RESUMEN
High-grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle-tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re-establishing endothelial barrier integrity. LIGHT-mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK-LIGHT with anti-vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T-cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide-binding activities in mouse and human primary brain tumour vessels. Thus, peptide-mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos , Glioblastoma/tratamiento farmacológico , Neovascularización Patológica , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/farmacología , Remodelación Vascular/efectos de los fármacos , Vénulas/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/metabolismo , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular , Composición de Medicamentos , Femenino , Glioblastoma/sangre , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Micelas , Persona de Mediana Edad , Terapia Molecular Dirigida , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Pericitos/patología , Fenotipo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Vénulas/metabolismo , Vénulas/patologíaRESUMEN
BACKGROUND: Epidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma. METHODS: Expression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown. RESULTS: EREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α. CONCLUSION: EREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the growth factor through ErbB1.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Endorribonucleasas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Glioma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Antracenos/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Comunicación Autocrina , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Cetuximab , Factor de Crecimiento Epidérmico/genética , Epirregulina , Expresión Génica , Glioma/tratamiento farmacológico , Glioma/patología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1alpha correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1beta, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness.
Asunto(s)
Endorribonucleasas/metabolismo , Glioma/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Experimentales/metabolismo , Neovascularización Patológica/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/metabolismo , Membrana Corioalantoides/patología , Endorribonucleasas/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/sangre , Glioma/patología , Humanos , Inmunohistoquímica , Interleucina-6/genética , Interleucina-6/metabolismo , Estimación de Kaplan-Meier , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Microscopía Fluorescente , Microscopía por Video , Invasividad Neoplásica , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Neovascularización Patológica/patología , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
Combined therapy with anti-BRAF plus anti-MEK is currently used as first-line treatment of patients with metastatic melanomas harboring the somatic BRAF V600E mutation. However, the main issue with targeted therapy is the acquisition of tumor cell resistance. In a majority of resistant melanoma cells, the resistant process consists in epithelial-to-mesenchymal transition (EMT). This process called phenotype switching makes melanoma cells more invasive. Its signature is characterized by MITF low, AXL high, and actin cytoskeleton reorganization through RhoA activation. In parallel of this phenotype switching phase, the resistant cells exhibit an anarchic cell proliferation due to hyper-activation of the MAP kinase pathway. We show that a majority of human melanoma overexpress discoidin domain receptor 2 (DDR2) after treatment. The same result was found in resistant cell lines presenting phenotype switching compared to the corresponding sensitive cell lines. We demonstrate that DDR2 inhibition induces a decrease in AXL expression and reduces stress fiber formation in resistant melanoma cell lines. In this phenotype switching context, we report that DDR2 control cell and tumor proliferation through the MAP kinase pathway in resistant cells in vitro and in vivo. Therefore, inhibition of DDR2 could be a new and promising strategy for countering this resistance mechanism.
Asunto(s)
Receptor con Dominio Discoidina 2 , Melanoma , Línea Celular Tumoral , Proliferación Celular/genética , Receptor con Dominio Discoidina 2/genética , Resistencia a Antineoplásicos/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Fenotipo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-rafRESUMEN
Inhibitors of VEGF (vascular endothelial growth factor)/VEGFR2 (vascular endothelial growth factor receptor 2) are commonly used in the clinic, but their beneficial effects are only observed in a subset of patients and limited by induction of diverse relapse mechanisms. We describe the up-regulation of an adaptive immunosuppressive pathway during antiangiogenic therapy, by which PD-L1 (programmed cell death ligand 1), the ligand of the negative immune checkpoint regulator PD-1 (programmed cell death protein 1), is enhanced by interferon-γ-expressing T cells in distinct intratumoral cell types in refractory pancreatic, breast, and brain tumor mouse models. Successful treatment with a combination of anti-VEGFR2 and anti-PD-L1 antibodies induced high endothelial venules (HEVs) in PyMT (polyoma middle T oncoprotein) breast cancer and RT2-PNET (Rip1-Tag2 pancreatic neuroendocrine tumors), but not in glioblastoma (GBM). These HEVs promoted lymphocyte infiltration and activity through activation of lymphotoxin ß receptor (LTßR) signaling. Further activation of LTßR signaling in tumor vessels using an agonistic antibody enhanced HEV formation, immunity, and subsequent apoptosis and necrosis in pancreatic and mammary tumors. Finally, LTßR agonists induced HEVs in recalcitrant GBM, enhanced cytotoxic T cell (CTL) activity, and thereby sensitized tumors to antiangiogenic/anti-PD-L1 therapy. Together, our preclinical studies provide evidence that anti-PD-L1 therapy can sensitize tumors to antiangiogenic therapy and prolong its efficacy, and conversely, antiangiogenic therapy can improve anti-PD-L1 treatment specifically when it generates intratumoral HEVs that facilitate enhanced CTL infiltration, activity, and tumor cell destruction.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/terapia , Femenino , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Recurrencia Local de Neoplasia , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal/efectos de los fármacos , Linfocitos T Citotóxicos/fisiología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
The G-protein-coupled receptors LGR4, LGR5 and LGR6 are Wnt signaling mediators, but their functions in squamous cell carcinoma (SCC) are unclear. Using lineage tracing in Lgr5-EGFP-CreERT2/Rosa26-Tomato and Lgr6-EGFP-CreERT2/Rosa26-Tomato reporter mice, we demonstrate that Lgr6, but not Lgr5, acts as an epithelial stem cell marker in SCCs in vivo. We identify, by single-molecule in situ hybridization and cell sorting, rare cells positive for Lgr6 expression in immortalized keratinocytes and show that their frequency increases in advanced SCCs. Lgr6 expression is enriched in cells with stem cell characteristics, and Lgr6 downregulation in vivo causes increased epidermal proliferation with expanded lineage tracing from epidermal stem cells positive for Lgr6 expression. Surprisingly, mice with germline knockout of Lgr6 are predisposed to SCC development, through a mechanism that includes compensatory upregulation of Lgr5. These data provide a model for human patients with germline loss-of-function mutations in Wnt pathway genes, including RSPO1 or LGR4, who show increased susceptibility to squamous tumor development.
Asunto(s)
Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica , Queratinocitos/metabolismo , Células Madre Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Neoplasias Cutáneas/genética , Animales , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Transformada , Epidermis/metabolismo , Epidermis/patología , Humanos , Queratinocitos/patología , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Trombospondinas/genética , Trombospondinas/metabolismoRESUMEN
IRE1α is an endoplasmic reticulum (ER)-resident transmembrane signaling protein and a cellular stress sensor. The protein harbors a cytosolic dual kinase/endoribonuclease activity required for adaptive responses to micro-environmental changes. In an orthotopic xenograft model of human glioma, invalidation of IRE1α RNase or/and kinase activities generated tumors with remarkably distinct phenotypes. Contrasting with the extensive angiogenesis observed in tumors derived from control cells, the double kinase/RNase invalidation reprogrammed mesenchymal differentiation of cancer cells and produced avascular and infiltrative glioblastomas with blood vessel co-option. In comparison, selective invalidation of IRE1α RNase did not compromise tumor angiogenesis but still elicited invasive features and vessel co-option. In vitro, IRE1α RNase deficient cells were also endowed with a higher ability to migrate. Constitutive activation of both enzymes led to wild-type-like lesions. The presence of IRE1α, but not its RNase activity, is therefore required for glioblastoma neovascularization, whereas invasion results only from RNase inhibition. In this model, two key mechanisms of tumor progression and cancer cell survival are functionally linked to IRE1α.