RESUMEN

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.

Asunto(s)

Glioblastoma , Humanos , Glioblastoma/metabolismo , Enzimas Convertidoras de Endotelina/genética , Enzimas Convertidoras de Endotelina/metabolismo , Línea Celular Tumoral , Células Madre Neoplásicas/patología , Fenotipo

RESUMEN

Glioblastoma is the most common and aggressive primary brain tumor, characterized by its high chemoresistance and the presence of a cell subpopulation that persists under hypoxic niches, called glioblastoma stem-like cells (GSCs). The chemoresistance of GSCs is mediated in part by adenosine signaling and ABC transporters, which extrude drugs outside the cell, such as the multidrug resistance-associated proteins (MRPs) subfamily. Adenosine promotes MRP1-dependent chemoresistance under normoxia. However, adenosine/MRPs-dependent chemoresistance under hypoxia has not been studied until now. Transcript and protein levels were determined by RT-qPCR and Western blot, respectively. MRP extrusion capacity was determined by intracellular 5 (6)-Carboxyfluorescein diacetate (CFDA) accumulation. Cell viability was measured by MTS assays. Cell cycle and apoptosis were determined by flow cytometry. Here, we show for the first time that MRP3 expression is induced under hypoxia through the A2B adenosine receptor. Hypoxia enhances MRP-dependent extrusion capacity and the chemoresistance of GSCs. Meanwhile, MRP3 knockdown decreases GSC viability under hypoxia. Downregulation of the A2B receptor decreases MRP3 expression and chemosensibilizes GSCs treated with teniposide under hypoxia. These data suggest that hypoxia-dependent activation of A2B adenosine receptor promotes survival of GSCs through MRP3 induction.

Asunto(s)

Neoplasias Encefálicas , Glioblastoma , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Adenosina/metabolismo , Neoplasias Encefálicas/metabolismo , Resistencia a Antineoplásicos , Glioblastoma/metabolismo , Humanos , Hipoxia/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Células Madre Neoplásicas/metabolismo , Receptor de Adenosina A2B/metabolismo , Receptores Purinérgicos P1/metabolismo

RESUMEN

Glioblastoma is the brain tumor with the worst prognosis. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine, which are increased even more under hypoxic conditions. Under hypoxia, adenosine signaling is related to HIF-2α expression, enhancing cell aggressiveness. Adenosine can be degraded using recombinant adenosine deaminase (ADA) to revert its pathological effects. The aim of this study was to degrade adenosine using ADA in order to decrease malignancy of GSCs. Adenosine depletion was performed using recombinant ADA. Migration and invasion were measured by transwell and matrigel-coated transwell assay, respectively. HIF-2α-dependent cell migration/invasion decreased in GSCs treated with ADA under hypoxia. MRPs-mediated chemoresistance and colony formation decreased in treatment with ADA. In conclusion, adenosine depletion using adenosine deaminase decreases GSCs aggressiveness.

Asunto(s)

Adenosina/deficiencia , Neoplasias Encefálicas/patología , Movimiento Celular , Proliferación Celular , Resistencia a Antineoplásicos , Glioblastoma/patología , Células Madre Neoplásicas/patología , Adenosina/metabolismo , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Adhesión Celular , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Humanos , Hipoxia , Invasividad Neoplásica , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Tumorales Cultivadas , Vincristina/farmacología

RESUMEN

Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production and activation of the A3 Adenosine Receptor (A3AR), therefore, the aim of this study was to determine the role of extracellular adenosine and A3AR on GSCs invasion under hypoxia. GSCs were obtained from a U87MG cell line and primary cultures of GBM patients, and then incubated under normoxia or hypoxia. Gene expression was evaluated by RNAseq, RT-qPCR, and western blot. Cell migration was measured by spreading and transwell boyden chamber assays; cell invasion was evaluated by Matrigel-coated transwell, ex vivo brain slice, and in vivo xenograft assays. The contribution of A3AR on cell migration/invasion was evaluated using the A3AR antagonist, MRS1220. Extracellular adenosine production was higher under hypoxia than normoxia, mainly by the catalytic action of the prostatic acid phosphatase (PAP), promoting cell migration/invasion in a HIF-2-dependent process. A3AR blockade decreased cell migration/invasion and the expression of Epithelial-Mesenchymal Transition markers. In conclusion, high levels of extracellular adenosine production enhance cell migration/invasion of GSCs, through HIF-2/PAP-dependent activation of A3AR under hypoxia.

Asunto(s)

Adenosina/metabolismo , Neoplasias Encefálicas/metabolismo , Movimiento Celular , Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Receptor de Adenosina A3/metabolismo , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Células Madre Neoplásicas/patología , Receptor de Adenosina A3/genética , Transducción de Señal , Células Tumorales Cultivadas , Hipoxia Tumoral , Microambiente Tumoral

RESUMEN

Poor response to current treatments for glioblastoma has been attributed to the presence of glioblastoma stem-like cells (GSCs). GSCs are able to expel antitumor drugs to the extracellular medium using the multidrug resistance-associated protein 1 (MRP1) transporter. Tacrolimus (FK506) has been identified as an MRP1 regulator in differentiated glioblastoma (GBM) cells (non-GSCs); however, the effect of FK506 on GSCs is currently unknown. The objective of the following research is to evaluate the effect of FK506 on the MRP1-related chemo-resistant phenotype of GSCs. For this, U87MG and C6 glioma cell lines were used to generate non-GSCs and GSCs. mRNA and MRP1-positive cells were evaluated by RT-qPCR and flow cytometry, respectively. A Carboxyfluorescein Diacetate (CFDA)-retention assay was performed to evaluate the MRP1 activity. Apoptosis and MTT assays were employed to evaluate the cytotoxic effects of FK506 plus Vincristine (MRP1 substrate). GSC-derived subcutaneous tumors were generated to evaluate the in vivo effect of FK506/Vincristine treatment. No differences in transcript levels and positive cells for MRP1 were observed in FK506-treated cells. Lesser cell viability, increased apoptosis, and CFDA-retention in the FK506/Vincristine-treated cells were observed. In vivo, the FK506/Vincristine treatment decreased the tumor size as well as ki67, Glial Fibrillary Acidic Protein (GFAP), and nestin expression. We conclude that FK506 confers a chemo-sensitive phenotype to MRP1-drug substrate in GSCs.

Asunto(s)

Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Células Madre Neoplásicas/efectos de los fármacos , Tacrolimus/uso terapéutico , Vincristina/uso terapéutico , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Ratas Sprague-Dawley , Tacrolimus/farmacología , Vincristina/farmacología

RESUMEN

Glioblastoma multiforme (GBM) is considered the most common and aggressive tumour of the central nervous system and is characterized for being highly chemoresistant. This property is mainly due to the activation of Multiple Drug Resistance (MDR) mechanisms that protect cancer cells from structurally and morphologically different drugs. Overexpression and increased ABC transporters activity is one of the most important MDR mechanisms at the clinical level, and both its expression and activity are elevated in GBM cells. Within the tumour, there is a subpopulation called glioblastoma stem-like cells (GSCs), which due to its high tumourigenic capacity and chemoresistance, have been postulated as the main responsible for tumour recurrence. The GSCs inhabit hypoxic tumour zones, niches that apart from maintaining and promoting stem phenotype have also been correlated with high chemoresistance. Of the signalling pathways activated during hypoxia, purinergic signalling has been highly associated to the induction of MDR mechanisms. Through its receptors, the nucleoside adenosine has been shown to promotes the chemoresistance mediated by ABC transporters. Therefore, targeting its components is a promising alternative for GBM treatment. In this review, we will discuss chemoresistance in GSCs and the effect of the hypoxic microenvironment and adenosine on MDR mechanisms.

Asunto(s)

Adenosina/genética , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Glioblastoma/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/genética , Adenosina/metabolismo , Hipoxia de la Célula/genética , Glioblastoma/genética , Glioblastoma/patología , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Transducción de Señal , Microambiente Tumoral/genética