Subject(s)
Dexamethasone/therapeutic use , Hematopoietic Stem Cell Transplantation , Lenalidomide/therapeutic use , Multiple Myeloma/therapy , Neoplasm, Residual/diagnosis , Oligopeptides/therapeutic use , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Female , Humans , Male , Middle Aged , Multiple Myeloma/blood , Multiple Myeloma/diagnosis , Neoplasm, Residual/blood , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transplantation, AutologousABSTRACT
The survival rate among children with relapsed neuroblastomas continues to be poor, and thus new therapeutic approaches identified by reliable preclinical drug testing models are urgently needed. Zebrafish are a powerful vertebrate model in preclinical cancer research. Here, we describe a zebrafish neuroblastoma yolk sac model to evaluate efficacy and toxicity of histone deacetylase (HDAC) inhibitor treatments. Larvae were engrafted with fluorescently labeled, genetically diverse, established cell lines and short-term cultures of patient-derived primary cells. Engrafted tumors progressed locally and disseminated remotely in an intact environment. Combination treatments involving the standard chemotherapy doxorubicin and HDAC inhibitors substantially reduced tumor volume, induced tumor cell death, and inhibited tumor cell dissemination to the tail region. Hence, this model allows for fast, cost-efficient, and reliable in vivo evaluation of toxicity and response of the primary and metastatic tumor sites to drug combinations.
ABSTRACT
In this phase 2 multicenter study, we evaluated the incorporation of autologous stem cell transplantation (ASCT) into a carfilzomib-lenalidomide-dexamethasone (KRd) regimen for patients with newly diagnosed multiple myeloma (NDMM). Transplant-eligible patients with NDMM received 4 cycles of KRd induction, ASCT, 4 cycles of KRd consolidation, and 10 cycles of KRd maintenance. The primary end point was rate of stringent complete response (sCR) after 8 cycles of KRd with a predefined threshold of ≥50% to support further study. Seventy-six patients were enrolled with a median age of 59 years (range, 40-76 years), and 35.5% had high-risk cytogenetics. The primary end point was met, with an sCR rate of 60% after 8 cycles. Depth of response improved over time. On intent-to-treat (ITT), the sCR rate reached 76%. The rate of minimal residual disease (MRD) negativity using modified ITT was 70% according to next-generation sequencing (<10-5 sensitivity). After median follow-up of 56 months, 5-year progression-free survival (PFS) and overall survival (OS) rates were 72% and 84% for ITT, 85% and 91% for MRD-negative patients, and 57% and 72% for patients with high-risk cytogenetics. For high-risk patients who were MRD negative, 5-year rates were 77% and 81%. Grade 3 to 4 adverse events included neutropenia (34%), lymphopenia (32%), infection (22%), and cardiac events (3%). There was no grade 3 to 4 peripheral neuropathy. Patients with NDMM treated with KRd with ASCT achieved high rates of sCR and MRD-negative disease at the end of KRd consolidation. Extended KRd maintenance after consolidation contributed to deepening of responses and likely to prolonged PFS and OS. Safety and tolerability were manageable. This trial was registered at www.clinicaltrials.gov as #NCT01816971.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Hematopoietic Stem Cell Transplantation , Multiple Myeloma , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Autografts , Dexamethasone/administration & dosage , Dexamethasone/adverse effects , Disease-Free Survival , Female , Humans , Lenalidomide/administration & dosage , Lenalidomide/adverse effects , Male , Middle Aged , Multiple Myeloma/diagnosis , Multiple Myeloma/mortality , Multiple Myeloma/therapy , Oligopeptides/administration & dosage , Oligopeptides/adverse effects , Progression-Free SurvivalSubject(s)
Attitude of Health Personnel , Clinical Decision-Making , Health Knowledge, Attitudes, Practice , Multiple Myeloma/pathology , Neoplasm, Residual/psychology , Physicians/psychology , Practice Patterns, Physicians' , Bone Marrow Examination/methods , Disease Progression , Flow Cytometry , Health Care Surveys , High-Throughput Nucleotide Sequencing , Humans , Multiple Myeloma/psychology , Multiple Myeloma/therapy , Neoplasm, Residual/diagnosis , Positron Emission Tomography Computed Tomography , Sensitivity and Specificity , Surveys and Questionnaires , Time FactorsABSTRACT
Selinexor, an oral Selective Inhibitor of Nuclear Export, targets Exportin 1 (XPO1, also termed CRM1). Non-clinical studies support combining selinexor with proteasome inhibitors (PIs) and corticosteroids to overcome resistance in relapsed/refractory multiple myeloma (RRMM). We conducted a phase I dose-escalation trial of twice-weekly selinexor in combination with carfilzomib and dexamethasone (SKd) to determine maximum tolerated dose in patients with RRMM (N = 21), with an expansion cohort to assess activity in carfilzomib-refractory disease and identify a recommended phase II dose (RP2D). During dose escalation, there was one dose-limiting toxicity (cardiac failure). The RP2D of twice-weekly SKd was selinexor 60 mg, carfilzomib 20/27 mg/m2 and dexamethasone 20 mg. The most common grade 3/4 treatment-emergent adverse events included thrombocytopenia (71%), anaemia (33%), lymphopenia (33%), neutropenia (33%) and infections (24%). Rates of ≥minimal response, ≥partial response and very good partial response were 71%, 48% and 14%, respectively; similar response outcomes were observed for dual-class refractory (PI and immunomodulatory drug)/quad-exposed (carfilzomib, bortezomib, lenalidomide and pomalidomide) patients (n = 17), and patients refractory to carfilzomib in last line of therapy (n = 13). Median progression-free survival was 3·7 months, and overall survival was 22·4 months in the overall population. SKd was tolerable and re-established disease control in RRMM patients, including carfilzomib-refractory patients. Registered at ClinicalTrials.gov (NCT02199665).
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Cytogenetic Analysis , Dexamethasone/administration & dosage , Drug Resistance, Neoplasm , Female , Humans , Hydrazines/administration & dosage , Male , Middle Aged , Multiple Myeloma/mortality , Neoplasm Staging , Oligopeptides/administration & dosage , Prognosis , Recurrence , Retreatment , Treatment Outcome , Triazoles/administration & dosageABSTRACT
The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3-6 µM PCI-34051 or 10 µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05 µM (ALK-amplified) to 0.8 µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n = 88) and the German Neuroblastoma Trial cohort (n = 649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.
Subject(s)
Anaplastic Lymphoma Kinase/genetics , Antineoplastic Agents/pharmacology , Neuroblastoma/drug therapy , Protein Kinase Inhibitors/pharmacology , RNA Interference , Repressor Proteins/antagonists & inhibitors , Anaplastic Lymphoma Kinase/antagonists & inhibitors , Anaplastic Lymphoma Kinase/metabolism , Animals , Cell Cycle Checkpoints/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Crizotinib/pharmacology , Drug Screening Assays, Antitumor , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/pharmacology , Indoles/pharmacology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Repressor Proteins/genetics , Repressor Proteins/metabolism , Tumor Cells, Cultured , ZebrafishABSTRACT
Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g., via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here we show that both static 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D-grown cells are substantially different from those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models more closely mimic tumor (patho-)physiology, including the upregulation of tumor relevant pathways, such as autophagy.
Subject(s)
Autophagy/physiology , Drug Resistance, Neoplasm/physiology , Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Humans , Neoplasms/drug therapyABSTRACT
Our understanding of the process of metastatic progression has improved markedly over the past decades, yet metastasis remains the most enigmatic component of cancer pathogenesis. This lack of knowledge has serious health-related implications, since metastasis is responsible for 90% of all cancer-related mortalities. The brain is considered a sanctuary site for metastatic tumor growth, where the blood-brain barrier (BBB) and other components of the brain microenvironment, provide protection to the tumor cells from immune surveillance, chemotherapeutics and other potentially harmful substances. The interactions between tumor cells and the brain microenvironment, principally brain vascular endothelium, are the critical determinants in their progression toward metastasis, dormancy, or clearance. This review discusses current knowledge of the biology of metastatic progression, with a particular focus on the tumor cell migration and colonization in the brain.
ABSTRACT
Selenium (Se) is an essential micronutrient that exerts multiple and complex effects on human health. Se is essential for human well-being largely due to its potent antioxidant, anti-inflammatory, and antiviral properties. The physiological functions of Se are carried out by selenoproteins, in which Se is specifically incorporated as the amino acid, selenocysteine. Importantly, both beneficial and toxic effects of Se have been reported suggesting that the mode of action of Se is strictly chemical form and concentration dependent. Additionally, there is a relatively narrow window between Se deficiency and toxicity and growing evidence suggests that Se health effects depend greatly on the baseline level of this micronutrient. Thus, Se supplementation is not an easy task and requires an individualized approach. It is essential that we continue to explore and better characterize Se containing compounds and mechanisms of action, which could be crucial for disease prevention and treatment.
Subject(s)
Antioxidants/metabolism , Micronutrients/metabolism , Selenium/metabolism , Selenoproteins/metabolism , Humans , Selenium Compounds/metabolism , Selenocysteine/metabolismSubject(s)
Antineoplastic Agents/adverse effects , Dasatinib/adverse effects , Lung Diseases/chemically induced , Respiratory Tract Infections/diagnosis , Diagnosis, Differential , Female , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Lung Diseases/diagnosis , Lung Diseases/microbiology , Middle AgedABSTRACT
Various dietary agents can modulate tumor invasiveness. The current study explored whether selenoglycoproteins (SeGPs) extracted from selenium-enriched yeast affect tumor cell homing and growth in the brain. Mice were fed diets enriched with specific SeGPs (SeGP40 or SeGP65, 1 mg/kg Se each), glycoproteins (GP40 or GP65, 0.2-0.3 mg/kg Se each) or a control diet (0.2-0.3 mg/kg Se) for 12 weeks. Then, murine Lewis lung carcinoma cells were infused into the brain circulation. Analyses were performed at early (48 h) and late stages (3 weeks) post tumor cell infusion. Imaging of tumor progression in the brain revealed that mice fed SeGP65-enriched diet displayed diminished metastatic tumor growth, fewer extravasating tumor cells and smaller metastatic lesions. While administration of tumor cells resulted in a significant upregulation of adhesion molecules in the early stage of tumor progression, overexpression of VCAM-1 (vascular call adhesion molecule-1) and ALCAM (activated leukocyte cell adhesion molecule) messenger RNA (mRNA) was diminished in SeGP65 supplemented mice. Additionally, mice fed SeGP65 showed decreased expression of acetylated NF-κB p65, 48 h post tumor cell infusion. The results indicate that tumor progression in the brain can be modulated by specific SeGPs. Selenium-containing compounds were more effective than their glycoprotein controls, implicating selenium as a potential negative regulator of metastatic process.
Subject(s)
Brain Neoplasms/pathology , Brain/blood supply , Brain/drug effects , Cell Adhesion Molecules/metabolism , Microvessels/drug effects , Microvessels/metabolism , Neoplasm Metastasis/drug therapy , Selenium/pharmacology , Activated-Leukocyte Cell Adhesion Molecule/genetics , Activated-Leukocyte Cell Adhesion Molecule/metabolism , Animals , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/secondary , Carcinoma, Lewis Lung/pathology , Dietary Supplements , Male , Mice , Mice, Inbred C57BL , RNA, Messenger/genetics , RNA, Messenger/metabolism , Selenium/administration & dosage , Selenium/therapeutic use , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Tumor cell extravasation into the brain requires passage through the blood-brain barrier, which is a highly protected microvascular environment fortified with tight junction (TJ) proteins. TJ integrity can be regulated under physiological and pathophysiological conditions. There is evidence that exercise can modulate oxidation status within the brain microvasculature and protect against tumor cell extravasation and metastasis formation. In order to study these events, mature male mice were given access to voluntary exercise on a running wheel (exercise) or access to a locked wheel (sedentary) for five weeks. The average running distance was 9.0 ± 0.2 km/day. Highly metastatic tumor cells (murine Lewis lung carcinoma) were then infused into the brain microvasculature through the internal carotid artery. Analyses were performed at early stage (48 h) and late stage (3 weeks) post tumor cell infusion. Immunohistochemical analysis revealed fewer isolated tumor cells extravasating into the brain at both 48 h and 3 weeks post surgery in exercised mice. Occludin protein levels were reduced in the sedentary tumor group, but maintained in the exercised tumor group at 48 h post tumor cell infusion. These results indicate that voluntary exercise may participate in modulating blood-brain barrier integrity thereby protecting the brain during metastatic progression.
Subject(s)
Blood-Brain Barrier , Brain Neoplasms/secondary , Physical Conditioning, Animal , Animals , Male , Mice , Mice, Inbred C57BL , Neoplasm MetastasisABSTRACT
Selenium-containing compounds and selenized yeast have anticancer properties. In order to address possible mechanisms involved in these effects, selenoglycoproteins (SGPs) were extracted from selenium-enriched yeast at pH 4.0 and 6.5 (the fractions are called SGP40 and SGP65, respectively), followed by evaluation of their impact on the interactions of lung and breast tumor cells with human brain microvascular endothelial cells (HBMECs). Extracted SGPs, especially SGP40, significantly inhibited adhesion of tumor cells to HBMECs and their transendothelial migration. Because the active components of SGPs are unknown, small selenium-containing compounds [leucyl-valyl-selenomethionyl-arginine (LVSe-MR) and methylselenoadenosine (M-Se-A)], which are normally present in selenized yeast, were introduced as additional treatment groups. Treatment of HBMECs with SGP40, LVSe-MR and M-Se-A induced changes in gene signatures, which suggested a central involvement of nuclear factor (NF)-κB-dependent pathway. These observations were confirmed in the subsequent analysis of NF-κB DNA binding activity, quantitative measurements of the expression of selected genes and proteins, and tumor cell adhesion assay with a specific NF-κB inhibitor as the additional treatment factor. These findings indicate that specific organic selenium-containing compounds have the ability to inhibit tumor cell adhesion to brain endothelial cells via down-regulation of NF-κB. SGPs appear to be more effective than small selenium-containing compounds, suggesting the role of not only selenium but also the glycoprotein component in the observed protective impact.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Endothelium, Vascular/drug effects , Glycoproteins/pharmacology , Lung Neoplasms/drug therapy , Saccharomyces cerevisiae Proteins/pharmacology , Selenoproteins/pharmacology , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/metabolism , Brain/blood supply , Brain/cytology , Brain/drug effects , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Adhesion/drug effects , Cell Line , Cell Line, Tumor , Endothelium, Vascular/cytology , Female , Gene Expression Regulation, Neoplastic/drug effects , Glycoproteins/biosynthesis , Glycoproteins/isolation & purification , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Microvessels/cytology , Microvessels/drug effects , NF-kappa B/agonists , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Proteins/agonists , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Organoselenium Compounds/isolation & purification , Organoselenium Compounds/metabolism , Organoselenium Compounds/pharmacology , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/isolation & purification , Selenium/metabolism , Selenomethionine/analogs & derivatives , Selenomethionine/isolation & purification , Selenomethionine/metabolism , Selenomethionine/pharmacology , Selenoproteins/biosynthesis , Selenoproteins/isolation & purification , Transendothelial and Transepithelial Migration/drug effectsABSTRACT
The treatment of newly diagnosed multiple myeloma has dramatically changed since the emergence of proteasome inhibitors and immunomodulatory drugs. Front-line combination regimens incorporating novel drugs such as thalidomide, bortezomib and lenalidomide, have significantly improved response rates and are the standard of care for induction regimens. Although the timing and role of autologous stem cell transplant are now being questioned, it remains an important part of the treatment paradigm in eligible patients. In addition, the concept of extended sequential therapy has recently emerged, including consolidation and/or maintenance in both the post-transplant setting and in nontransplant candidates. In this article we focus on management strategies in newly diagnosed multiple myeloma, including choice of induction regimens in transplant-eligible and -ineligible patients, as well as the role of autologous stem cell transplant, consolidation therapy and maintenance therapy.
ABSTRACT
The endothelial barrier strictly maintains vascular and tissue homeostasis, and therefore modulates many physiological processes such as angiogenesis, immune responses, and dynamic exchanges throughout organs. Consequently, alteration of this finely tuned function may have devastating consequences for the organism. This is particularly obvious in cancers, where a disorganized and leaky blood vessel network irrigates solid tumors. In this context, vascular permeability drives tumor-induced angiogenesis, blood flow disturbances, inflammatory cell infiltration, and tumor cell extravasation. This can directly restrain the efficacy of conventional therapies by limiting intravenous drug delivery. Indeed, for more effective anti-angiogenic therapies, it is now accepted that not only should excessive angiogenesis be alleviated, but also that the tumor vasculature needs to be normalized. Recovery of normal state vasculature requires diminishing hyperpermeability, increasing pericyte coverage, and restoring the basement membrane, to subsequently reduce hypoxia, and interstitial fluid pressure. In this review, we will introduce how vascular permeability accompanies tumor progression and, as a collateral damage, impacts on efficient drug delivery. The molecular mechanisms involved in tumor-driven vascular permeability will next be detailed, with a particular focus on the main factors produced by tumor cells, especially the emblematic vascular endothelial growth factor. Finally, new perspectives in cancer therapy will be presented, centered on the use of anti-permeability factors and normalization agents.
ABSTRACT
Metastases are the leading cause of cancer mortality and their development may be affected by diet. The aim of this study was to compare the effects of dietary supplementation with different selenium (Se) compounds on the dynamics of brain metastasis development in a novel mouse model. Mice were fed experimental diets enriched (1 mg/kg) with sodium selenite (Se-S), seleno-1-methionine (Se-Meth), a yeast-derived organic form of selenium (Se-Yeast), or a control diet (Se < 0.05 mg/kg) for 20 wk. At the end of the feeding period, animals were injected with luciferase-tagged K1735 (K1735-Luc) melanoma cells into the brain vasculature. The development of brain metastatic tumors was monitored for 2 wk following injection. Mice bearing brain metastatic tumors and fed Se-Yeast- or Se-S-enriched diets displayed a higher survival rate compared with other experimental and control groups. Importantly, Se-Yeast supplementation decreased the growth of brain metastatic tumors as determined by the measurement of the intensity of the bioluminescent signal emitted by K1735-Luc cells upon reaction with luciferin. Different chemical forms of Se have distinct effects on the development of brain metastases. Organic Se in the form of Se-Yeast may be a valuable agent in suppression of brain metastatic disease.
Subject(s)
Brain Neoplasms/diet therapy , Brain Neoplasms/pathology , Selenium/pharmacology , Yeast, Dried/pharmacology , Animals , Antineoplastic Agents/pharmacology , Body Weight/drug effects , Brain Neoplasms/mortality , Cell Movement/drug effects , Dietary Supplements , Male , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/diet therapy , Neoplasms, Experimental/pathologyABSTRACT
BACKGROUND: The endothelial specific cell-cell adhesion molecule, VE-cadherin, modulates barrier function and vascular homeostasis. In this context, we have previously characterized that VEGF (vascular endothelial growth factor) leads to VE-cadherin phosphorylation, ß-arrestin2 recruitment and VE-cadherin internalization in mouse endothelial cells. However, exactly how this VE-cadherin/ß-arrestin complex contributes to VEGF-mediated permeability in human endothelial cells remains unclear. In this study, we investigated in-depth the VE-cadherin/ß-arrestin interactions in human endothelial cells exposed to VEGF. FINDINGS: First, we demonstrated that VEGF induces VE-cadherin internalization in a clathrin-dependent manner in human umbilical vein endothelial cells (HUVEC). In addition to the classical components of endocytic vesicles, ß-arrestin1 was recruited and bound to phosphorylated VE-cadherin. Molecular mapping of this interaction uncovered that the C-terminus tail of ß-arrestin1, that comprises amino acids 375 to 418, was sufficient to directly interact with the phosphorylated form of VE-cadherin. Interestingly, the expression of the C-terminus tail of ß-arrestin1 induced loss of surface exposed-VE-cadherin, promoted monolayer disorganization and enhanced permeability. Finally, this effect relied on decreased VE-cadherin expression at the transcriptional level, through inhibition of its promoter activity. CONCLUSIONS: Altogether, our results demonstrate that ß-arrestin1 might play multiple functions collectively contributing to endothelial barrier properties. Indeed, in addition to a direct implication in VE-cadherin endocytosis, ß-arrestin1 could also control VE-cadherin transcription and expression. Ultimately, understanding the molecular mechanisms involved in VE-cadherin function might provide therapeutic tools for many human diseases where the vascular barrier is compromised.
ABSTRACT
Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis.
Subject(s)
Brain Neoplasms/metabolism , Capillary Permeability , Endothelial Cells/metabolism , Glioblastoma/metabolism , Interleukin-8/metabolism , Receptors, Interleukin-8B/metabolism , Brain Neoplasms/genetics , Capillary Permeability/drug effects , Cell Line, Tumor , Culture Media, Conditioned/pharmacology , Endothelial Cells/drug effects , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Humans , Interleukin-8/pharmacology , Receptors, Interleukin-8B/geneticsABSTRACT
DEP-1/CD148 is a receptor-like protein tyrosine phosphatase with antiproliferative and tumor-suppressive functions. Interestingly, it also positively regulates Src family kinases in hematopoietic and endothelial cells, where we showed it promotes VE-cadherin-associated Src activation and endothelial cell survival upon VEGF stimulation. However, the molecular mechanism involved and its biologic functions in endothelial cells remain ill-defined. We demonstrate here that DEP-1 is phosphorylated in a Src- and Fyn-dependent manner on Y1311 and Y1320, which bind the Src SH2 domain. This allows DEP-1-catalyzed dephosphorylation of Src inhibitory Y529 and favors the VEGF-induced phosphorylation of Src substrates VE-cadherin and Cortactin. Accordingly, RNA interference (RNAi)-mediated knockdown of DEP-1 or expression of DEP-1 Y1311F/Y1320F impairs Src-dependent biologic responses mediated by VEGF including permeability, invasion, and branching capillary formation. In addition, our work further reveals that above a threshold expression level, DEP-1 can also dephosphorylate Src Y418 and attenuate downstream signaling and biologic responses, consistent with the quiescent behavior of confluent endothelial cells that express the highest levels of endogenous DEP-1. Collectively, our findings identify the VEGF-dependent phosphorylation of DEP-1 as a novel mechanism controlling Src activation, and show this is essential for the proper regulation of permeability and the promotion of the angiogenic response.
Subject(s)
Capillaries/metabolism , Cell Membrane Permeability , Endothelium, Vascular/cytology , Neovascularization, Pathologic , Tyrosine/metabolism , src-Family Kinases/metabolism , Antigens, CD/metabolism , Blotting, Western , Cadherins/metabolism , Cell Adhesion , Cell Movement , Cell Proliferation , Cells, Cultured , Cortactin/metabolism , Endothelium, Vascular/metabolism , Fluorescent Antibody Technique , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunoprecipitation , Mutation/genetics , Neoplasm Invasiveness , Phosphorylation , Receptor-Like Protein Tyrosine Phosphatases, Class 3/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 3/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/metabolismABSTRACT
VE-cadherin-mediated cell-cell junction weakening increases paracellular permeability in response to both angiogenic and inflammatory stimuli. Although Semaphorin 3A has emerged as one of the few known anti-angiogenic factors to exhibit pro-permeability activity, little is known about how it triggers vascular leakage. Here we report that Semaphorin 3A induced VE-cadherin serine phosphorylation and internalisation, cell-cell junction destabilisation, and loss of barrier integrity in brain endothelial cells. In addition, high-grade glioma-isolated tumour-initiating cells were found to secrete Semaphorin 3A, which promoted brain endothelial monolayer permeability. From a mechanistic standpoint, Semaphorin 3A impinged upon the basal activity of the serine phosphatase PP2A and disrupted PP2A interaction with VE-cadherin, leading to cell-cell junction disorganization and increased permeability. Accordingly, both pharmacological inhibition and siRNA-based knockdown of PP2A mimicked Semaphorin 3A effects on VE-cadherin. Hence, local Semaphorin 3A production impacts on the PP2A/VE-cadherin equilibrium and contributes to elevated vascular permeability.