RESUMEN
The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-based cytoprotective mechanism acting to prevent pathologies accompanying protein aggregation. It is frequently active in tumors, but relatively unstudied in gliomas. We hypothesized that UPR stress effects on glioma cells might protect tumors from additional exogenous stress (ie, chemotherapeutics), postulating that protection was concurrent with altered tumor cell metabolism. Using human brain tumor cell lines, xenograft tumors, human samples and gene expression databases, we determined molecular features of glioma cell UPR induction/activation, and here report a detailed analysis of UPR transcriptional/translational/metabolic responses. Immunohistochemistry, Western and Northern blots identified elevated levels of UPR transcription factors and downstream ER chaperone targets in gliomas. Microarray profiling revealed distinct regulation of stress responses between xenograft tumors and parent cell lines, with gene ontology and network analyses linking gene expression to cell survival and metabolic processes. Human glioma samples were examined for levels of the ER chaperone GRP94 by immunohistochemistry and for other UPR components by Western blotting. Gene and protein expression data from patient gliomas correlated poor patient prognoses with increased expression of ER chaperones, UPR target genes, and metabolic enzymes (glycolysis and lipogenesis). NMR-based metabolomic studies revealed increased metabolic outputs in glucose uptake with elevated glycolytic activity as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol were also evident upon UPR stress; in particular, recurrent tumors had overall higher lipid outputs and elevated specific UPR arms. Clonogenicity studies following temozolomide treatment of stressed or unstressed cells demonstrated UPR-induced chemoresistance. Our data characterize the UPR in glioma cells and human tumors, and link the UPR to chemoresistance possibly via enhanced metabolism. Given the role of the UPR in the balance between cell survival and apoptosis, targeting the UPR and/or controlling metabolic activity may prove beneficial for malignant glioma therapeutics.
Asunto(s)
Resistencia a Antineoplásicos , Glioma/metabolismo , Respuesta de Proteína Desplegada , Animales , Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Estrés del Retículo Endoplásmico/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Glioma/genética , Glioma/mortalidad , Glioma/patología , Humanos , Lipogénesis , Ratones , Chaperonas Moleculares/metabolismo , Clasificación del Tumor , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Temozolomida , Factores de Transcripción/metabolismo , Transcripción Genética , Respuesta de Proteína Desplegada/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: This paper presents the treatment of a 12-year-old female spayed Great Dane who presented with vestibular signs (ataxia, nystagmus, hind end collapse). Thoracic radiographs revealed a discrete pulmonary nodule in the right cranial lung lobe. Ultrasound-guided fine needle aspirate detected primary bronchoalveolar adenocarcinoma, verified via computed tomography, with a second smaller nodule discovered in the right cranial lung lobe. MATERIALS AND METHODS: A lateral thoracotomy with right cranial lung lobectomy was performed. Histopathological analysis of the nodules and an excised lymph node identified grade III bronchoalveolar adenocarcinoma with vascular infiltration and lymph node metastasis - a grim diagnosis with a reported median survival time of 6-27 days. A 10-g sample of the tumour was processed into a chaperone-rich cell lysate (CRCL) vaccine, which was administered weekly to the patient. Imiquimod - a Toll-like receptor 7 (TLR7) agonist - was applied topically for the first 12 treatments to stimulate local Langerhans cells. A single injection of bacillus Calmette-Guerin (BCG) was administered for additional immune stimulation at week 30 of treatment. RESULTS: The dog remained stable and in otherwise good health until diffuse relapse occurred 44 weeks after the initial treatment; following gastrointestinal bleeding, the dog was euthanised 50+ weeks post diagnosis. CONCLUSION: To the authors' knowledge, this is the first report of significantly prolonged survival following a diagnosis of grade III/stage III bronchoalveolar adenocarcinoma in a canine patient. This case report suggests that CRCL vaccine combined with topical imiquimod is a safe, effective treatment for canine tumours.
Asunto(s)
Adenocarcinoma Bronquioloalveolar/terapia , Vacunas contra el Cáncer/uso terapéutico , Enfermedades de los Perros/terapia , Neoplasias Pulmonares/terapia , Chaperonas Moleculares/inmunología , Adenocarcinoma Bronquioloalveolar/diagnóstico por imagen , Adenocarcinoma Bronquioloalveolar/patología , Adenocarcinoma Bronquioloalveolar/veterinaria , Animales , Enfermedades de los Perros/diagnóstico por imagen , Enfermedades de los Perros/patología , Perros , Femenino , Pulmón/diagnóstico por imagen , Pulmón/patología , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/veterinaria , RadiografíaRESUMEN
Medulloblastomas are the most prevalent malignant pediatric brain tumors. Survival for these patients has remained largely the same for approximately 20 years, and our therapies for these cancers cause significant health, cognitive, behavioral and developmental sequelae for those who survive the tumor and their treatments. We obviously need a better understanding of the biology of these tumors, particularly with regard to their migratory/invasive behaviors, their proliferative propensity, and their abilities to deflect immune responses. Exosomes, virus-sized membrane vesicles released extracellularly from cells after formation in, and transit thru, the endosomal pathway, may play roles in medulloblastoma pathogenesis but are as yet unstudied in this disease. Here we characterized exosomes from a medulloblastoma cell line with biochemical and proteomic analyses, and included characterization of patient serum exosomes. Further scrutiny of the proteomic data suggested functional properties of the exosomes that are relevant to medulloblastoma tumor biology, including their roles as proliferation stimulants, their activities as attractants for tumor cell migration, and their immune modulatory impacts on lymphocytes. Aspects of this held true for exosomes from other medulloblastoma cell lines as well. Additionally, pathway analyses suggested a possible role for the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A); however, inhibition of the protein's activity actually increased D283MED cell proliferation/clonogenecity, suggesting that HNF4A may act as a tumor suppressor in this cell line. Our work demonstrates that relevant functional properties of exosomes may be derived from appropriate proteomic analyses, which translate into mechanisms of tumor pathophysiology harbored in these extracellular vesicles.