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BACKGROUND: CC-90011 is an oral, potent, selective, reversible inhibitor of lysine-specific demethylase 1 (LSD1) that was well tolerated, with encouraging activity in patients who had advanced solid tumors or relapsed/refractory marginal zone lymphoma. The authors present long-term safety and efficacy and novel pharmacodynamic and pharmacokinetic data from the first-in-human study of CC-90011. METHODS: CC-90011-ST-001 (ClincalTrials.gov identifier NCT02875223; Eudract number 2015-005243-13) is a phase 1, multicenter study in which patients received CC-90011 once per week in 28-day cycles. The objectives were to determine the safety, maximum tolerated dose, and/or recommended phase 2 dose (primary) and to evaluate preliminary efficacy and pharmacokinetics (secondary). RESULTS: Sixty-nine patients were enrolled, including 50 in the dose-escalation arm and 19 in the dose-expansion arm. Thrombocytopenia was the most common treatment-related adverse event and was successfully managed with dose modifications. Clinical activity with prolonged, durable responses were observed, particularly in patients who had neuroendocrine neoplasms. In the dose-escalation arm, one patient with relapsed/refractory marginal zone lymphoma achieved a complete response (ongoing in cycle 58). In the dose-expansion arm, three patients with neuroendocrine neoplasms had stable disease after nine or more cycles, including one patient who was in cycle 46 of ongoing treatment. CC-90011 decreased levels of secreted neuroendocrine peptides chromogranin A, progastrin-releasing peptide, and RNA expression of the blood pharmacodynamic marker monocyte-to-macrophage differentiation-associated. CONCLUSIONS: The safety profile of CC-90011 suggested that its reversible mechanism of action may provide an advantage over other irreversible LSD1 inhibitors. The favorable tolerability profile, clinical activity, durable responses, and once-per-week dosing support further exploration of CC-90011 as monotherapy and in combination with other treatments for patients with advanced solid tumors and other malignancies.
Assuntos
Linfoma de Zona Marginal Tipo Células B , Neoplasias , Histona Desmetilases , Humanos , Dose Máxima Tolerável , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Compostos OrgânicosRESUMO
Inhibition of B-cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) provides significant clinical benefit to patients, mainly by blocking adhesion of CLL cells in the lymph node microenvironment. The currently applied inhibitors ibrutinib and idelalisib have limited capacity however to induce cell death as monotherapy and are unlikely to eradicate the disease. Acquired resistance to therapy in CLL is often caused by mutations in the response network being targeted, both for DNA damage or BCR signaling pathways. Thus, drugs with dual targeting capacity could offer improved therapeutic value. Here, the potency of CC-115, a novel inhibitor of mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK), was evaluated in primary CLL cells in vitro and in CLL patients. Combined TORK and DNA-PK inhibition in vitro resulted in caspase-dependent cell killing irrespective of p53, ATM, NOTCH1, or SF3B1 status. Proliferation induced by CD40(+) interleukin-21 stimulation was completely blocked by CC-115, and CD40-mediated resistance to fludarabine and venetoclax could be reverted by CC-115. BCR-mediated signaling was inhibited by CC-115 and also in CLL samples obtained from patients with acquired resistance to idelalisib treatment. Clinical efficacy of CC-115 was demonstrated in 8 patients with relapsed/refractory CLL/small lymphocytic lymphoma harboring ATM deletions/mutations; all but 1 patient had a decrease in lymphadenopathy, resulting in 1 IWCLL partial response (PR) and 3 PRs with lymphocytosis. In conclusion, these preclinical results, along with early promising clinical activity, suggest that CC-115 may be developed further for treatment of CLL. The trial was registered at www.clinicaltrials.gov as #NCT01353625.
Assuntos
Proteína Quinase Ativada por DNA/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Pirazinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína Quinase Ativada por DNA/metabolismo , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Purinas/farmacologia , Quinazolinonas/farmacologia , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Early data suggested that CC-115, a clinical molecule, already known to inhibit the mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK) may have additional targets beyond TORK and DNA-PK. Therefore, we aimed to identify such target(s) and investigate a potential therapeutic applicability. Functional profiling of 141 cancer cell lines revealed inhibition of kinase suppressor of morphogenesis in genitalia 1 (SMG1), a key regulator of the RNA degradation mechanism nonsense-mediated mRNA decay (NMD), as an additional target of CC-115. CC-115 treatment showed a dose-dependent increase of SMG1-mediated NMD transcripts. A subset of cell lines, including multiple myeloma (MM) cell lines sensitive to the endoplasmic reticulum stress-inducing compound thapsigargin, were highly susceptible to SMG1 inhibition. CC-115 caused the induction of UPR transcripts and cell death by mitochondrial apoptosis, requiring the presence of BAX/BAK and caspase activity. Superior antitumor activity of CC-115 over TORK inhibitors in primary human MM cells and three xenograft mouse models appeared to be via inhibition of SMG1. Our data support further development of SMG1 inhibitors as possible therapeutics in MM.
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Mieloma Múltiplo , Degradação do RNAm Mediada por Códon sem Sentido , Animais , Humanos , Camundongos , Linhagem Celular , DNA/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
PURPOSE: Lysine-specific demethylase 1 (LSD1) is implicated in multiple tumor types, and its expression in cancer stem cells is associated with chemoresistance. CC-90011 is a potent, selective, and reversible oral LSD1 inhibitor. We examined CC-90011 in advanced solid tumors and relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: CC-90011-ST-001 (NCT02875223; 2015-005243-13) is a phase I, multicenter, first-in-human dose-escalation study. Nine dose levels of CC-90011 (1.25-120 mg) given once per week were explored. Primary objectives were to determine safety, maximum tolerated dose (MTD), and/or recommended phase II dose (RP2D). Secondary objectives were to evaluate preliminary efficacy and pharmacokinetics. RESULTS: Fifty patients were enrolled, 49 with solid tumors (27 neuroendocrine tumors/carcinomas) and 1 with R/R NHL. Median age was 61 years (range, 22-75). Patients received a median of three (range, 1-9) prior anticancer regimens. The RP2D was 60 mg once per week; the nontolerated dose (NTD) and MTD were 120 mg once per week and 80 mg once per week, respectively. Grade 3/4 treatment-related toxicities were thrombocytopenia (20%; an on-target effect unassociated with clinically significant bleeding), neutropenia (8%; in the context of thrombocytopenia at the highest doses), and fatigue (2%). The patient with R/R NHL had a complete response, currently ongoing in cycle 34, and 8 patients with neuroendocrine tumors/carcinomas had stable disease ≥6 months, including bronchial neuroendocrine tumors, kidney tumor, and paraganglioma. CONCLUSIONS: CC-90011 is well tolerated, with the RP2D established as 60 mg once per week. The MTD and NTD were determined to be 80 mg once per week and 120 mg once per week, respectively. Further evaluation of CC-90011 is warranted.
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Linfoma não Hodgkin/tratamento farmacológico , Neoplasias/tratamento farmacológico , Compostos Orgânicos/uso terapêutico , Adulto , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Área Sob a Curva , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Fadiga/induzido quimicamente , Feminino , Humanos , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Neoplasias/patologia , Compostos Orgânicos/efeitos adversos , Compostos Orgânicos/farmacocinética , Trombocitopenia/induzido quimicamente , Resultado do Tratamento , Adulto JovemRESUMO
Whole-body MRI combined with a semiautomated hierarchical multispectral image analysis technique was evaluated as a method for detecting viable tumor tissue in a murine model of metastatic breast cancer (4T1 cell line). Whole-body apparent diffusion coefficient, T(2), and proton density maps were acquired in this study. The viable tumor tissue segmentation included three-stage k-means clustering of the parametric maps, morphologic operations, application of a size threshold, and reader discrimination of the segmented objects. The segmentation results were validated by histologic evaluation, and the detection accuracy of the technique was evaluated at three size thresholds (15, 100, and 500 voxels). The accuracy was 88.9% for a 500-voxel size threshold, and the area under receiver operating characteristic curve was 0.84. The regions of segmented viable tumor tissue within the primary tumors were found mostly on the periphery of the tumors in agreement with the histologic findings. The presented technique was found capable of detecting metastases and segmenting the viable tumor from necrotic regions within tumors found in this model. It offers a noninvasive, whole-body, viable tumor tissue detection method for preclinical and potentially clinical applications such as tumor screening and evaluating therapeutic efficacy.
Assuntos
Algoritmos , Inteligência Artificial , Neoplasias da Mama/diagnóstico , Interpretação de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Reconhecimento Automatizado de Padrão/métodos , Imagem Corporal Total/métodos , Animais , Neoplasias da Mama/secundário , Linhagem Celular Tumoral , Aumento da Imagem/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Second-generation mammalian target of rapamycin (mTOR) inhibitors such as CC-223 may have theoretical advantages over first-generation drugs by inhibiting TOR kinase in mTOR complex 1 (mTORC1) and 2 (mTORC2), potentially improving clinical efficacy for well-differentiated neuroendocrine tumors (NET).Enrolled patients had metastatic, well-differentiated NET of non-pancreatic gastrointestinal or unknown origin, with/without carcinoid symptoms, had failed ≥1 systemic chemotherapy, and were taking a somatostatin analog (SSA). Oral once-daily CC-223 was administered in 28-day cycles starting at 45 mg (n = 24), with a subsequent cohort starting at 30 mg (n = 23). Objectives were to evaluate tolerability, preliminary efficacy, and pharmacokinetic and biomarker profiles of CC-223. Forty-seven patients completed the study, with mean treatment duration of 378 days and mean dose of 26 mg; 26% of patients remained on the starting dose. Most frequent grade ≥3 toxicities were diarrhea (38%), fatigue (21%), and stomatitis (11%). By investigator, 3 of 41 evaluable patients (7%) showed partial response (PR) and 34 (83%) had stable disease (SD) for a disease control rate (DCR) of 90% (95% confidence interval [CI] 76.9-97.3%). Duration of PR was 125-401 days; median SD duration was 297 days (min-max, 50-1519 days). Median progression-free survival was 19.5 months (95% CI 10.4-28.5 months). Carcinoid symptoms of flushing, diarrhea, or both improved in 50%, 41%, and 39% of affected patients, respectively. For the first time, this study describes that a second-generation mTOR pathway inhibitor can result in highly durable tumor regression and control of NET carcinoid symptoms. The manageable safety profile, high DCR, and durable response, coupled with reduction in carcinoid symptoms refractory to SSA, make CC-223 a promising agent for further development.
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Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Tumores Neuroendócrinos/tratamento farmacológico , Pirazinas/administração & dosagem , Administração Oral , Adulto , Idoso , Tumor Carcinoide/sangue , Tumor Carcinoide/tratamento farmacológico , Estudos de Coortes , Feminino , Neoplasias Gastrointestinais/sangue , Neoplasias Gastrointestinais/tratamento farmacológico , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Tumores Neuroendócrinos/sangue , Pirazinas/efeitos adversos , Pirazinas/sangueRESUMO
As a critical signaling node, ERK1/2 are attractive drug targets, particularly in tumors driven by activation of the MAPK pathway. Utility of targeting the MAPK pathway has been demonstrated by clinical responses to inhibitors of MEK1/2 or RAF kinases in some mutant BRAF-activated malignancies. Unlike tumors with mutations in BRAF, those with mutations in KRAS (>30% of all cancers and >90% of certain cancer types) are generally not responsive to inhibitors of MEK1/2 or RAF. Here, a covalent ERK1/2 inhibitor, CC-90003, was characterized and shown to be active in preclinical models of KRAS-mutant tumors. A unique occupancy assay was used to understand the mechanism of resistance in a KRAS-mutant patient-derived xenograft (PDX) model of colorectal cancer. Finally, combination of CC-90003 with docetaxel achieved full tumor regression and prevented tumor regrowth after cessation of treatment in a PDX model of lung cancer. This effect corresponded to changes in a stemness gene network, revealing a potential effect on tumor stem cell reprograming. IMPLICATIONS: Here, a covalent ERK1/2 inhibitor (CC-90003) is demonstrated to have preclinical efficacy in models of KRAS-mutant tumors, which present a therapeutic challenge for currently available therapies.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , MutaçãoRESUMO
PURPOSE: This first-in-human Phase I study investigated the safety, pharmacokinetics (PK), pharmacodynamic profile, and preliminary efficacy of CC-115, a dual inhibitor of mammalian target of rapamycin (mTOR) kinase and DNA-dependent protein kinase. PATIENTS AND METHODS: Patients with advanced solid or hematologic malignancies were enrolled in dose-finding and cohort expansion phases. In dose-finding, once-daily or twice-daily (BID) ascending oral doses of CC-115 (range: 0.5-40 mg/day) in 28-day continuous cycles identified the maximum-tolerated dose for cohort expansion in 5 specified tumor types. Twelve additional patients with mixed solid tumors participated in a bioavailability substudy. RESULTS: Forty-four patients were enrolled in the dose-finding cohort. Dose-limiting toxicity included thrombocytopenia, stomatitis, hyperglycemia, asthenia/fatigue, and increased transaminases. CC-115 10 mg BID was selected for cohort expansion (n=74) in which fatigue, nausea, and decreased appetite were the most frequent toxicities. Dose-proportional PK was found. CC-115 distributed to glioblastoma tissue (mean tumor/plasma concentration ratio: 0.713). Total exposure of CC-115 was similar under fasting and fed conditions. A patient with endometrial carcinoma remained in complete remission >4 years. Partial response (PR; n=2) and stable disease (SD; n=4) were reported in the bioavailability substudy; SD was reached in 53%, 22%, 21%, and 64% of patients with head and neck squamous cell carcinoma, Ewing sarcoma, glioblastoma multiforme, and castration-resistant prostate cancer, respectively. Chronic lymphocytic leukemia/small lymphocytic lymphoma showed 38% PR and 25% SD. CONCLUSION: CC-115 was well-tolerated, with toxicities consistent with mTOR inhibitors. Together with biomarker inhibition and preliminary efficacy, oral CC-115 10 mg BID is a promising novel anticancer treatment. CLINICAL TRIAL REGISTRATION: NCT01353625.
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PURPOSE: DNA-dependent protein kinase catalytic subunit (DNA-PK) is a pleiotropic kinase involved in DNA repair and transcriptional regulation. DNA-PK is deregulated in selected cancer types and is strongly associated with poor outcome. The underlying mechanisms by which DNA-PK promotes aggressive tumor phenotypes are not well understood. Here, unbiased molecular investigation in clinically relevant tumor models reveals novel functions of DNA-PK in cancer.Experimental Design: DNA-PK function was modulated using both genetic and pharmacologic methods in a series of in vitro models, in vivo xenografts, and patient-derived explants (PDE), and the impact on the downstream signaling and cellular cancer phenotypes was discerned. Data obtained were used to develop novel strategies for combinatorial targeting of DNA-PK and hormone signaling pathways. RESULTS: Key findings reveal that (i) DNA-PK regulates tumor cell proliferation; (ii) pharmacologic targeting of DNA-PK suppresses tumor growth both in vitro, in vivo, and ex vivo; (iii) DNA-PK transcriptionally regulates the known DNA-PK-mediated functions as well as novel cancer-related pathways that promote tumor growth; (iv) dual targeting of DNA-PK/TOR kinase (TORK) transcriptionally upregulates androgen signaling, which can be mitigated using the androgen receptor (AR) antagonist enzalutamide; (v) cotargeting AR and DNA-PK/TORK leads to the expansion of antitumor effects, uncovering the modulation of novel, highly relevant protumorigenic cancer pathways; and (viii) cotargeting DNA-PK/TORK and AR has cooperative growth inhibitory effects in vitro and in vivo. CONCLUSIONS: These findings uncovered novel DNA-PK transcriptional regulatory functions and led to the development of a combinatorial therapeutic strategy for patients with advanced prostate cancer, currently being tested in the clinical setting.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Neoplasias/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Androgênicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Protein kinases are known to play a prominent role in oncogenic progression across multiple cancer subtypes, yet their role in prostate cancer progression remains underexplored. The purpose of this study was to identify kinases that drive prostate cancer progression.Experimental Design: To discover kinases that drive prostate cancer progression, we investigated the association between gene expression of all known kinases and long-term clinical outcomes in tumor samples from 545 patients with high-risk disease. We evaluated the impact of genetic and pharmacologic inhibition of the most significant kinase associated with metastatic progression in vitro and in vivo. RESULTS: DNA-dependent protein kinase (DNAPK) was identified as the most significant kinase associated with metastatic progression in high-risk prostate cancer. Inhibition of DNAPK suppressed the growth of both AR-dependent and AR-independent prostate cancer cells. Gene set enrichment analysis nominated Wnt as the top pathway associated with DNAPK. We found that DNAPK interacts with the Wnt transcription factor LEF1 and is critical for LEF1-mediated transcription. CONCLUSIONS: Our data show that DNAPK drives prostate cancer progression through transcriptional regulation of Wnt signaling and is an attractive therapeutic target in aggressive prostate cancer.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Transcrição Gênica , Via de Sinalização Wnt , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/genética , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Masculino , Camundongos , Metástase Neoplásica , Fenótipo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
The Drosophila Fused (Fu) kinase is an integral component of the Hedgehog (Hh) pathway that helps promote Hh-dependent gene transcription. Vertebrate homologues of Fu function in the Hh pathway in vitro, suggesting that Fu is evolutionarily conserved. We have generated fused (stk36) knockout mice to address the in vivo function of the mouse Fu (mFu) homologue. fused knockouts develop normally, being born in Mendelian ratios, but fail to thrive within 2 weeks, displaying profound growth retardation with communicating hydrocephalus and early mortality. The fused gene is expressed highly in ependymal cells and the choroid plexus, tissues involved in the production and circulation of cerebral spinal fluid (CSF), suggesting that loss of mFu disrupts CSF homeostasis. Similarly, fused is highly expressed in the nasal epithelium, where fused knockouts display bilateral suppurative rhinitis. No obvious defects were observed in the development of organs where Hh signaling is required (limbs, face, bones, etc.). Specification of neuronal cell fates by Hh in the neural tube was normal in fused knockouts, and induction of Hh target genes in numerous tissues is not affected by the loss of mFu. Furthermore, stimulation of fused knockout cerebellar granule cells to proliferate with Sonic Hh revealed no defect in Hh signal transmission. These results show that the mFu homologue is not required for Hh signaling during embryonic development but is required for proper postnatal development, possibly by regulating the CSF homeostasis or ciliary function.
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Líquido Cefalorraquidiano/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hidrocefalia/etiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Animais , Proteína Axina , Linhagem da Célula , Proliferação de Células , Relação Dose-Resposta a Droga , Genes Reporter , Genótipo , Heterozigoto , Hidrocefalia/genética , Hidrocefalia/metabolismo , Hibridização In Situ , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Modelos Genéticos , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rinite/genética , Transdução de Sinais , Fatores de Tempo , Distribuição Tecidual , Transcrição Gênica , beta-Galactosidase/metabolismoRESUMO
UNLABELLED: The role of angiogenesis during mechanically induced bone formation is incompletely understood. The relationship between the mechanical environment, angiogenesis, and bone formation was determined in a rat distraction osteogenesis model. Disruption of either the mechanical environment or endothelial cell proliferation blocked angiogenesis and bone formation. This study further defines the role of the mechanical environment and angiogenesis during distraction osteogenesis. INTRODUCTION: Whereas successful fracture repair requires a coordinated and complex transcriptional program that integrates mechanotransductive signaling, angiogenesis, and osteogenesis, the interdependence of these processes is not fully understood. In this study, we use a system of bony regeneration known as mandibular distraction osteogenesis (DO) in which a controlled mechanical stimulus promotes bone induction after an osteotomy and gradual separation of the osteotomy edges to examine the relationship between the mechanical environment, angiogenesis, and osteogenesis. MATERIALS AND METHODS: Adult Sprague-Dawley rats were treated with gradual distraction, gradual distraction plus the angiogenic inhibitor TNP-470, or acute distraction (a model of failed bony regeneration). Animals were killed at the end of distraction (day 13) or at the end of consolidation (day 41) and examined with muCT, histology, and immunohistochemistry for angiogenesis and bone formation (n = 4 per time-point per group). An additional group of animals (n = 6 per time-point per group) was processed for microarray analysis at days 5, 9, 13, 21, and 41. RESULTS AND CONCLUSIONS: Either TNP-470 administration or disruption of the mechanical environment prevented normal osteogenesis and resulted in a fibrous nonunion. Subsequent analysis of the regenerate showed an absence of angiogenesis by gross histology and immunohistochemical localization of platelet endothelial cell adhesion molecule in the groups that failed to heal. Microarray analysis revealed distinct patterns of expression of genes associated with osteogenesis, angiogenesis, and hypoxia in each of the three groups. Our findings confirm the interdependence of the mechanical environment, angiogenesis, and osteogenesis during DO, and suggest that induction of proangiogenic genes and the proper mechanical environment are both necessary to support new vasculature for bone induction in DO.
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Regeneração Óssea/fisiologia , Osso e Ossos/irrigação sanguínea , Neovascularização Fisiológica , Osteogênese por Distração , Inibidores da Angiogênese/farmacologia , Animais , Regeneração Óssea/genética , Osso e Ossos/citologia , Cicloexanos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Perfilação da Expressão Gênica , Masculino , Mandíbula/irrigação sanguínea , Mandíbula/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , O-(Cloroacetilcarbamoil)fumagilol , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sesquiterpenos/farmacologia , Veias Umbilicais/citologiaRESUMO
A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.
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Antineoplásicos/química , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Linfoma/tratamento farmacológico , Neoplasias/tratamento farmacológico , Animais , Biomarcadores , Linhagem Celular Tumoral , Ciclofosfamida/análogos & derivados , Ciclofosfamida/química , Cães , Doxorrubicina/química , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Prednisolona/química , Serina-Treonina Quinases TOR/metabolismo , Vincristina/químicaRESUMO
The intimate connection, both physical and biochemical, between blood vessels and bone cells has long been recognized. Genetic, biochemical, and pharmacological studies have identified and characterized factors involved in the conversation between endothelial cells (EC) and osteoblasts (OB) during both bone formation and repair. The long-awaited FDA approval of two growth factors, BMP-2 and OP-1, with angiogenic and osteogenic activity confirms the importance of these two processes in human skeletal healing. In this review, the role of osteogenic factors in the adaptive response and interactive function of OB and EC during the multi-step process of bone repair will be discussed.
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Osso e Ossos/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas Morfogenéticas Ósseas/uso terapêutico , Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/fisiologia , Comunicação Celular , Células Endoteliais/fisiologia , Consolidação da Fratura/efeitos dos fármacos , Humanos , Neovascularização Fisiológica/fisiologia , Osteoblastos/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Fish stanniocalcin (STC) inhibits uptake of calcium and stimulates phosphate reabsorption. To determine the role of the highly homologous mammalian protein, STC-1, we created and characterized transgenic mice that express STC-1 under control of a muscle-specific promoter. STC-1 transgenic mice were smaller than wild-type littermates and had normal growth plate cartilage morphology but increased cartilage matrix synthesis. In STC-1 mice, the rate of bone formation, but not bone mineralization, was decreased. Increased cortical bone thickness and changes in trabeculae number, density, and thickness in STC-1 mice indicated a concomitant suppression of osteoclast activity, which was supported by microcomputed tomography analyses and histochemistry. Skeletal muscles were disproportionately small and showed altered function and response to injury in STC-1 mice. Electron microscopy indicated that muscle mitochondria were dramatically enlarged in STC-1 mice. These changes in STC-1 mice could not be explained by deficits in blood vessel formation, as vascularity in organs and skeletal tissues was increased as was induction of vascularity in response to femoral artery ligation. Our results indicate that STC-1 can affect calcium homeostasis, bone and muscle mass and structure, and angiogenesis through effects on osteoblasts, osteoclasts, myoblasts/myocytes, and endothelial cells.
Assuntos
Osso e Ossos/anatomia & histologia , Osso e Ossos/fisiologia , Glicoproteínas/fisiologia , Hormônios/fisiologia , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/fisiologia , Animais , Composição Corporal , Constituição Corporal , Densidade Óssea , Desenvolvimento Ósseo , Matriz Óssea/metabolismo , Calcificação Fisiológica , Cálcio/sangue , Cartilagem/metabolismo , Feminino , Expressão Gênica , Glicoproteínas/genética , Crescimento/genética , Lâmina de Crescimento/anatomia & histologia , Hormônios/genética , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Neovascularização Fisiológica , Osteoclastos/fisiologia , Crânio/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
While the histological and ultrastructural changes associated with distraction osteogenesis have been extensively characterized using various animal models, the molecular mechanisms governing this technique remain poorly understood. In the current study, for the first time, we describe a mouse mandibular distraction osteogenesis model. Development of this model will allow assessment of factors involved in normal vs. abnormal healing (especially in non-unions) of craniofacial skeletal elements. Complete osteotomies were created on the right hemimandibles of 51 adult male CD-1 mice and customized distraction devices attached. Thirty-three animals underwent gradual distraction (5 days latency, distraction at 0.2 mm BID x 8 days, 28 days consolidation), while the remaining 18 mice underwent acute lengthening (immediate distraction to 3.2 mm) at the time of surgery. Mandibles were harvested at time points corresponding to the latent (POD 5), distraction (POD 9, 13), and consolidation (POD 28, 41) periods and processed for histological or quantitative real-time RT-PCR analysis. Specimens from each group were processed for microCT analysis. Histological and radiological data demonstrated that all mandibles undergoing gradual distraction achieved complete bony union by the end of consolidation, while those undergoing acute lengthening formed a fibrous non-union. Quantitative real-time RT-PCR demonstrated upregulation of mRNA for VEGF, FGF-2, collagen I, and osteopontin during gradual distraction but not during acute lengthening. These data validate our novel mouse mandibular distraction model and demonstrate its utility in elucidating the molecular mechanisms regulating bone formation during distraction osteogenesis as compared to those that are expressed during the formation of fibrous non-unions.
Assuntos
Mandíbula/metabolismo , Mandíbula/ultraestrutura , Modelos Animais , Osteogênese por Distração/métodos , Animais , Colágeno Tipo I/biossíntese , Masculino , Camundongos , Osteogênese/fisiologia , Osteogênese por Distração/instrumentação , Osteopontina , Sialoglicoproteínas/biossínteseRESUMO
Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.
Assuntos
Metástase Neoplásica/prevenção & controle , Neoplasias/patologia , Neuropilina-2/antagonistas & inibidores , Animais , Anticorpos Bloqueadores/farmacologia , Especificidade de Anticorpos/efeitos dos fármacos , Bacteriófagos , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/secundário , Linfonodos/patologia , Linfangiogênese/efeitos dos fármacos , Metástase Linfática/prevenção & controle , Sistema Linfático/efeitos dos fármacos , Sistema Linfático/patologia , Camundongos , Neuropilina-2/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The characteristic toughness and strength of bone result from the nature of bone matrix, the mineralized extracellular matrix produced by osteoblasts. The mechanical properties and composition of bone matrix, along with bone mass and architecture, are critical determinants of a bone's ability to resist fracture. Several regulators of bone mass and architecture have been identified, but factors that regulate the mechanical properties and composition of bone matrix are largely unknown. We used a combination of high-resolution approaches, including atomic-force microscopy, x-ray tomography, and Raman microspectroscopy, to assess the properties of bone matrix independently of bone mass and architecture. Properties were evaluated in genetically modified mice with differing levels of TGF-beta signaling. Bone matrix properties correlated with the level of TGF-beta signaling. Smad3+/- mice had increased bone mass and matrix properties, suggesting that the osteopenic Smad3-/- phenotype may be, in part, secondary to systemic effects of Smad3 deletion. Thus, a reduction in TGF-beta signaling, through its effector Smad3, enhanced the mechanical properties and mineral concentration of the bone matrix, as well as the bone mass, enabling the bone to better resist fracture. Our results provide evidence that bone matrix properties are controlled by growth factor signaling.
Assuntos
Osso e Ossos/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologia , Animais , Densidade Óssea , Matriz Óssea , Matriz Extracelular/metabolismo , Consolidação da Fratura , Fraturas Ósseas/patologia , Deleção de Genes , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Força Atômica , Osteoblastos/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Análise Espectral Raman , Estresse Mecânico , Tomografia por Raios X , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: To investigate the role of nitric oxide (NO) in basal and cytokine induced cartilage matrix breakdown and synthesis across different species and in chondrocytes cultured as isolated cells or as tissue explants. METHODS: Articular cartilage from bovine, porcine, or human joints was cultured as explants in serum-free media. Explants or monolayer cultures of primary chondrocytes were treated with cytokines in the absence or presence of inhibitors [antibodies to leukemia inhibitory factor (anti-LIF) or tumor necrosis factor-alpha, dexamethasone, or inhibitors of aggrecanase or NO synthase]. NO production and matrix breakdown and synthesis were measured. RESULTS: At low concentrations, a novel interleukin 17 (IL-17) family member induced matrix breakdown without altering NO production. Treatment of articular cartilage explants with dexamethasone or anti-LIF blocked NO production by IL-17, but not by IL-1alpha. Inhibition of NO production in cytokine treated cartilage explants enhanced matrix breakdown and partially overcame suppression of matrix synthesis. In isolated chondrocytes, inhibition of NO production decreased expression of gelatinase and increased expression of stromelysin. CONCLUSION: Endogenous NO serves a dual function in cartilage: to protect the tissue from matrix breakdown and to mediate suppression of proteoglycan synthesis by cytokines. Despite the similarities in biological function between IL-I and IL-17, their downstream signaling pathways are distinct and appear to be affected by extracellular matrix degradation.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Interleucina-17/farmacologia , Óxido Nítrico/biossíntese , Idoso , Idoso de 80 Anos ou mais , Animais , Artrite Reumatoide/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Bovinos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Meios de Cultivo Condicionados/metabolismo , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/metabolismo , Feminino , Gelatinases/metabolismo , Humanos , Interleucina-17/antagonistas & inibidores , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Proteoglicanas/metabolismo , SuínosRESUMO
Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing. To investigate the role of WISP-1 in osteogenic processes, we characterized its tissue and cellular expression and evaluated its activity in osteoblastic and chondrocytic cell culture models. During embryonic development, WISP-1 expression was restricted to osteoblasts and to osteoblastic progenitor cells of the perichondral mesenchyme. In vitro, we showed that WISP-1 expression in differentiating osteoblasts promotes BMP-2-induced osteoblastic differentiation. Using in situ and cell binding analysis, we demonstrated WISP-1 interaction with perichondral mesenchyme and undifferentiated chondrocytes. We evaluated the effect of WISP-1 on chondrocytes by generating stably transfected mouse chondrocytic cell lines. In these cells, WISP-1 increased proliferation and saturation density but repressed chondrocytic differentiation. Because of the similarity between skeletogenesis and bone healing, we also analyzed WISP-1 spatiotemporal expression in a fracture repair model. We found that WISP-1 expression recapitulates the pattern observed during skeletal development. Our data demonstrate that WISP-1 is an osteogenic potentiating factor promoting mesenchymal cell proliferation and osteoblastic differentiation while repressing chondrocytic differentiation. Therefore, we propose that WISP-1 plays an important regulatory role during bone development and fracture repair.