex virus (oHSV) on NOTCH signaling in central nervous system tumors. EXPERIMENTAL DESIGN: Bioluminescence imaging, reverse phase protein array proteomics, fluorescence microscopy, reporter assays, and molecular biology approaches were used to evaluate NOTCH signaling. Orthotopic glioma-mouse models were utilized to evaluate effects in vivo. RESULTS: We have identified that herpes simplex virus-1 (HSV-1; oncolytic and wild-type)-infected glioma cells induce NOTCH signaling, from inside of infected cells into adjacent tumor cells (inside out signaling). This was canonical NOTCH signaling, which resulted in activation of RBPJ-dependent transcriptional activity that could be rescued with dnMAML. High-throughput screening of HSV-1-encoded cDNA and miRNA libraries further uncovered that HSV-1 miR-H16 induced NOTCH signaling. We further identified that factor inhibiting HIF-1 (FIH-1) is a direct target of miR-H16, and that FIH-1 downregulation by virus encoded miR-H16 induces NOTCH activity. FIH-1 binding to Mib1 has been reported, but this is the first report that shows FIH-1 sequester Mib1 to suppress NOTCH activation. We observed that FIH-1 degradation induced NOTCH ligand ubiquitination and NOTCH activity. REMBRANDT and The Cancer Genome Atlas data analysis also uncovered a significant negative regulation between FIH-1 and NOTCH. Furthermore, combination of oHSV with NOTCH-blocking gamma secretase inhibitor (GSI) had a therapeutic advantage in two different intracranial glioma models treated with oncolytic HSV, without affecting safety profile of the virus in vivo. CONCLUSIONS: To our knowledge this is the first report to identify impact of HSV-1 on NOTCH signaling and highlights the significance of combining oHSV and GSI for glioblastoma therapy.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Herpesvirus Humano 1/genética , Viroterapia Oncolítica/métodos , Receptores Notch/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Benzazepinas/farmacología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Diaminas/farmacología , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Desnudos , MicroARNs/genética , Oxigenasas de Función Mixta/metabolismo , Distribución Aleatoria , Proteínas Represoras/metabolismo , Transducción de Señal , Tiazoles/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Integrin ß1 receptor, expressed on the surface of tumor cells and macrophages in the tumor microenvironment (TME), has been implicated in both tumor progression and resistance to multiple modalities of therapy. OS2966 is the first clinical-ready humanized monoclonal antibody to block integrin ß1 and was recently orphan designated by the FDA Office of Orphan Products Development. Here, we tested therapeutic potential of OS2966-mediated integrin ß1 blockade to enhance the efficacy of oncolytic herpes simplex virus-1 (oHSV) through evaluation of virus replication, tumor cell killing efficiency, effect on the antiviral signaling pathway, co-culture assays of oHSV-infected cells with macrophages, and in vivo bioluminescence imaging on mammary fat pad triple-negative breast cancer xenograft and subcutaneous and intracranial glioma xenografts. OS2966 treatment decreased interferon signaling and proinflammatory cytokine induction in oHSV-treated tumor cells and inhibited migration of macrophages, resulting in enhanced oHSV replication and cytotoxicity. OS2966 treatment also significantly enhanced oHSV replication and oHSV-mediated antitumor efficacy in orthotopic xenograft models, including triple-negative breast cancer and glioblastoma. The results demonstrated the synergistic potential of the combinatory treatment approach with OS2966 to improve antitumor efficacy of conventional oHSV therapy.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Herpesvirus Humano 1/fisiología , Integrina beta1/inmunología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Movimiento Celular/inmunología , Técnicas de Cocultivo , Terapia Combinada/métodos , Femenino , Glioma/metabolismo , Glioma/patología , Glioma/terapia , Humanos , Macrófagos/metabolismo , Ratones , Ratones Desnudos , Células RAW 264.7 , Replicación Viral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Hyperactivation of the RAS-RAF-MEK-ERK signaling pathway is exploited by glioma cells to promote their growth and evade apoptosis. MEK activation in tumor cells can increase replication of ICP34.5-deleted herpes simplex virus type 1 (HSV-1), but paradoxically its activation in tumor-associated macrophages promotes a pro-inflammatory signaling that can inhibit virus replication and propagation. Here we investigated the effect of blocking MEK signaling in conjunction with oncolytic HSV-1 (oHSV) for brain tumors. METHODS: Infected glioma cells co-cultured with microglia or macrophages treated with or without trametinib were used to test trametinib effect on macrophages/microglia. Enzyme-linked immunosorbent assay, western blotting, and flow cytometry were utilized to evaluate the effect of the combination therapy. Pharmacokinetic (PK) analysis of mouse plasma and brain tissue was used to evaluate trametinib delivery to the CNS. Intracranial human and mouse glioma-bearing immune deficient and immune competent mice were used to evaluate the antitumor efficacy. RESULT: Oncolytic HSV treatment rescued trametinib-mediated feedback reactivation of the mitogen-activated protein kinase signaling pathway in glioma. In vivo, PK analysis revealed enhanced blood-brain barrier penetration of trametinib after oHSV treatment. Treatment by trametinib, a MEK kinase inhibitor, led to a significant reduction in microglia- and macrophage-derived tumor necrosis factor alpha (TNFα) secretion in response to oHSV treatment and increased survival of glioma-bearing mice. Despite the reduced TNFα production observed in vivo, the combination treatment activated CD8+ T-cell mediated immunity and increased survival in a glioma-bearing immune-competent mouse model. CONCLUSION: This study provides a rationale for combining oHSV with trametinib for the treatment of brain tumors.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/terapia , Linfocitos T CD8-positivos/efectos de los fármacos , Glioblastoma/terapia , Herpesvirus Humano 1 , Macrófagos/efectos de los fármacos , Microglía/efectos de los fármacos , Viroterapia Oncolítica/métodos , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Animales , Neoplasias Encefálicas/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Glioblastoma/inmunología , Glioma/inmunología , Glioma/terapia , Humanos , Inmunocompetencia , Macrófagos/inmunología , Ratones , Microglía/inmunología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Células RAW 264.7 , Tasa de Supervivencia , Factor de Necrosis Tumoral alfa/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Engineered oncolytic viruses are used clinically to destroy cancer cells and have the ability to boost anticancer immunity. Phosphatase and tensin homolog deleted on chromosome 10 loss is common across a broad range of malignancies, and is implicated in immune escape. The N-terminally extended isoform, phosphatase and tensin homolog deleted on chromosome 10 alpha (PTENα), regulates cellular functions including protein kinase B signaling and mitochondrial adenosine triphosphate production. Here we constructed HSV-P10, a replicating, PTENα expressing oncolytic herpesvirus, and demonstrate that it inhibits PI3K/AKT signaling, increases cellular adenosine triphosphate secretion, and reduces programmed death-ligand 1 expression in infected tumor cells, thus priming an adaptive immune response and overcoming tumor immune escape. A single dose of HSV-P10 resulted in long term survivors in mice bearing intracranial tumors, priming anticancer T-cell immunity leading to tumor rejection. This implicates HSV-P10 as an oncolytic and immune stimulating therapeutic for anticancer therapy.
Asunto(s)
Herpesviridae/metabolismo , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Virus Oncolíticos/metabolismo , Fosfohidrolasa PTEN/metabolismo , Linfocitos T/metabolismo , Animales , Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Femenino , Humanos , Inmunidad , Cinética , Ratones , Modelos Biológicos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
PURPOSE: Alternative strategies to EGFR blockage by mAbs is necessary to improve the efficacy of therapy in patients with locally advanced or metastatic pancreatic cancer. One such strategy includes the use of NK cells to clear cetuximab-coated tumor cells, as need for novel therapeutic approaches to enhance the efficacy of cetuximab is evident. We show that IL-21 enhances NK cell-mediated effector functions against cetuximab-coated pancreatic tumor cells irrespective of KRAS mutation status. EXPERIMENTAL DESIGN: NK cells from normal donors or donors with pancreatic cancer were used to assess ADCC, IFN-γ release, and T-cell chemotaxis toward human pancreatic cancer cell lines. The in vivo efficacy of IL-21 in combination with cetuximab was evaluated in a subcutaneous and intraperitoneal model of pancreatic cancer. RESULTS: NK cell lysis of cetuximab-coated wild-type and mutant kras pancreatic cancer cell lines were significantly higher following NK cell IL-21 treatment. In response to cetuximab-coated pancreatic tumor cells, IL-21-treated NK cells secreted significantly higher levels of IFN-γ and chemokines, increased chemotaxis of T cells, and enhanced NK cell signal transduction via activation of ERK and STAT1. Treatment of mice bearing subcutaneous or intraperitoneal EGFR-positive pancreatic tumor xenografts with mIL-21 and cetuximab led to significant inhibition of tumor growth, a result further enhanced by the addition of gemcitabine. CONCLUSIONS: These results suggest that cetuximab treatment in combination with IL-21 adjuvant therapy in patients with EGFR-positive pancreatic cancer results in significant NK cell activation, irrespective of KRAS mutation status, and may be a potential therapeutic strategy. Clin Cancer Res; 23(2); 489-502. ©2016 AACR.
Asunto(s)
Interleucinas/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Pancreáticas/terapia , Proteínas Proto-Oncogénicas p21(ras)/genética , Linfocitos T/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Cetuximab/administración & dosificación , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Citometría de Flujo , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucinas/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Linfocitos T/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Natural killer (NK) cells are innate immune effector cells that play a crucial role in immune surveillance and the destruction of cancer cells. NK cells express a low-affinity receptor for the Fc or constant region of immunoglobulin G (FcγRIIIa) and multiple cytokine receptors that respond to antibody-coated targets and cytokines in the tumor microenvironment. In the present work, microarray gene expression analysis revealed that the IL-21 receptor (IL-21R) was strongly upregulated following FcR stimulation. The IL-21R was found to be upregulated on FcR-stimulated NK cells at the transcript level as determined by reverse transcription polymerase chain reaction (RT-PCR). Immunoblot analysis revealed that protein expression of the IL-21R peaked at 8 h post-stimulation of the FcR. Inhibition of the mitogen-activated protein kinase (MAPK) pathway downstream of the FcR blocked the induction of IL-21R expression. Increased expression of the IL-21R sensitized NK cells to IL-21 stimulation, as treatment of FcR-stimulated NK cells led to significantly increased phosphorylation of STAT1 and STAT3, as measured by intracellular flow cytometry and immunoblot analysis. Following FcR-stimulation, IL-21-activated NK cells were better able to mediate the lysis of trastuzumab-coated human epidermal growth factor receptor 2 (HER2+) SK-BR-3 tumor cells as compared to control-treated cells. Likewise, IL-21-induced NK cell secretion of IFNγ following exposure to antibody-coated tumor cells was enhanced following FcR-stimulation. The analysis of NK cells from patients receiving trastuzumab therapy for HER2+ cancer exhibited increased levels of the IL-21R following the administration of antibody suggesting that the presence of monoclonal antibody-coated tumor cells in vivo can stimulate the increased expression of IL-21R on NK cells.
RESUMEN
The ubiquitin-proteasome signaling pathway is critical for cell cycle regulation and neoplastic growth. Proteasome inhibition can activate apoptotic pathways. Bortezomib, a selective proteasome inhibitor, has anti-melanoma activity. MLN2238 (ixazomib), an oral proteasome inhibitor, has improved pharmacotherapeutic parameters compared to bortezomib. Interferon-alpha (IFN-α), an immune boosting agent, is FDA-approved for treatment of melanoma. In this study in vitro and in vivo evaluation of the antitumor potential of ixazomib and combination treatments with ixazomib and IFN-α were performed. Apoptosis induced by ixazomib was first observed at 12 hours and was maximal at 48 hours with similar levels of cell death compared to bortezomib. IFN-α alone had little effect on cell viability in vitro. However, the combination of ixazomib with IFN-α significantly enhanced ixazomib's ability to induce apoptotic cell death in BRAF V600E mutant and BRAF wild-type human melanoma tumor cells. The combination of ixazomib and IFN-α also enhanced inhibition of cell proliferation in BRAF V600E mutant melanoma tumor cells; however, this was not seen in BRAF wild-type cells. Ixazomib-induced apoptosis was associated with processing of the pro-apoptotic proteins procaspase-3, -7, -8, and -9, and cleavage of poly-ADP-ribose polymerase (PARP). In an in vivo xenograft model of human melanoma, combination treatment with IFN-α-2b and ixazomib demonstrated a significant reduction in tumor volume when compared to vehicle (p = 0.005) and single therapy ixazomib (p = 0.017) and IFN-α-2b (p = 0.036). These pre-clinical results support further evaluation of combination treatment with ixazomib and IFN-α for the treatment of advanced BRAF V600E mutant melanoma.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Compuestos de Boro/farmacología , Glicina/análogos & derivados , Interferón-alfa/farmacología , Melanoma/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Glicina/farmacología , Humanos , Interferón alfa-2 , Melanoma/enzimología , Melanoma/genética , Melanoma/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Recombinantes/farmacología , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multiple myeloma remains incurable and the majority of patients die within 5 years of diagnosis. Reolysin, the infusible form of human reovirus (RV), is a novel viral oncolytic therapy associated with antitumor activity likely resulting from direct oncolysis and a virus-mediated antitumor immune response. Results from our phase I clinical trial investigating single agent Reolysin in patients with relapsed multiple myeloma confirmed tolerability, but no objective responses were evident, likely because the virus selectively entered the multiple myeloma cells but did not actively replicate. To date, the precise mechanisms underlying the RV infectious life cycle and its ability to induce oncolysis in patients with multiple myeloma remain unknown. Here, we report that junctional adhesion molecule 1 (JAM-1), the cellular receptor for RV, is epigenetically regulated in multiple myeloma cells. Treatment of multiple myeloma cells with clinically relevant histone deacetylase inhibitors (HDACi) results in increased JAM-1 expression as well as increased histone acetylation and RNA polymerase II recruitment to its promoter. Furthermore, our data indicate that the combination of Reolysin with HDACi, potentiates RV killing activity of multiple myeloma cells in vitro and in vivo This study provides the molecular basis to use these agents as therapeutic tools to increase the efficacy of RV therapy in multiple myeloma. Mol Cancer Ther; 15(5); 830-41. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Vectores Genéticos , Inhibidores de Histona Desacetilasas/farmacología , Mieloma Múltiple/patología , Mieloma Múltiple/terapia , Viroterapia Oncolítica , Virus Oncolíticos , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Terapia Combinada , Modelos Animales de Enfermedad , Epigénesis Genética , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Masculino , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Virus Oncolíticos/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Both the proteasome inhibitor bortezomib and an oncolytic herpes simplex virus-1 (oHSV)-expressing GM-CSF are currently FDA approved. Although proteasome blockade can increase oHSV replication, immunologic consequences, and consequent immunotherapy potential are unknown. In this study, we investigated the impact of bortezomib combined with oHSV on tumor cell death and sensitivity to natural killer (NK) cell immunotherapy. EXPERIMENTAL DESIGN: Western blot, flow cytometry, and caspase 3/7 activity assays were used to evaluate the induction of apoptosis/autophagy and/or necroptotic cell death. Cellular and mitochondrial reactive oxygen species (ROS) production was measured using CellROX and MitoSOX. Inhibitors/shRNA-targeting ROS, JNK and RIP1 kinase (RIPK1) were used to investigate the mechanism of cell killing. The synergistic interaction between oHSV and bortezomib was calculated using a Chou-Talalay analysis. NK cells isolated from normal human blood were co-cultured with tumor cells to evaluate cellular interactions. Q-PCR, ELISA, and FACS analysis were used to evaluate NK cell activation. Intracranial tumor xenografts were used to evaluate antitumor efficacy. RESULTS: Combination treatment with bortezomib- and oHSV-induced necroptotic cell death and increased the production of mitochondrial ROS and JNK phosphorylation. Inhibitors/shRNA of RIPK1 and JNK rescued synergistic cell killing. Combination treatment also significantly enhanced NK cell activation and adjuvant NK cell therapy of mice treated with bortezomib and oHSV improved antitumor efficacy. CONCLUSIONS: This study provides a significant rationale for triple combination therapy with bortezomib, oHSV, and NK cells to improve efficacy, in glioblastoma patients. Clin Cancer Res; 22(21); 5265-76. ©2016 AACRSee related commentary by Suryadevara et al., p. 5164.
Asunto(s)
Bortezomib/farmacología , Herpesvirus Humano 1/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Virus Oncolíticos/inmunología , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Línea Celular Tumoral , Terapia Combinada/métodos , Femenino , Humanos , Inmunoterapia/métodos , Células Asesinas Naturales/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Desnudos , Neoplasias/inmunología , Neoplasias/metabolismo , Viroterapia Oncolítica/métodos , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
PURPOSE: Oncolytic herpes simplex viruses (oHSV) represent a promising therapy for glioblastoma (GBM), but their clinical success has been limited. Early innate immune responses to viral infection reduce oHSV replication, tumor destruction, and efficacy. Here, we characterized the antiviral effects of macrophages and microglia on viral therapy for GBM. EXPERIMENTAL DESIGN: Quantitative flow cytometry of mice with intracranial gliomas (±oHSV) was used to examine macrophage/microglia infiltration and activation. In vitro coculture assays of infected glioma cells with microglia/macrophages were used to test their impact on oHSV replication. Macrophages from TNFα-knockout mice and blocking antibodies were used to evaluate the biologic effects of TNFα on virus replication. TNFα blocking antibodies were used to evaluate the impact of TNFα on oHSV therapy in vivo. RESULTS: Flow-cytometry analysis revealed a 7.9-fold increase in macrophage infiltration after virus treatment. Tumor-infiltrating macrophages/microglia were polarized toward a M1, proinflammatory phenotype, and they expressed high levels of CD86, MHCII, and Ly6C. Macrophages/microglia produced significant amounts of TNFα in response to infected glioma cells in vitro and in vivo. Using TNFα-blocking antibodies and macrophages derived from TNFα-knockout mice, we discovered TNFα-induced apoptosis in infected tumor cells and inhibited virus replication. Finally, we demonstrated the transient blockade of TNFα from the tumor microenvironment with TNFα-blocking antibodies significantly enhanced virus replication and survival in GBM intracranial tumors. CONCLUSIONS: The results of these studies suggest that FDA approved TNFα inhibitors may significantly improve the efficacy of oncolytic virus therapy.
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Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Viroterapia Oncolítica/métodos , Microambiente Tumoral/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Antineoplásicos/inmunología , Western Blotting , Neoplasias Encefálicas/patología , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Glioblastoma/patología , Herpesvirus Humano 1/inmunología , Macrófagos/inmunología , Ratones , Ratones Noqueados , Ratones Desnudos , Microglía/inmunología , Virus Oncolíticos/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The 2-year survival rate of patients with breast cancer brain metastases is less than 2%. Treatment options for breast cancer brain metastases are limited, and there is an unmet need to identify novel therapies for this disease. Brain angiogenesis inhibitor 1 (BAI1) is a GPCR involved in tumor angiogenesis, invasion, phagocytosis, and synaptogenesis. For the first time, we identify that BAI1 expression is significantly reduced in breast cancer and higher expression is associated with better patient survival. Nestin is an intermediate filament whose expression is upregulated in several cancers. We found that higher Nestin expression significantly correlated with breast cancer lung and brain metastases, suggesting both BAI1 and Nestin can be therapeutic targets for this disease. Here, we demonstrate the ability of an oncolytic virus, 34.5ENVE, to target and kill high Nestin-expressing cells and deliver Vstat120 (extracellular fragment of BAI1). Finally, we created two orthotopic immune-competent murine models of breast cancer brain metastases and demonstrated 34.5ENVE extended the survival of immune-competent mice bearing intracranial breast cancer tumors.
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Proteínas Angiogénicas/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Nestina/metabolismo , Virus Oncolíticos/genética , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Ratones , Metástasis de la Neoplasia , Viroterapia Oncolítica , Pronóstico , Receptores Acoplados a Proteínas G , Células VeroRESUMEN
Metastatic melanoma is the most aggressive form of this cancer. It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma. Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity. It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma. It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21. Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls. This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39. The proliferation and migration of miR-21 over-expressing cell lines was not affected. Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression. Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice. Intra-tumoral injections of anti-miR-21 produced similar effects. This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.
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Melanoma/genética , MicroARNs/genética , Invasividad Neoplásica/genética , Neoplasias Cutáneas/genética , Inhibidor Tisular de Metaloproteinasa-3/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/metabolismo , Melanoma/patología , MicroARNs/metabolismo , Invasividad Neoplásica/patología , ARN Interferente Pequeño , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
Pancreatic cancer is a devastating disease, with a median survival of around 6 months for patients with stage IV disease. The epidermal growth factor receptor (EGFR, or HER1) belongs to the erbB receptor tyrosine kinase family. HER1-mediated cell signaling has been shown to play a major role in promoting tumor proliferation, angiogenesis, metastasis, and evasion of apoptosis. Over-expression of HER1 is observed in multiple human malignancies, including colorectal, lung, breast and pancreatic cancers. In pancreatic carcinoma, over-expression of HER1 is observed in greater than 70% of patients and is associated with a poor prognosis and a significant decrease in survival. Cetuximab (Erbitux) is a chimeric monoclonal antibody (mAb) that binds to the extracellular domain of the HER1 molecule preventing ligand binding and promoting internalization and subsequent degradation of the HER1 receptor. Cetuximab has shown anti-tumor activity either alone or in combination with other agents and is currently FDA approved for use in both squamous cell carcinoma of the head and neck (SCCHN) and colorectal carcinoma. Research efforts continue to elucidate a possible role for cetuximab in the treatment of pancreatic cancer. Despite promising preclinical work, phase II and phase III clinical trials have failed to consistently show efficacy of cetuximab treatment in advanced pancreatic cancer either alone or in combination with cytotoxic agents. Alternative approaches to HER1 blockade and mAbs including immune modulation with cytokines might be necessary in order to improve the efficacy of mAbs in pancreatic cancer therapy.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Pancreáticas/terapia , Anticuerpos Monoclonales Humanizados , Apoptosis/efectos de los fármacos , Cetuximab , Ensayos Clínicos como Asunto , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoterapia , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Transducción de Señal , Tasa de Supervivencia , Insuficiencia del TratamientoRESUMEN
BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide. Greater than 90% of SCCHN of the oropharynx overexpress the epidermal growth factor receptor (EGFR or HER1). Cetuximab (Erbitux-TM) is a humanized anti-HER1 monoclonal antibody (mAb) that binds to HER1 overexpressing tumor cells. Cetuximab has a direct effect on HER1-positive cancer cells, but it also can activate immune cells that bear receptors for the Fc (constant portion) of IgG such as natural killer (NK) cells. NK cells have an activating Fc receptor for IgG (FcγRIIIa), which mediates Ab dependent cellular cytotoxicity (ADCC) and enhances production of interferon-γ (IFN-γ) in response to Ab-coated targets. Interleukin-12 (IL-12) is a cytokine produced by antigen-presenting cells that stimulates IFN-γ production from NK cells. We hypothesized that IL-12 would enhance the anti-tumor activity of cetuximab by activating the FcR effector mechanisms of NK cells. METHODS: Expression of HER1 was measured on human papilloma virus (HPV)-positive (UD-SCC2, UM-SCC47) and HPV-negative (Cal27, UM-SCC74B) SCCHN cell lines by immunoblot analysis and flow cytometry. NK cells from normal donors were treated overnight with IL-2 (100 U), IL-12, IL-15, or IL-21 (all 10 ng/mL) and tested for ADCC versus cetuximab-coated cancer cells in a 4 hr (51)Cr assay. Release of cytokines by NK cells in response to cetuximab-coated cells was measured by ELISA. Phosphorylation of the ERK transcription factor in NK cells was measured by flow cytometry. The efficacy of combination therapy with cetuximab plus IL-12 was evaluated in a murine tumor model of head and neck cancer. RESULTS: All cell lines showed >99% expression of HER1 by flow cytometry and immunoblot analysis except UM-SCC74B (73%). Normal NK cells mediated 49.4% lysis of cetuximab-coated SCCHN cell lines as compared to 7.6% lysis of cells treated with control IgG (P = .0002). NK cell lysis of cetuximab-coated SCCHN cells was markedly enhanced by 12 hr pre-treatment of NK cells with IL-12 (71.6% lysis, P = .005 vs cetuximab alone). As a control, IL-12-activated NK cells were tested against IgG-treated cells. ADCC under these conditions was just 21.7%. Similar levels of lysis were noted for both HPV-positive and HPV-negative and cell lines. Other NK cell activating factors such as IL-2, IL-15, and IL-21 were also able to enhance NK cell ADCC. The stimulus of IL-12 and cetuximab-coated tumor cells induced the synergistic production of nanogram levels of IFN-γ (>6-fold increase over controls) (P < .001). A similar effect was seen for NK cell production of the chemokines RANTES, MIP-1α, and IL-8. Phosphorylation of ERK (which is critical for FcR-mediated ADCC and cytokine production) was enhanced in NK cells exposed to IL-12 and IgG as compared to control conditions. The combination of cetuximab plus IL-12 resulted in a reduction in tumor burden when compared to either agent alone in a murine xenograft model of SCCHN. CONCLUSION: Cytokine stimulation of NK cells in the presence of cetuximab-coated head and neck cancer cells leads to enhanced NK cell mediated ADCC and cytokine secretion independent of tumor cell HPV-status. Cytokine administration could be a useful adjuvant in the cetuximab treatment of HER1-positive head and neck cancer.