RESUMEN
BACKGROUND: VTP-50469 is a potent inhibitor of the menin-MLL1 interaction and is implicated in signaling downstream of EWSR1-FLI1. PROCEDURE: VTP-50469 was evaluated against seven Ewing sarcoma (EwS) xenograft models and in vitro against EwS cell lines. RESULTS: VTP-50469 showed limited antitumor activity, statistically significantly slowing tumor progression in four tumor models but with no evidence of tumor regression. In vitro, the IC50 concentration was 10 nM for the mixed lineage leukemia (MLL)-rearranged leukemia cell line MV4;11, but > 3 µM for EwS cell lines. CONCLUSIONS: In contrast to its high level of activity against MLL1-rearranged leukemia xenografts, VTP-50469 shows little activity against EwS models.
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Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , N-Metiltransferasa de Histona-Lisina/efectos de los fármacos , Proteína de la Leucemia Mieloide-Linfoide/efectos de los fármacos , Proteínas Proto-Oncogénicas/efectos de los fármacos , Sarcoma de Ewing/tratamiento farmacológico , Animales , Antineoplásicos/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Femenino , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Pediatría , Proteínas Proto-Oncogénicas/metabolismo , Sarcoma de Ewing/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Entinostat, a selective class I histone deacetylase inhibitor, has been reported to enhance the activity of cytotoxic agents and suppress expression of PAX3-FOXO1 in alveolar rhabdomyosarcoma (ARMS). PROCEDURES: Entinostat was tested against three rhabdomyosarcoma cell lines using 96-hour drug exposure. Entinostat alone or in binary combination with vincristine, actinomycin D or cyclophosphamide was tested in ARMS and two embryonal rhabdomyosarcoma (ERMS) xenograft models. Tumor growth was measured at weekly intervals. Drug-induced changes in acetylated histone H3(K9) and entinostat pharmacokinetics were determined. RESULTS: In vitro, the IC50 concentration of entinostat ranged from 280 to 1300 nM. In vivo, entinostat significantly inhibited the growth of only Rh10 xenografts. For most studies, entinostat did not potentiate the activity of the cytotoxic agent. Exceptions included the vincristine and entinostat combination for Rh10 and the entinostat and actinomycin D combination for Rh10 and Rh18, although the effects were modest. For Rh18, the combination of entinostat with vincristine showed evidence of an antagonistic interaction compared with single-agent vincristine. Pharmacokinetic studies showed the average Cmax was 569.4 ng/mL (1.51 µM) with Tmax at 15 minutes, and total exposure (AUC0-12 h ) was 435.6 h × ng/mL. Entinostat treatment increased acetylated histone H3. CONCLUSIONS: Entinostat demonstrated modest antitumor activity in only one of four models at dose and shedule that gave drug exposures relevant to human treatment. The addition of entinostat to standard-of-care cytotoxic agents was in most instances no more effective than the cytotoxic agents used alone. Entinostat demonstrated target inhibition with increased histone 2A acetylation.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Nivel de Atención , Animales , Benzamidas/administración & dosificación , Neoplasias de la Mama/patología , Ciclofosfamida/administración & dosificación , Dactinomicina/administración & dosificación , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Piridinas/administración & dosificación , Células Tumorales Cultivadas , Vincristina/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Integrating molecularly targeted agents with cytotoxic drugs used in curative treatment of pediatric cancers is complex. An evaluation was undertaken with the ERBB3/Her3-specific antibody patritumab (P) either alone or with the ERBB1/epidermal growth factor receptor inhibitor erlotinib (E) in combination with standard cytotoxic agents, cisplatin, vincristine, and cyclophosphamide, in pediatric sarcoma xenograft models that express receptors and ligands targeted by these agents. PROCEDURES: Tumor models were selected based upon ERBB3 expression and phosphorylation, and ligand (heregulin) expression. Patritumab, E, or these agents combined was evaluated without or with concomitant cytotoxic agents using procedures developed by the Pediatric Preclinical Testing Program. RESULTS: Full doses of cytotoxic agents were tolerated when combined with P, whereas dose reductions of 25% (vincristine, cisplatin) or 50% (cyclophosphamide) were required when combined with P + E. Patritumab, E alone, or in combination did not significantly inhibit growth of any tumor model, except for Rh18 xenografts (E alone). Patritumab had no single-agent activity and marginally enhanced the activity of vincristine and cisplatin only in Ewing sarcoma ES-4. P + E did not increase the antitumor activity of vincristine or cisplatin, whereas dose-reduced cyclophosphamide was significantly less active than cyclophosphamide administered at its maximum tolerated dose when combined with P + E. CONCLUSIONS: P had no single-agent activity, although it marginally potentiated the activity of vincristine and cisplatin in one of three models studied. However, the addition of E necessitated dose reduction of each cytotoxic agent, abrogating the enhancement observed with P alone.
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Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Óseas/tratamiento farmacológico , Clorhidrato de Erlotinib/farmacología , Sarcoma de Ewing/tratamiento farmacológico , Animales , Anticuerpos Monoclonales Humanizados , Neoplasias Óseas/metabolismo , Anticuerpos ampliamente neutralizantes , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones SCID , Receptor ErbB-3/antagonistas & inhibidores , Receptor ErbB-3/metabolismo , Sarcoma de Ewing/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma. EXPERIMENTAL DESIGN: The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose 10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. RESULTS: Inhibition of MEK signaling by selumetinib suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity. CONCLUSIONS: Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae.
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Astrocitoma/genética , Astrocitoma/radioterapia , Bencimidazoles/efectos adversos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Radioterapia/efectos adversos , Animales , Western Blotting , Línea Celular Tumoral , Femenino , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones SCID , Complejos Multiproteicos/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/radioterapia , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF-1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (â¼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R-targeted antibodies tested against this model.
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Proteínas Nucleares , Sarcoma , Niño , Humanos , Factores de Transcripción , Receptor IGF Tipo 1 , Sarcoma/tratamiento farmacológico , Receptores de Somatomedina , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Proteínas de Ciclo Celular , Factores de Transcripción SOXFRESUMEN
BACKGROUND: The EWSR1/FLI1 gene fusion is the most common rearrangement leading to cell transformation in Ewing sarcoma (ES). Previous studies have indicated that expression at the cellular level is heterogeneous, and that levels of expression may oscillate, conferring different cellular characteristics. In ES the role of EWSR1/FLI1 in regulating subpopulation dynamics is currently unknown. METHODS: We used siRNA to transiently suppress EWSR1/FLI1 expression and followed population dynamics using both single cell expression profiling, CyTOF and functional assays to define characteristics of exponentially growing ES cells and of ES cells in which EWSR1/FLI1 had been downregulated. Novel transcriptional states with distinct features were assigned using random forest feature selection in combination with machine learning. Cells isolated from ES xenografts in immune-deficient mice were interrogated to determine whether characteristics of specific subpopulations of cells in vitro could be identified. Stem-like characteristics were assessed by primary and secondary spheroid formation in vitro, and invasion/motility was determined for each identified subpopulation. Autophagy was determined by expression profiling, cell sorting and immunohistochemical staining. RESULTS: We defined a workflow to study EWSR1/FLI1 driven transcriptional states and phenotypes. We tracked EWSR1/FLI1 dependent proliferative activity over time to discover sources of intra-tumoral diversity. Single-cell RNA profiling was used to compare expression profiles in exponentially growing populations (si-Control) or in two dormant populations (D1, D2) in which EWSR1/FLI1 had been suppressed. Three distinct transcriptional states were uncovered contributing to ES intra-heterogeneity. Our predictive model identified ~1% cells in a dormant-like state and ~ 2-4% cells with stem-like and neural stem-like features in an exponentially proliferating ES cell line and in ES xenografts. Following EWSR1/FLI1 knockdown, cells re-entering the proliferative cycle exhibited greater stem-like properties, whereas for those cells remaining quiescent, FAM134B-dependent dormancy may provide a survival mechanism. CONCLUSIONS: We show that time-dependent changes induced by suppression of oncogenic EWSR1/FLI1 expression induces dormancy, with different subpopulation dynamics. Cells re-entering the proliferative cycle show enhanced stem-like characteristics, whereas those remaining dormant for prolonged periods appear to survive through autophagy. Cells with these characteristics identified in exponentially growing cell populations and in tumor xenografts may confer drug resistance and could potentially contribute to metastasis.
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Sarcoma de Ewing , Animales , Carcinogénesis , Línea Celular Tumoral , Regulación hacia Abajo/genética , Humanos , Ratones , Proteínas de Fusión Oncogénica/genética , ARN , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologíaRESUMEN
Novel therapeutic strategies are needed for the treatment of rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. By using a combination of cell surface proteomics and transcriptomic profiling of RMS and normal muscle, we generated a catalog of targetable cell surface proteins enriched in RMS tumors. Among the top candidates, we identified B7-H3 as the major immunoregulatory molecule expressed by RMS tumors. By using a large cohort of tissue specimens, we demonstrated that B7-H3 is expressed in a majority of RMS tumors while not detected in normal human tissues. Through a deconvolution analysis of the RMS tumor RNA-seq data, we showed that B7-H3-rich tumors are enriched in macrophages M1, NK cells, and depleted in CD8+-T cells. Furthermore, in vitro functional assays showed that B7-H3 knockout in RMS tumor cells increases T-cell mediated cytotoxicity. Altogether, our study uncovers new potential targets for the treatment of RMS and provides the first biological insights into the role of B7-H3 in RMS biology, paving the way for the development of next-generation immunotherapies.
RESUMEN
PURPOSE: Vincristine combined with camptothecin derivatives showed synergy in preclinical pediatric cancer models, and the combinations are effective in treatment of childhood solid tumors. We determined whether the synergy between vincristine and irinotecan extends to eribulin, another microtubule inhibitor. EXPERIMENTAL DESIGN: Vincristine or eribulin, alone or combined with irinotecan, was studied in 12 xenograft models. Tumor regression and time to event were used to assess antitumor activity. Pharmacodynamic studies and RNA sequencing (RNA-seq) were conducted 24 and 144 hours after single-agent or combination treatment. Effects on vascular development were studied in Matrigel plugs implanted in mice. The interaction between binary combinations was examined in vitro. RESULTS: Eribulin combined with irinotecan was more effective than vincristine-irinotecan in 6 of 12 models. Pharmacodynamic markers induced by eribulin (phospho-histone H3) and irinotecan (γ-H2A.X) were abrogated in combination-treated tumors. The predominant RNA-seq signature in combination-treated tumors was activation of the TP53 pathway with increased nuclear TP53. Massive apoptosis was observed 24 hours only after treatment with the eribulin combination. In vitro, neither combination showed interaction using combination index analysis. Eribulin alone and the combination caused alterations in developing vasculature. CONCLUSIONS: The eribulin combination is very active in these xenograft models, but not synergistic in vitro. The combination reduced pharmacodynamic markers indicative of single-agent mechanisms but in tumors, dramatically activated the TP53 pathway. Although a mechanism for in vivo synergy requires further study, it is possible that eribulin-induced inhibition of microtubule dynamics enhances irinotecan-induced nuclear accumulation of TP53, leading to rapid cell death.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Renales/tratamiento farmacológico , Rabdomiosarcoma/tratamiento farmacológico , Tumor de Wilms/tratamiento farmacológico , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Femenino , Furanos/administración & dosificación , Perfilación de la Expresión Génica , Humanos , Irinotecán/administración & dosificación , Cetonas/administración & dosificación , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Ratones SCID , Pronóstico , Rabdomiosarcoma/genética , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Células Tumorales Cultivadas , Vincristina/administración & dosificación , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Tumor de Wilms/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Alvespimycin (17-DMAG, KOS-1022), a potent small-molecule inhibitor of the protein chaperone Hsp90, is being developed as an anticancer agent because of the multiple Hsp90 client proteins involved in cancer cell growth and survival. PROCEDURES: Alvespimycin was tested against the in vitro panel of the Pediatric Preclinical Testing Program (PPTP) at concentrations from 1 nM to 10 microM and was tested against the PPTP's in vivo tumor panels by intraperitoneal administration using a 50 mg/kg BID twice weekly x 6 weeks dose and schedule. Hsp70 induction in tumor and liver tissue was used as a pharmacodynamic measure of Hsp90 inhibition and stress response induction. RESULTS: Alvespimycin had a median IC(50) of 68 nM against the PPTP's in vitro panel, with a trend for lower IC(50) values for the rhabdomyosarcoma panel (median IC(50) 32 nM) and for higher IC(50) values for the neuroblastoma panel (median IC(50) 380 nM). Using the time to event activity measure, alvespimycin had intermediate or high activity against 4 of 28 evaluable solid tumor xenografts, including 3 of 4 alveolar rhabdomyosarcoma xenografts (one with a partial response). Hsp70 induction was observed in tumor tissue from both responding and non-responding xenografts. CONCLUSIONS: Alvespimycin demonstrated little in vivo antitumor activity against most of the PPTP's preclinical models. The greatest drug effect was observed for the alveolar rhabdomyosarcoma xenografts in the rhabdomyosarcoma panel. Hsp70 induction was observed in responding and non-responding xenografts, suggesting that tumor-specific events subsequent to HSP90 inhibition are primary determinants of antitumor activity.
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Antineoplásicos/farmacocinética , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Benzoquinonas/farmacocinética , Benzoquinonas/uso terapéutico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Lactamas Macrocíclicas/farmacocinética , Lactamas Macrocíclicas/uso terapéutico , Ratones , Ratones SCID , Neoplasias Experimentales/tratamiento farmacológico , Trasplante Heterólogo , Carga Tumoral/efectos de los fármacosRESUMEN
Osteosarcoma (OS), a malignant tumor of bone, kills through aggressive metastatic spread almost exclusively to the lung. Mechanisms driving this tropism for lung tissue remain unknown, though likely invoke specific interactions between tumor cells and other cells within the lung metastatic niche. Aberrant overexpression of ΔNp63 in OS cells directly drives production of IL-6 and CXCL8. All these factors were expressed at higher levels in OS lung metastases than in matched primary tumors from the same patients. Expression in cell lines correlated strongly with lung colonization efficiency in murine xenograft models. Lentivirus-mediated expression endowed poorly metastatic OS cells with increased metastatic capacity. Disruption of IL-6 and CXCL8 signaling using genetic or pharmaceutical inhibitors had minimal effects on tumor cell proliferation in vitro or in vivo, but combination treatment inhibited metastasis across multiple models of metastatic OS. Strong interactions occurred between OS cells and both primary bronchial epithelial cells and bronchial smooth muscle cells that drove feed-forward amplification of IL-6 and CXCL8 production. These results identify IL-6 and CXCL8 as primary mediators of OS lung tropism and suggest pleiotropic, redundant mechanisms by which they might effect metastasis. Combination therapy studies demonstrate proof of concept for targeting these tumor-lung interactions to affect metastatic disease.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Óseas/patología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Neoplasias Pulmonares/patología , Adolescente , Adulto , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Huesos/patología , Línea Celular Tumoral , Proliferación Celular , Niño , Receptor gp130 de Citocinas/antagonistas & inhibidores , Receptor gp130 de Citocinas/metabolismo , Evaluación Preclínica de Medicamentos , Estudios de Seguimiento , Humanos , Hidrazinas/farmacología , Hidrazinas/uso terapéutico , Pulmón/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Masculino , Ratones , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/prevención & control , Osteosarcoma/secundario , Cultivo Primario de Células , Quinoxalinas/farmacología , Quinoxalinas/uso terapéutico , Receptores de Interleucina-8A/antagonistas & inhibidores , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
The bromodomain and extra-terminal domain inhibitor JQ1 has marked antitumor activity against several hematologic malignancies as well as solid tumor models. Here, we investigated its activity in vitro and in vivo against models of childhood rhabdomyosarcoma and Ewing sarcoma. In vitro, JQ1 (but not the inactive enantiomer JQ1R) inhibited cell proliferation and increased G1 fraction of cells, although there was no correlation between cell line sensitivity and suppression of c-MYC or MYCN. In vivo, xenografts showed significant inhibition of growth during the period of treatment, and rapid regrowth after treatment was stopped, activity typical of antiangiogenic agents. Furthermore, xenografts derived from cell lines intrinsically resistant or sensitive to JQ1 in vitro had similar sensitivity in vivo as xenografts. Further investigation showed that JQ1 reduced tumor vascularization. This was secondary to both drug-induced downregulation of tumor-derived growth factors and direct effects of JQ1 on vascular elements. JQ1 suppressed VEGF-stimulated vascularization of Matrigel plugs in mice, and in vitro suppressed differentiation, proliferation, and invasion of human umbilical cord vascular endothelial cells (HUVEC). In HUVECs, JQ1 partially suppressed c-MYC levels, but dramatically reduced AP-1 levels and activity through suppression of the AP-1-associated protein FOSL1. Our data suggest that the antitumor activity of JQ1 in these sarcoma models is largely a consequence of its antiangiogenic activity. Mol Cancer Ther; 15(5); 1018-28. ©2016 AACR.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Azepinas/farmacología , Neovascularización Patológica/metabolismo , Sarcoma/metabolismo , Sarcoma/patología , Triazoles/farmacología , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Niño , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Neovascularización Patológica/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Sarcoma/tratamiento farmacológico , Factor de Transcripción AP-1/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The BT-40 low-grade childhood astrocytoma xenograft model expresses mutated BRAF(V600E) and is highly sensitive to the MEK inhibitor selumetinib (AZD6244). In this study, we developed and characterized selumetinib resistance and explored approaches to circumventing the mechanisms of acquired resistance. EXPERIMENTAL DESIGN: BT-40 xenografts were selected in vivo for selumetinib resistance. Resistant tumors were obtained and characterized, as were tumors that reverted to sensitivity. Characterization included expression profiling, assessment of MEK signature and compensatory pathways, MEK inhibition, BRAF expression, and cytokine levels. Combination treatment of BT-40/AZD-resistant tumors with the MEK inhibitor and a STAT3 inhibitor (LLL12) was assessed. RESULTS: Resistance was unstable, tumors reverting to selumetinib sensitivity when passaged in untreated mice, and MEK was equally inhibited in sensitive and resistant tumors by selumetinib. Drug resistance was associated with an enhanced MEK signature and increased interleukin (IL)-6 and IL-8 expression. Selumetinib treatment induced phosphorylation of STAT3 (Y705) only in resistant xenografts, and similar results were observed in BRAF(V600E) astrocytic cell lines intrinsically resistant to selumetinib. Treatment of BT-40-resistant tumors with selumetinib or LLL12 had no significant effect, whereas combined treatment induced complete regressions of BT-40/AZD-resistant xenografts. CONCLUSIONS: Resistance to selumetinib selected in vivo in BT-40 tumor xenografts was unstable. In resistant tumors, selumetinib activated STAT3, and combined treatment with selumetinib and LLL12 induced complete responses in resistant BT-40 tumors. These results suggest dual targeting BRAF (V600E) signaling and STAT3 signaling may be effective in selumetinib-resistant tumors or may retard or prevent onset of resistance.
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Astrocitoma/tratamiento farmacológico , Astrocitoma/genética , Bencimidazoles/administración & dosificación , MAP Quinasa Quinasa 1/genética , Animales , Antraquinonas/farmacología , Astrocitoma/patología , Línea Celular Tumoral , Niño , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Humanos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Ratones , Proteínas Proto-Oncogénicas B-raf/genética , Factor de Transcripción STAT3/genética , Sulfonamidas/farmacologíaRESUMEN
Previously, we reported that a predominant action of a type-1 insulin-like growth factor receptor (IGF-1R)-targeted antibody was through inhibiting tumor-derived VEGF, and indirectly, angiogenesis. Here, we examined the direct antiangiogenic activity of the IGF-1R-targeted antibody SCH717454 that inhibits ligand-receptor binding and the mechanism by which tumors circumvent its antiangiogenic activity. Inhibition of ligand-stimulated activation of IGF-1R, insulin receptor (IN-R), or downstream signaling [phosphorylation of Akt (Ser473)] was determined by receptor-specific immunoprecipitation and immunoblotting. Inhibition of angiogenesis was determined by proliferation and tube formation using human umbilical vein endothelial cells (HUVEC) in vitro and in Matrigel plugs implanted in mice. SCH717454 blocked IGF-1-stimulated but not IGF-2-stimulated phosphorylation of Akt in sarcoma cells. Immunoprecipitation using anti-IGF-1R and anti-IN-R antibodies revealed that SCH717454 equally blocked IGF-1-stimulated and IGF-2-stimulated IGF-1R phosphorylation, but not IGF-2-stimulated phosphorylation of IN-R. SCH717454 completely blocked VEGF-stimulated proliferation and tube formation of HUVECs, but exogenous IGF-2 and insulin circumvented these inhibitory effects. Coculture of HUVECs with IGF-2-secreting tumor cells completely abrogated SCH717454 inhibition of VEGF-stimulated HUVEC tube formation. In mice, SCH717454 inhibited angiogenesis in VEGF-infused Matrigel plugs, but had no inhibitory activity when plugs contained both VEGF + IGF-2. These results reveal for the first time, a role for IGF-1R signaling in VEGF-mediated angiogenesis in vitro and indicate direct antiangiogenic activity of SCH717454. Both in vitro and in vivo IGF-2 circumvented these effects through IN-R signaling. Many childhood cancers secrete IGF-2, suggesting that tumor-derived IGF-2 in the microenvironment maintains angiogenesis in the presence of IGF-1R-targeted antibodies allowing tumor progression.
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Anticuerpos Monoclonales/farmacología , Vasos Sanguíneos/efectos de los fármacos , Factor II del Crecimiento Similar a la Insulina/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Inhibidores de la Angiogénesis/inmunología , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados , Secuencia de Bases , Vasos Sanguíneos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Datos de Secuencia Molecular , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/inmunología , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/antagonistas & inhibidores , Receptor de Insulina/inmunología , Receptor de Insulina/metabolismo , Sarcoma/irrigación sanguínea , Sarcoma/patología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/alpha-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/alpha-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets.
Asunto(s)
Actinas/metabolismo , Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Actinina/metabolismo , Animales , Antígenos CD/fisiología , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/fisiología , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Ratones , Ratones SCID , Metástasis de la Neoplasia , Estadificación de Neoplasias , Fenotipo , Unión Proteica , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered. METHODS AND FINDINGS: We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups). CONCLUSIONS: The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available.