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Given that, even after multimodal therapy, early-stage lung cancer (LC) often recurs, novel prognostic markers to help guide therapy are highly desired. The mRNA levels of cell division cycle 25C (CDC25C), a phosphatase that regulates G2/M cell cycle transition in malignant cells, correlate with poor clinical outcomes in lung adenocarcinoma (LUAD). However, whether CDC25C protein detected by immunohistochemistry can serve as a prognostic marker in LUAD is yet unknown. We stained an LC tissue array and a cohort of 61 LUAD tissue sections for CDC25C and searched for correlations between CDC25C staining score and the pathological characteristics of the tumors and the patients' clinical outcomes. Clinical data were retrieved from our prospectively maintained departmental database. We found that high expression of CDC25C was predominant among poorly differentiated LUAD (p < 0.001) and in LUAD > 1cm (p < 0.05). Further, high expression of CDC25C was associated with reduced disease-free survival (p = 0.03, median follow-up of 39 months) and with a trend for reduced overall survival (p = 0.08). Therefore, high expression of CDC25C protein in LUAD is associated with aggressive histological features and with poor outcomes. Larger studies are required to further validate CDC25C as a prognostic marker in LUAD.
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BACKGROUND: We assessed the mechanism by which multiple myeloma (MM) shapes the bone marrow (BM) microenvironment and affects MΦ polarization. METHODS: In vivo xenograft model of BM-disseminated human myeloma, as well as analysis of MM cell lines, stromal components, and primary samples from patients with MM, was utilized. RESULTS: Analysis of the BM from MM-bearing mice inoculated with human CXCR4-expressing RPMI8226 cells revealed a significant increase in M2 MΦ cell numbers (p < 0.01). CXCL13 was one of the most profoundly increased factors upon MM growth with increased levels in the blood of MM-bearing animals. Myeloid cells were the main source of the increased murine CXCL13 detected in MM-infiltrated BM. MM cell lines induced CXCL13 and concurrent expression of M2 markers (MERTK, CD206, CD163) in co-cultured human MΦ in vitro. Interaction with MΦ reciprocally induced CXCL13 expression in MM cell lines. Mechanistically, TGFß signaling was involved in CXCL13 induction in MM cells, while BTK signaling was implicated in MM-stimulated increase of CXCL13 in MΦ. Recombinant CXCL13 increased RANKL expression and induced TRAP+ osteoclast (OC) formation in vitro, while CXCL13 neutralization blocked these activities. Moreover, mice inoculated with CXCL13-silenced MM cells developed significantly lower BM disease. Reduced tumor load correlated with decreased numbers of M2 MΦ in BM, decreased bone disease, and lower expression of OC-associated genes. Finally, higher levels of CXCL13 were detected in the blood and BM samples of MM patients in comparison with healthy individuals. CONCLUSIONS: Altogether, our findings suggest that bidirectional interactions of MΦ with MM tumor cells result in M2 MΦ polarization, CXCL13 induction, and subsequent OC activation, enhancing their ability to support bone resorption and MM progression. CXCL13 may thus serve as a potential novel target in MM.
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Quimiocina CXCL13 , Macrófagos , Mieloma Múltiple , Animales , Quimiocina CXCL13/metabolismo , Humanos , Macrófagos/metabolismo , Ratones , Mieloma Múltiple/patología , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral , Tirosina Quinasa c-Mer/metabolismoRESUMEN
BACKGROUND: CXCR4 mediates the retention and survival of acute myelogenous leukemia blasts in bone marrow and contributes to their resistance to chemotherapy. The authors evaluated a combination of the high-affinity CXCR4 antagonist BL-8040 with high-dose cytarabine (HiDAC) chemotherapy in a phase 2a study of patients with relapsed and refractory AML. METHODS: Forty-two patients received treatment with BL-8040 monotherapy for 2 days followed by a combination of BL-8040 with HiDAC for 5 days. Six escalating BL-8040 dose levels were investigated (0.5, 0.75, 1.0, 1.25, 1.5, and 2.0 mg/kg), and 1.5 mg/kg was selected as the dose for the expansion phase (n = 23). RESULTS: BL-8040 in combination with HiDAC was safe and well tolerated at all dose levels. Clinical response was observed with BL-8040 doses ≥1.0 mg/kg. The composite response rate (complete remissions plus complete remissions with incomplete hematologic recovery of platelets or neutrophils) was 29% (12 of 42) in all patients and 39% (9 of 23) in the 1.5-mg/kg phase. The median overall survival was 8.4 months for all patients, 10.8 months in the 1.5-mg/kg phase, and 21.8 months for responding patients in the 1.5-mg/kg cohort. Two days of BL-8040 monotherapy triggered the mobilization of blasts into peripheral blood, with significantly higher mean fold-changes in responders versus nonresponders. This was accompanied by a decrease in bone marrow blasts. CONCLUSIONS: The current results demonstrate the efficacy of CXCR4 targeting with BL-8040 and support continued clinical development in acute myelogenous leukemia.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Citarabina/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Péptidos/administración & dosificación , Receptores CXCR4/antagonistas & inhibidores , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Células de la Médula Ósea/efectos de los fármacos , Esquema de Medicación , Resistencia a Antineoplásicos , Femenino , Movilización de Célula Madre Hematopoyética , Humanos , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Péptidos/efectos adversos , Péptidos/farmacocinética , Recurrencia , Inducción de RemisiónRESUMEN
Graft versus host disease (GvHD) remains a limiting factor for successful hematopoietic stem cell transplantation (HSCT). T cells and antigen-presenting cells (APCs) are major components of the hematopoietic G-CSF mobilized peripheral blood cell (MPBC) graft. Here we show that a short incubation (2 h) of MPBCs with hexameric Fas ligand (FasL) selectively induces apoptosis of specific donor T cell subsets and APCs but not of CD34+ cells. FasL treatment preferentially induces apoptosis in mature T cell subsets which express high levels of Fas (CD95), such as T stem cell memory, T central memory, and T effector memory cells, as well as TH1 and TH17 cells. Anti-CD3/CD28 stimulated T cells derived from FasL-treated-MPBCs express lower levels of CD25 and secrete lower levels of IFN-γ as compared to control cells not treated with FasL. FasL treatment also induces apoptosis of transitional, naïve, memory and plasmablastoid B cells leading to a reduction in their numbers in the graft and following engraftment in transplanted mice. Most importantly, ex vivo treatment of MPBCs with FasL prior to transplant in conditioned NOD-scid IL2Rγnull (NSG) mice prevented GvHD while preserving graft versus leukemia (GvL) effects, and leading to robust stem cell engraftment.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Animales , Proteína Ligando Fas , Enfermedad Injerto contra Huésped/prevención & control , Ligandos , Ratones , Ratones Endogámicos NOD , Células MadreRESUMEN
BACKGROUND: Age-related macular degeneration (AMD), the most common cause of blindness in the developed world, usually affects individuals older than 60 years of age. The majority of visual loss in this disease is attributable to the development of choroidal neovascularization (CNV). Mononuclear phagocytes, including monocytes and their tissue descendants, macrophages, have long been implicated in the pathogenesis of neovascular AMD (nvAMD). Current therapies for nvAMD are based on targeting vascular endothelial growth factor (VEGF). This study is aimed at assessing if perturbation of chemokine signaling and mononuclear cell recruitment may serve as novel complementary therapeutic targets for nvAMD. METHODS: A promiscuous chemokine antagonist (BKT130), aflibercept treatment, or combined BKT130+aflibercept treatment was tested in an in vivo laser-induced model of choroidal neovascularization (LI-CNV) and in an ex vivo choroidal sprouting assay (CSA). Quantification of CD11b+ cell in the CNV area was performed, and mRNA levels of genes implicated in CNV growth were measured in the retina and RPE-choroid. RESULTS: BKT130 reduced the CNV area and recruitment of CD11b+ cells by 30-35%. No effect of BKT130 on macrophages' proangiogenic phenotype was demonstrated ex vivo, but a lower VEGFA and CCR2 expression was found in the RPE-choroid and a lower expression of TNFα and NOS1 was found in both RPE-choroid and retinal tissues in the LI-CNV model under treatment with BKT130. CONCLUSIONS: Targeting monocyte recruitment via perturbation of chemokine signaling can reduce the size of experimental CNV and should be evaluated as a potential novel therapeutic modality for nvAMD.
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Quimiocinas/antagonistas & inhibidores , Neovascularización Coroidal/tratamiento farmacológico , Monocitos/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Animales , Antígeno CD11b/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocinas/metabolismo , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Femenino , Humanos , Rayos Láser , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Monocitos/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ratas , Ratas Long-Evans , Receptores CCR2/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Retina/metabolismo , Retina/patología , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Although having promising anti-myeloma properties, the pan-histone deacetylase inhibitor (HDACi) panobinostat lacks therapeutic activity as a single agent. The aim of the current study was to elucidate the mechanisms underlying multiple myeloma (MM) resistance to panobinostat monotherapy and to define strategies to overcome it. Sensitivity of MM cell lines and primary CD138+ cells from MM patients to panobinostat correlated with reduced expression of the chemokine receptor CXCR4, whereas overexpression of CXCR4 in MM cell lines increased their resistance to panobinostat. Decreased sensitivity to HDACi was associated with reversible G0/G1 cell growth arrest while response was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators revealed the pro-survival mTOR pathway to be regulated by CXCR4 overexpression. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance to HDACi and induced synergistic cell death. The combination of panobinostat/everolimus resulted in sustained DNA damage and irreversible suppression of proliferation accompanied by robust apoptosis. Gene expression analysis revealed distinct genetic profiles of single versus combined agent exposure. Whereas panobinostat increased the expression of the cell cycle inhibitor p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated the expression of DNA repair genes and mitotic checkpoint regulators. Importantly, the combination of panobinostat with everolimus effectively targeted CXCR4-expressing resistant MM cells in vivo in the BM niche. In summary, our results uncover the mechanism responsible for the strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel potential therapeutic approach to treat MM.
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Antineoplásicos/administración & dosificación , Everolimus/administración & dosificación , Inhibidores de Histona Desacetilasas/administración & dosificación , Mitosis/efectos de los fármacos , Panobinostat/administración & dosificación , Receptores CXCR4/antagonistas & inhibidores , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Células Cultivadas , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Humanos , Ratones , Mitosis/fisiología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/biosíntesis , Serina-Treonina Quinasas TOR/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Proteínas de Unión al GTP rho/biosíntesis , Proteínas de Unión al GTP rho/genéticaRESUMEN
The chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1/CXCL12) are important players in the cross-talk among lymphoma, myeloma and leukemia cells and their microenvironments. In hematological malignancies and solid tumors, the overexpression of CXCR4 on the cell surface has been shown to be responsible for disease progression, increasing tumor cell survival and chemoresistance and metastasis to organs with high CXCL12 levels (e.g., lymph nodes and bone marrow (BM)). Furthermore, the overexpression of CXCR4 has been found to have prognostic significance for disease progression in many type of tumors including lymphoma, leukemia, glioma, and prostate, breast, colorectal, renal, and hepatocellular carcinomas. In leukemia, CXCR4 expression granted leukemic blasts a higher capacity to seed into BM niches, thereby protecting leukemic cells from chemotherapy-induced apoptosis, and was correlated with shorter disease-free survival. In contrast, neutralizing the interaction of CXCL12/CXCR4 with a variety of antagonists induced apoptosis and differentiation and increased the chemosensitivity of lymphoma, myeloma, and leukemia cells. The role of CXCL12 and CXCR4 in the pathogenesis of hematological malignancies and the clinical therapeutic potential of CXCR4 antagonists in these diseases is discussed.
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Quimiocina CXCL12/metabolismo , Neoplasias Hematológicas/patología , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Mieloide Aguda/patología , Mieloma Múltiple/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores CXCR4/metabolismo , Apoptosis/inmunología , Supervivencia Celular/fisiología , Progresión de la Enfermedad , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Pronóstico , Microambiente Tumoral/fisiologíaRESUMEN
OBJECTIVE: Past studies are inconsistent with regard to the role of matrix metalloproteinase 12 in lung tumorigenesis. This is due, in part, to differential tumorigenesis based on tumor-derived versus immune-derived matrix metalloproteinase 12 expression. Our study aims to thoroughly dissect the role of matrix metalloproteinase 12 in lung tumorigenesis. METHODS: We tested matrix metalloproteinase 12 expression and the association with prognosis using a tissue array and a published non-small cell lung cancer gene expression database. In addition, we characterized the contribution of matrix metalloproteinase 12 to tumor propagation in the lung using a series of in vitro and in vivo studies. RESULTS: Tumor cells of a diverse set of human lung cancers stained positive for matrix metalloproteinase 12, and high matrix metalloproteinase 12 mRNA levels in the tumor were associated with reduced survival. The lung microenvironment stimulated endogenous production of matrix metalloproteinase 12 in lung cancer cells (human 460 lung cancer cell line, Lewis lung carcinoma). In vitro, matrix metalloproteinase 12 knockout Lewis lung carcinoma and Lewis lung carcinoma cells had the same proliferation rate, but Lewis lung carcinoma showed increased invasiveness. In vivo, deficiency of matrix metalloproteinase 12 in Lewis lung carcinoma cells, but not in the host, reduced tumor growth and invasiveness. CONCLUSIONS: We suggest that tumor cell-derived matrix metalloproteinase 12 promotes tumor propagation in the lung and that in the context of pulmonary malignancies matrix metalloproteinase 12 should further be tested as a potential novel therapeutic target.
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Biomarcadores de Tumor/metabolismo , Carcinoma Pulmonar de Lewis/enzimología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Movimiento Celular , Neoplasias Pulmonares/enzimología , Metaloproteinasa 12 de la Matriz/metabolismo , Animales , Biomarcadores de Tumor/genética , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Metaloproteinasa 12 de la Matriz/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica , Transducción de SeñalRESUMEN
CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.Significance: These results provide a mechanistic rationale for combination therapy of CXCR4 and BCL-2 inhibitors to treat a common and commonly aggressive pediatric cancer.Cancer Res; 78(6); 1471-83. ©2017 AACR.
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Neoplasias Encefálicas/patología , MicroARNs/metabolismo , Neuroblastoma/patología , Receptores CXCR4/metabolismo , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , MicroARNs/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chemokines and their receptors play critical roles in the progression of autoimmunity and inflammation. Typically, multiple chemokines are involved in the development of these pathologies. Indeed, targeting single chemokines or chemokine receptors has failed to achieve significant clinical benefits in treating autoimmunity and inflammation. Moreover, the binding of host atypical chemokine receptors to multiple chemokines as well as the binding of chemokine-binding proteins secreted by various pathogens can serve as a strategy for controlling inflammation. In this work, promiscuous chemokine-binding peptides that could bind and inhibit multiple inflammatory chemokines, such as CCL2, CCL5, and CXCL9/10/11, were selected from phage display libraries. These peptides were cloned into human mutated immunoglobulin Fc-protein fusions (peptibodies). The peptibodies BKT120Fc and BKT130Fc inhibited the ability of inflammatory chemokines to induce the adhesion and migration of immune cells. Furthermore, BKT120Fc and BKT130Fc also showed a significant inhibition of disease progression in a variety of animal models for autoimmunity and inflammation. Developing a novel class of antagonists that can control the courses of diseases by selectively blocking multiple chemokines could be a novel way of generating effective therapeutics.
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Th-17 type immune response that occurs in multiple sclerosis (MS) is linked to CCR6-CCL20 interaction. We confirmed the dependency on CCR6 in EAE development. Vaccination of mice with hCCL20, but not mCCL20, produced anti-murine CCL20 and ameliorated EAE. The EAE clinical score negatively correlated with anti CCL20 levels. A beneficial effect was transferred by sera from hCCL20-immunized mice. Immunized mice with cyclic peptide that include a bacterial outer membrane protein A (ompA), that share homology sequence with hCCL20 produced anti CCL20, anti ompA and anti-cyclic peptide. Immunization of mice with ompA or the cyclic peptide ameliorated EAE. The cyclic peptide inhibited CCL20 activity in an adhesion assay. A significantly higher level of anti CCL20 were found in healthy individuals compared to RR-MS patients. There was no similar difference for anti-CXCL10. Natural or induced immunization against CCL20 confer protection against EAE and may be beneficial in MS.
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Quimiocina CCL20/inmunología , Encefalitis/prevención & control , Enfermedad de Hashimoto/prevención & control , Esclerosis Múltiple/prevención & control , Animales , Adhesión Celular , Quimiocina CCL20/administración & dosificación , Biología Computacional , Femenino , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores CCR6/genética , Receptores CCR6/metabolismo , Linfocitos TRESUMEN
Purpose: The potential of the high-affinity CXCR4 antagonist BL-8040 as a monotherapy-mobilizing agent and its derived graft composition and quality were evaluated in a phase I clinical study in healthy volunteers (NCT02073019).Experimental Design: The first part of the study was a randomized, double-blind, placebo-controlled dose escalation phase. The second part of the study was an open-label phase, in which 8 subjects received a single injection of BL-8040 (1 mg/kg) and approximately 4 hours later underwent a standard leukapheresis procedure. The engraftment potential of the purified mobilized CD34+ cells was further evaluated by transplanting the cells into NSG immunodeficient mice.Results: BL-8040 was found safe and well tolerated at all doses tested (0.5-1 mg/kg). The main treatment-related adverse events were mild to moderate. Transient injection site and systemic reactions were mitigated by methylprednisolone, paracetamol, and promethazine pretreatment. In the first part of the study, BL-8040 triggered rapid and substantial mobilization of WBCs and CD34+ cells in all tested doses. Four hours postdose, the count rose to a mean of 8, 37, 31, and 35 cells/µL (placebo, 0.5, 0.75, and 1 mg/kg, respectively). FACS analysis revealed substantial mobilization of immature dendritic, T, B, and NK cells. In the second part, the mean CD34+ cells/kg collected were 11.6 × 106 cells/kg. The graft composition was rich in immune cells.Conclusions: The current data demonstrate that BL-8040 is a safe and effective monotherapy strategy for the collection of large amounts of CD34+ cells and immune cells in a one-day procedure for allogeneic HSPC transplantation. Clin Cancer Res; 23(22); 6790-801. ©2017 AACR.
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Antígenos CD34/metabolismo , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Péptidos/administración & dosificación , Receptores CXCR4/antagonistas & inhibidores , Animales , Biomarcadores , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Voluntarios Sanos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Leucaféresis , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Animales , Trasplante HomólogoRESUMEN
Purpose: To explore the functional consequences of possible cross-talk between the CXCR4/CXCL12 and the sphingosine-1-phosphate (S1P) pathways in multiple myeloma (MM) cells and to evaluate the effect of S1P targeting with the FTY720 modulator as a potential anti-MM therapeutic strategy.Experimental Design and Results: S1P targeting with FTY720 induces MM cell apoptosis. The combination of FTY720 with the SPHK1 inhibitor SKI-II results in synergistic inhibition of MM growth. CXCR4/CXCL12-enhanced expression correlates with reduced MM cell sensitivity to both FTY720 and SKI-II inhibitors, and with SPHK1 coexpression in both cell lines and primary MM bone marrow (BM) samples, suggesting regulative cross-talk between the CXCR4/CXCL12 and SPHK1 pathways in MM cells. FTY720 was found to directly target CXCR4. FTY720 profoundly reduces CXCR4 cell-surface levels and abrogates the CXCR4-mediated functions of migration toward CXCL12 and signaling pathway activation. Moreover, FTY720 cooperates with bortezomib, inducing its cytotoxic activity and abrogating the bortezomib-mediated increase in CXCR4 expression. FTY720 effectively targets bortezomib-resistant cells and increases their sensitivity to bortezomib, promoting DNA damage. Finally, in a recently developed novel xenograft model of CXCR4-dependent systemic MM with BM involvement, FTY720 treatment effectively reduces tumor burden in the BM of MM-bearing mice. FTY720 in combination with bortezomib demonstrates superior tumor growth inhibition and abrogates bortezomib-induced CXCR4 increase on MM cells.Conclusions: Altogether, our work identifies a cross-talk between the S1P and CXCR4 pathways in MM cells and provides a preclinical rationale for the therapeutic application of FTY720 in combination with bortezomib in patients with MM. Clin Cancer Res; 23(7); 1733-47. ©2016 AACR.
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Quimiocina CXCL12/genética , Mieloma Múltiple/tratamiento farmacológico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptores CXCR4/genética , Animales , Apoptosis/efectos de los fármacos , Bortezomib/administración & dosificación , Daño del ADN/efectos de los fármacos , Sinergismo Farmacológico , Clorhidrato de Fingolimod/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lisofosfolípidos/genética , Lisofosfolípidos/metabolismo , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/genética , Esfingosina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The existing shortage of animal models that properly mimic the progression of early-stage human lung cancer from a solitary confined tumor to an invasive metastatic disease hinders accurate characterization of key interactions between lung cancer cells and their stroma. We herein describe a novel orthotopic animal model that addresses these concerns and consequently serves as an attractive platform to study tumor-stromal cell interactions under conditions that reflect early-stage lung cancer. METHODS: Unlike previous methodologies, we directly injected small numbers of human or murine lung cancer cells into murine's left lung and longitudinally monitored disease progression. Next, we used green fluorescent protein-tagged tumor cells and immuno-fluorescent staining to determine the tumor's microanatomic distribution and to look for tumor-infiltrating immune cells and stromal cells. Finally, we compared chemokine gene expression patterns in the tumor and lung microenvironment. RESULTS: We successfully generated a solitary pulmonary nodule surrounded by normal lung parenchyma that grew locally and spread distally over time. Notably, we found that both fibroblasts and leukocytes are recruited to the tumor's margins and that distinct myeloid cell attracting and CCR2-binding chemokines are specifically induced in the tumor microenvironment. CONCLUSION: Our orthotopic lung cancer model closely mimics the pathologic sequence of events that characterizes early-stage human lung cancer propagation. It further introduces new means to monitor tumor-stromal cell interactions and offers unique opportunities to test therapeutic targets under conditions that reflect early-stage lung cancer. We argue that for such purposes our model is superior to lung cancer models that are based either on genetic induction of epithelial transformation or on ectopic transplantation of malignant cells.
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Carcinoma Pulmonar de Lewis/patología , Carcinoma Pulmonar de Lewis/terapia , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Modelos Animales de Enfermedad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Trasplante Heterólogo , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multiple myeloma (MM) cells specifically attract peripheral-blood monocytes, while interaction of MM with bone marrow stromal cells (BMSCs) significantly increased monocyte recruitment (p<0.01). The CXCL12 chemokine, produced by both the MM and BMSCs, was found to be a critical regulator of monocyte migration. CXCL12 production was up-regulated under MM-BMSCs co-culture conditions, whereas blockage with anti-CXCR4 antibodies significantly abrogated monocyte recruitment toward a MM-derived conditioned medium (p<0.01). Furthermore, elevated levels of CXCL12 were detected in MM, but not in normal BM samples, whereas malignant MM cells often represented the source of increased CXCL12 in the BM. Blood-derived macrophages effectively supported MM cells proliferation and protected them from chemotherapy-induced apoptosis. Importantly, MM cells affected macrophage polarization, elevating the expression of M2-related scavenger receptor CD206 in macrophages and blocking LPS-induced TNFα secretion (a hallmark of M1 response). Of note, MM-educated macrophages suppressed T-cell proliferation and IFNγ production in response to activation. Finally, increased numbers of CXCR4-expressing CD163+CD206+ macrophages were detected in the BM of MM patients (n=25) in comparison to MGUS (n=11) and normal specimens (n=8). Taken together, these results identify macrophages as important players in MM tumorogenicity, and recognize the CXCR4/CXCL12 axis as a critical regulator of MM-stroma interactions and microenvironment formation.
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Quimiocina CXCL12/metabolismo , Macrófagos/patología , Mieloma Múltiple/patología , Receptores CXCR4/metabolismo , Adulto , Anciano , Apoptosis/fisiología , Línea Celular Tumoral , Polaridad Celular/fisiología , Proliferación Celular/fisiología , Femenino , Humanos , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Mieloma Múltiple/metabolismo , Fenotipo , Transducción de SeñalRESUMEN
Functional role of CXCR4 in chronic myelogenous leukemia (CML) progression was evaluated. Elevated CXCR4 significantly increased the in vitro survival and proliferation in response to CXCL12. CXCR4 stimulation resulted in activation of extracellular signal-regulated kinase (Erk)-1/2, Akt, S6K, STAT3, and STAT5 prosurvival signaling pathways. In accordance, we found that in vitro treatment with CXCR4 antagonist BKT140 directly inhibited the cell growth and induced cell death of CML cells. Combination of BKT140 with suboptimal concentrations of imatinib significantly increased the anti-CML effect. BKT140 induced apoptotic cell death, decreasing the levels of HSP70 and HSP90 chaperones and antiapoptotic proteins BCL-2 and BCL-XL, subsequently promoting the release of mitochondrial factors cytochrome c and SMAC/Diablo. Bone marrow (BM) stromal cells (BMSC) markedly increased the proliferation of CML cells and protected them from imatinib-induced apoptosis. Furthermore, BMSCs elevated proto-oncogene BCL6 expression in the CML cells in response to imatinib treatment, suggesting the possible role of BCL6 in stroma-mediated TKI resistance. BKT140 reversed the protective effect of the stroma, effectively promoted apoptosis, and decreased BCL6 levels in CML cells cocultured with BMSCs. BKT140 administration in vivo effectively reduced the growth of subcutaneous K562-produced xenografts. Moreover, the combination of BKT140 with low-dose imatinib markedly inhibited tumor growth, achieving 95% suppression. Taken together, our data indicate the importance of CXCR4/CXCL12 axis in CML growth and CML-BM stroma interaction. CXCR4 inhibition with BKT140 antagonist efficiently cooperated with imatinib in vitro and in vivo. These results provide the rational basis for CXCR4-targeted therapy in combination with TKI to override drug resistance and suppress residual disease.
Asunto(s)
Benzamidas/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Oligopéptidos/farmacología , Piperazinas/farmacología , Pirimidinas/farmacología , Receptores CXCR4/antagonistas & inhibidores , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Expresión Génica , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proto-Oncogenes Mas , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: CXCR4 plays an important role in the retention of stem cells within the bone marrow. BKT140 (4F-benzoyl-TN14003) is a 14-residue bio stable synthetic peptide, which binds CXCR4 with a greater affinity compared with plerixafor (4 vs. 84 nmol/L). Studies in mice demonstrated the efficient and superior mobilization and transplantation of stem cells collected with GCSF-BKT140, compared with those obtained when using stem cells obtained with each one of these mobilizing agent alone. These results have served as a platform for the present clinical phase I study. EXPERIMENTAL DESIGN: Eighteen patients with multiple myeloma who were preparing for their first autologous stem cell transplantation were included. Patients received a standard multiple myeloma mobilization regimen, consisting of 3 to 4 g/m(2) cyclophosphamide (day 0), followed by granulocyte colony-stimulating factor (G-CSF) at 5 µg/kg/d starting on day 5 and administered between 8 and 10 pm until the end of stem cell collection. A single injection of BKT140 (0.006, 0.03, 0.1, 0.3, and 0.9 mg/kg) was administered subcutaneously on day 10 in the early morning, followed by G-CSF 12 hours later. RESULTS: BKT140 was well tolerated at all concentrations, and none of the patients developed grade 3 and 4 toxicity. A single administration of BKT140 at the highest dose, 0.9 mg/kg, resulted in a robust mobilization and collection of CD34(+) cells (20.6 ± 6.9 × 10(6)/kg), which were obtained through a single apheresis. All transplanted patients received â¼5.3 × 10(6) CD34(+) cells/kg, which rapidly engrafted (n = 17). The median time to neutrophil and platelet recovery was 12 and 14 days, respectively, at the highest dose (0.9 mg/kg). CONCLUSIONS: When combined with G-CSF, BKT140 is a safe and efficient stem cell mobilizer that enabled the collection of a high number of CD34(+) cells in 1 and 2 aphaeresis procedures, resulting in successful engraftment.
Asunto(s)
Antineoplásicos/farmacología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Mieloma Múltiple/metabolismo , Mieloma Múltiple/terapia , Oligopéptidos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Adulto , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Recuento de Células Sanguíneas , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Factor Estimulante de Colonias de Granulocitos/farmacología , Trasplante de Células Madre Hematopoyéticas , Humanos , Masculino , Ratones , Persona de Mediana Edad , Oligopéptidos/administración & dosificación , Oligopéptidos/efectos adversos , Unión Proteica , Receptores CXCR4/metabolismo , Resultado del TratamientoRESUMEN
Platelets are the terminal differentiation product of megakaryocytes (MKs). Cytokines, such as thrombopoietin (TPO), are known to influence different steps in MK development; however, the complex differentiation and platelet localization processes are not fully understood. MKs express the receptor CXCR4 and have been shown to migrate in response to CXCL12 and to increase their platelet production. In this study, we studied the role of CXCR4 in platelet production with the high affinity CXCR4 antagonist, BKT140. Single and sequential administration of BKT140 significantly increased the number of MKs and haematopoietic progenitors (HPCs) within the bone marrow (BM). Increased megakaryopoiesis was associated with increased platelet production. Single and sequential administration of BKT140 also increased the number of HPCs in the blood. In a model of 5-fluorouracil-induced thrombocytopenia, BKT140 significantly reduced the severity and duration of thrombocytopenia and cytopenia when administered before and after chemotherapy. Our results demonstrated that the CXCR4 antagonist, BKT140, mediated unique beneficial effects by stimulating megakaryopoiesis and platelet production. These results provide evidence for the possible therapeutic use of BKT140 for modulating platelet numbers in thrombocytopenic conditions.
Asunto(s)
Plaquetas/citología , Plaquetas/efectos de los fármacos , Oligopéptidos/administración & dosificación , Receptores CXCR4/antagonistas & inhibidores , Trombopoyesis/efectos de los fármacos , Trombopoyesis/fisiología , Animales , Antineoplásicos/efectos adversos , Plaquetas/metabolismo , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Recuento de Leucocitos , Células Progenitoras de Megacariocitos/citología , Células Progenitoras de Megacariocitos/efectos de los fármacos , Ratones , Recuento de Plaquetas , Trombocitopenia/inducido químicamente , Trombocitopenia/tratamiento farmacológico , Trombopoyetina/farmacologíaRESUMEN
PURPOSE: Chemokine axis CXCR4/CXCL12 is critically involved in the survival and trafficking of normal and malignant B lymphocytes. Here, we investigated the effect of high-affinity CXCR4 antagonist BKT140 on lymphoma cell growth and rituximab-induced cytotoxicity in vitro and in vivo. EXPERIMENTAL DESIGN: In vitro efficacy of BKT140 alone or in combination with rituximab was determined in non-Hodgkin lymphoma (NHL) cell lines and primary samples from bone marrow aspirates of patients with NHL. In vivo efficacy was evaluated in xenograft models of localized and disseminated NHL with bone marrow involvement. RESULTS: Antagonizing CXCR4 with BKT140 resulted in significant inhibition of CD20+ lymphoma cell growth and in the induction of cell death, respectively. Combination of BKT140 with rituximab significantly enhanced the apoptosis against the lymphoma cells in a dose-dependent manner. Moreover, rituximab induced CXCR4 expression in lymphoma cell lines and primary lymphoma cells, suggesting the possible interaction between CD20 and CXCR4 pathways in NHL. Primary bone marrow stromal cells (BMSC) further increased CXCR4 expression and protected NHL cells from rituximab-induced apoptosis, whereas BKT140 abrogated this protective effect. Furthermore, BKT140 showed efficient antilymphoma activity in vivo in the xenograft model of disseminated NHL with bone marrow involvement. BKT140 treatment inhibited the local tumor progression and significantly reduced the number of NHL cells in the bone marrow. Combined treatment of BKT140 with rituximab further decreased the number of viable lymphoma cells in the bone marrow, achieving 93% reduction. CONCLUSIONS: These findings suggest the possible role of CXCR4 in NHL progression and response to rituximab and provide the scientific basis for the development of novel CXCR4-targeted therapies for refractory NHL.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Antígenos CD20/metabolismo , Antineoplásicos/farmacología , Linfoma no Hodgkin/metabolismo , Oligopéptidos/farmacología , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Humanos , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/patología , Ratones , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: Human hepatocellular carcinoma (HCC) is an inflammation-induced cancer, which is the third-leading cause of cancer mortality worldwide. We investigated the role of the chemokine receptors, CCR5 and CCR1, in regulating inflammation and tumorigenesis in an inflammation-induced HCC model in mice. Multidrug resistance 2 gene (Mdr2)-knockout (Mdr2-KO) mice spontaneously develop chronic cholestatic hepatitis and fibrosis that is eventually followed by HCC. We generated two new strains from the Mdr2-KO mouse, the Mdr2:CCR5 and the Mdr2:CCR1 double knockouts (DKOs), and set out to compare inflammation and tumorigenesis among these strains. We found that in Mdr2-KO mice lacking the chemokine receptor, CCR5 (Mdr2:CCR5 DKO mice), but not CCR1 (Mdr2:CCR1 DKO), macrophage recruitment and trafficking to the liver was significantly reduced. Furthermore, in the absence of CCR5, reduced inflammation was also associated with reduced periductal accumulation of CD24(+) oval cells and abrogation of fibrosis. DKO mice for Mdr2 and CCR5 exhibited a significant decrease in tumor incidence and size. CONCLUSIONS: Our results indicate that CCR5 has a critical role in both the development and progression of liver cancer. Therefore, we propose that a CCR5 antagonist can serve for HCC cancer prevention and treatment.