RESUMEN
Neural progenitor cells (NPCs) are self-renewing cells that give rise to the major cells in the nervous system and are considered to be the possible cell of origin of glioblastoma. The gap junction protein connexin43 (Cx43) is expressed by NPCs, exerting channel-dependent and -independent roles. We focused on one property of Cx43-its ability to inhibit Src, a key protein in brain development and oncogenesis. Because Src inhibition is carried out by the sequence 266-283 of the intracellular C terminus in Cx43, we used a cell-penetrating peptide containing this sequence, TAT-Cx43266-283, to explore its effects on postnatal subventricular zone NPCs. Our results show that TAT-Cx43266-283 inhibited Src activity and reduced NPC proliferation and survival promoted by epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). In differentiation conditions, TAT-Cx43266-283 increased astrocyte differentiation at the expense of neuronal differentiation, which coincided with a reduction in Src activity and ß-catenin expression. We propose that Cx43, through the region 266-283, reduces Src activity, leading to disruption of EGF and FGF-2 signaling and to down-regulation of ß-catenin with effects on proliferation and differentiation. Our data indicate that the inhibition of Src might contribute to the complex role of Cx43 in NPCs and open new opportunities for further research in gliomagenesis.
Asunto(s)
Conexina 43/genética , Factor de Crecimiento Epidérmico/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Glioblastoma/genética , Animales , Astrocitos/metabolismo , Astrocitos/patología , Carcinogénesis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/patología , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células-Madre Neurales/efectos de los fármacos , Ratas , Células Madre/efectos de los fármacos , beta Catenina/genética , Familia-src Quinasas/genéticaRESUMEN
This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below.
Asunto(s)
Uniones Comunicantes/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Animales , Uniones Comunicantes/genética , Uniones Comunicantes/patología , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologíaRESUMEN
BACKGROUND: Glioblastoma (GBM) commonly displays epidermal growth factor receptor (EGFR) alterations (mainly amplification and EGFRvIII) and TAT-Cx43266-283 is a Src-inhibitory peptide with antitumor properties in preclinical GBM models. Given the link between EGFR and Src, the aim of this study was to explore the role of EGFR in the antitumor effects of TAT-Cx43266-283. METHODS: The effect of TAT-Cx43266-283, temozolomide (TMZ), and erlotinib (EGFR inhibitor) was studied in patient-derived GBM stem cells (GSCs) and murine neural stem cells (NSCs) with and without EGFR alterations, in vitro and in vivo. EGFR alterations were analyzed by western blot and fluorescence in situ hybridization in these cells, and compared with Src activity and survival in GBM samples from The Cancer Genome Atlas. RESULTS: The effect of TAT-Cx43266-283 correlated with EGFR alterations in a set of patient-derived GSCs and was stronger than that exerted by TMZ and erlotinib. In fact, TAT-Cx43266-283 only affected NSCs with EGFR alterations, but not healthy NSCs. EGFR alterations correlated with Src activity and poor survival in GBM patients. Finally, tumors generated from NSCs with EGFR alterations showed a decrease in growth, invasiveness, and vascularization after treatment with TAT-Cx43266-283, which enhanced the survival of immunocompetent mice. CONCLUSIONS: Clinically relevant EGFR alterations are predictors of TAT-Cx43266-283 response and part of its mechanism of action, even in TMZ- and erlotinib-resistant GSCs. TAT-Cx43266-283 targets NSCs with GBM-driver mutations, including EGFR alterations, in an immunocompetent GBM model in vivo, suggesting a promising effect on GBM recurrence. Together, this study represents an important step toward the clinical application of TAT-Cx43266-283.
Asunto(s)
Neoplasias Encefálicas , Receptores ErbB , Amplificación de Genes , Glioblastoma , Temozolomida , Ensayos Antitumor por Modelo de Xenoinjerto , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Animales , Humanos , Ratones , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Temozolomida/farmacología , Clorhidrato de Erlotinib/farmacología , Células Tumorales Cultivadas , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismoRESUMEN
Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer. The Src inhibitor, TAT-Cx43266-283, exerts antitumor effects in in vitro and in vivo models of GBM. Because addressing the mechanism of action is essential to translate these results to a clinical setting, in this study we carried out an unbiased proteomic approach. Data-independent acquisition mass spectrometry proteomics allowed the identification of 190 proteins whose abundance was modified by TAT-Cx43266-283. Our results were consistent with the inhibition of Src as the mechanism of action of TAT-Cx43266-283 and unveiled antitumor effectors, such as p120 catenin. Changes in the abundance of several proteins suggested that TAT-Cx43266-283 may also impact the brain microenvironment. Importantly, the proteins whose abundance was reduced by TAT-Cx43266-283 correlated with an improved GBM patient survival in clinical datasets and none of the proteins whose abundance was increased by TAT-Cx43266-283 correlated with shorter survival, supporting its use in clinical trials.
RESUMEN
Introduction: The subventricular zone (SVZ) is a brain region that contains neural stem cells and progenitor cells (NSCs/NPCs) from which new neurons and glial cells are formed during adulthood in mammals. Recent data indicate that SVZ NSCs are the cell type that acquires the initial tumorigenic mutation in glioblastoma (GBM), the most aggressive form of malignant glioma. NSCs/NPCs of the SVZ present hemichannel activity whose function has not yet been fully elucidated. In this work, we aimed to analyze whether hemichannel-mediated communication affects proliferation of SVZ NPCs and GBM cells. Methods and Results: For that purpose, we used boldine, an alkaloid derived from the boldo tree (Peumus boldus), that inhibits connexin and pannexin hemichannels, but without affecting gap junctional communication. Boldine treatment (50 µM) of rat SVZ NPCs grown as neurospheres effectively inhibited dye uptake through hemichannels and induced a significant reduction in neurosphere diameter and in bromodeoxyuridine (BrdU) incorporation. However, the differentiation pattern was not modified by the treatment. Experiments with specific blockers for hemichannels formed by connexin subunits (D4) or pannexin 1 (probenecid) revealed that probenecid, but not D4, produced a decrease in BrdU incorporation similar to that obtained with boldine. These results suggest that inhibition of pannexin 1 hemichannels could be partially responsible for the antiproliferative effect of boldine on SVZ NPCs. Analysis of the effect of boldine (25-600 µM) on different types of primary human GBM cells (GBM59, GBM96, and U87-MG) showed a concentration-dependent decrease in GBM cell growth. Boldine treatment also induced a significant inhibition of hemichannel activity in GBM cells. Discussion: Altogether, we provide evidence of an antimitotic action of boldine in SVZ NPCs and in GBM cells which may be due, at least in part, to its hemichannel blocking function. These results could be of relevance for future possible strategies in GBM aimed to suppress the proliferation of mutated NSCs or glioma stem cells that might remain in the brain after tumor resection.
RESUMEN
In diverse brain pathologies, astrocytes become reactive and undergo profound phenotypic changes. Connexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is one of the proteins modified in reactive astrocytes. Downregulation of Cx43 in cultured astrocytes activates c-Src, promotes proliferation, and increases the rate of glucose uptake; however, so far there have been no studies examining whether this cascade of events takes place in reactive astrocytes. In this work, we analyzed this pathway after a cortical lesion induced by a kainic acid injection. As previously described, astrocytes reacted to the lesion with an increase in glial fibrillary acidic protein and a decrease in Cx43 expression. Some of these reactive astrocytes proliferated, as estimated by bromodeoxyuridine incorporation and cyclins D1 and D3 upregulation. In addition, the expression of the glucose transporter GLUT-3 and the enzyme responsible for glucose phosphorylation, Type II hexokinase (Hx-2), were induced in reactive astrocytes, suggesting an increased glucose uptake. Previous in vitro studies reported that c-Src is the link between Cx43 and glucose uptake and proliferation in astrocytes. Here, we found that c-Src activity increased in the lesioned area. c-Src activation and Cx43 downregulation preceded the peak of Hx-2 and cyclin D3 expression, suggesting that c-Src could mediate the effect of Cx43 on glucose uptake and proliferation in reactive astrocytes after an excitotoxic insult. Interestingly, we identify c-Src, GLUT-3, and Hx-2 in the signaling mechanisms involved in the reaction of astroglia to injury. Altogether these data contribute to identify new therapeutical targets to enhance astrocyte neuroprotective activities.
Asunto(s)
Astrocitos/metabolismo , Astrocitos/patología , Proliferación Celular/efectos de los fármacos , Conexina 43/antagonistas & inhibidores , Agonistas de Aminoácidos Excitadores/toxicidad , Genes src/fisiología , Glucosa/metabolismo , Animales , Astrocitos/efectos de los fármacos , Conexina 43/biosíntesis , Conexina 43/genética , Ácido Kaínico/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Metabolism must be tightly regulated to fulfil the dynamic requirements of cancer cells during proliferation, migration, stemness and differentiation. Src is a node of several signals involved in many of these biological processes, and it is also an important regulator of cell metabolism. Glucose uptake, glycolysis, the pentose-phosphate pathway and oxidative phosphorylation are among the metabolic pathways that can be regulated by Src. Therefore, this oncoprotein is in an excellent position to coordinate and finely tune cell metabolism to fuel the different cancer cell activities. Here, we provide an up-to-date summary of recent progress made in determining the role of Src in glucose metabolism as well as the link of this role with cancer cell metabolic plasticity and tumour progression. We also discuss the opportunities and challenges facing this field.
Asunto(s)
Neoplasias , Vía de Pentosa Fosfato , Humanos , Glucólisis , Fosforilación Oxidativa , Neoplasias/genética , Glucosa/metabolismoRESUMEN
Autophagy is a physiological process by which various damaged or non-essential cytosolic components are recycled, contributing to cell survival under stress conditions. In cancer, autophagy can have antitumor or protumor effects depending on the developmental stage. Here, we use Western blotting, immunochemistry, and transmission electron microscopy to demonstrate that the antitumor peptide TAT-Cx43266-283, a c-Src inhibitor, blocks autophagic flux in glioblastoma stem cells (GSCs) under basal and nutrient-deprived conditions. Upon nutrient deprivation, GSCs acquired a dormant-like phenotype that was disrupted by inhibition of autophagy with TAT-Cx43266-283 or chloroquine (a classic autophagy inhibitor), leading to GSC death. Remarkably, dasatinib, a clinically available c-Src inhibitor, could not replicate TAT-Cx43266-283 effect on dormant GSCs, revealing for the first time the possible involvement of pathways other than c-Src in TAT-Cx43266-283 effect. TAT-Cx43266-283 exerts an antitumor effect both in nutrient-complete and nutrient-deprived environments, which constitutes an advantage over chloroquine and dasatinib, whose effects depend on nutrient environment. Finally, our analysis of the levels of autophagy-related proteins in healthy and glioma donors suggests that autophagy is upregulated in glioblastoma, further supporting the interest in inhibiting this process in the most aggressive brain tumor and the potential use of TAT-Cx43266-283 as a therapy for this type of cancer.
RESUMEN
Our previous works showed that oleic acid synthesized in vitro by astrocytes in response to albumin behaves as a neurotrophic factor in neurons, up-regulating several proteins, such as the axonal growth marker growth-associated protein 43(GAP-43). Although the molecular mechanism of this process is fairly known, there is no evidence pinpointing the region/s in which oleic acid is synthesized. In this study, we show that the rate-limiting enzyme in oleic acid synthesis, stearoyl-CoA desaturase (SCD-1), is located in the periventricular zone of the brain of newborn rats, simultaneously to an increase in the amount of free oleic acid in the forebrain. In addition, the spatio-temporal presence of albumin - the signal that promotes oleic acid synthesis - and that of GAP-43 are correlated with that of SCD-1. Using organotypic slice cultures, we found that albumin up-regulates SCD-1 and stimulates the growth of GAP-43-positive axons in the striatum. The effect of albumin on GAP-43 was reduced when SCD-1 was silenced by siRNA. In conclusion, our results suggest that albumin up-regulates axonogenesis in the striatum by increasing the amount of the neurotrophic factor oleic acid synthesized by SCD-1 in the periventricular zone of the newborn brain.
Asunto(s)
Axones/fisiología , Ventrículos Cerebrales/metabolismo , Cuerpo Estriado/metabolismo , Ácido Oléico/biosíntesis , Albúminas/metabolismo , Animales , Animales Recién Nacidos , Ventrículos Cerebrales/crecimiento & desarrollo , Ventrículos Cerebrales/ultraestructura , Cuerpo Estriado/crecimiento & desarrollo , Cuerpo Estriado/ultraestructura , Proteína GAP-43/biosíntesis , Regulación del Desarrollo de la Expresión Génica , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Malignant gliomas are the most frequent primary brain tumors and remain among the most incurable cancers. Although the role of the gap junction protein, connexin43 (Cx43), has been deeply investigated in malignant gliomas, no compounds have been reported with the ability to recapitulate the tumor suppressor properties of this protein in in vivo glioma models. METHODS: TAT-Cx43266-283 a cell-penetrating peptide which mimics the effect of Cx43 on c-Src inhibition, was studied in orthotopic immunocompetent and immunosuppressed models of glioma. The effects of this peptide in brain cells were also analyzed. RESULTS: While glioma stem cell malignant features were strongly affected by TAT-Cx43266-283, these properties were not significantly modified in neurons and astrocytes. Intraperitoneally administered TAT-Cx43266-283 decreased the invasion of intracranial tumors generated by GL261 mouse glioma cells in immunocompetent mice. When human glioma stem cells were intracranially injected with TAT-Cx43266-283 into immunodeficient mice, there was reduced expression of the stemness markers nestin and Sox2 in human glioma cells at 7 days post-implantation. Consistent with the role of Sox2 as a transcription factor required for tumorigenicity, TAT-Cx43266-283 reduced the number and stemness of human glioma cells at 30 days post-implantation. Furthermore, TAT-Cx43266-283 enhanced the survival of immunocompetent mice bearing gliomas derived from murine glioma stem cells. CONCLUSION: TAT-Cx43266-283 reduces the growth, invasion, and progression of malignant gliomas and enhances the survival of glioma-bearing mice without exerting toxicity in endogenous brain cells, which suggests that this peptide could be considered as a new clinical therapy for high-grade gliomas.
Asunto(s)
Neoplasias Encefálicas , Glioma , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Conexina 43 , Modelos Animales de Enfermedad , Glioma/tratamiento farmacológico , Ratones , PéptidosRESUMEN
BACKGROUND: Glioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and primary glioblastoma cells by the peptide TAT-Cx43266-283 reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43266-283. METHODS: Metabolic impairment induced by the c-Src inhibitor TAT-Cx43266-283 in vitro was assessed by fluorometry, western blotting, immunofluorescence, qPCR, enzyme activity assays, electron microscopy, Seahorse analysis, time-lapse imaging, siRNA, and MTT assays. Protein expression in tumours from a xenograft orthotopic glioblastoma mouse model was evaluated by immunofluorescence. FINDINGS: TAT-Cx43266-283 decreased glucose uptake in human GSCs and reduced oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on brain cell metabolism, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43266-283 impaired metabolic plasticity, reducing GSC growth and survival under different nutrient environments. Finally, GSCs intracranially implanted with TAT-Cx43266-283 showed decreased levels of important metabolic targets for cancer therapy, such as hexokinase-2 and GLUT-3. INTERPRETATION: The reduced ability of TAT-Cx43266-283-treated GSCs to survive in metabolically challenging settings, such as those with restricted nutrient availability or the ever-changing in vivo environment, allows us to conclude that the advantageous metabolic plasticity of GSCs can be therapeutically exploited through the specific and cell-selective inhibition of c-Src by TAT-Cx43266-283. FUNDING: Spanish Ministerio de Economía y Competitividad (FEDER BFU2015-70040-R and FEDER RTI2018-099873-B-I00), Fundación Ramón Areces. Fellowships from the Junta de Castilla y León, European Social Fund, Ministerio de Ciencia and Asociación Española Contra el Cáncer (AECC).
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Glioma/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Péptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Espacio Extracelular , Fluorocarburos/metabolismo , Glioma/tratamiento farmacológico , Glioma/patología , Glucosa/metabolismo , Glucólisis , Humanos , Hidrocarburos Bromados/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , RatasRESUMEN
In previous studies, we showed that endothelin-1 increased astrocyte proliferation and glucose uptake. These effects were similar to those observed with other gap junction inhibitors, such as carbenoxolone (CBX). Because 24-h treatment with endothelin-1 or CBX downregulates the expression of connexin43, the main protein forming astrocytic gap junctions, which can also be involved in proliferation, in this study, we addressed the possible role of connexin43 in the effects of endothelin-1. To do so, connexin43 was silenced in astrocytes by siRNA. The knock down of connexin43 increased the rate of glucose uptake, characterized by the upregulation of GLUT-1 and type I hexokinase. Neither endothelin-1 nor CBX were able to further increase the rate of glucose uptake in connexin43-silenced astrocytes. In agreement, no effects of endothelin-1 and CBX on GLUT-1 and type I hexokinase were observed in connexin-43 silenced astrocytes or in astrocytes from connexin43 knock-out (KO) mice. Our previous studies suggested a close relationship between glucose uptake and astrocyte proliferation. Consistent with this, connexin43-silenced astrocytes exhibited an increase in Ki-67, a marker of proliferation. The effects of ET-1 on retinoblastoma phosphorylation on Ser780 and on the upregulation of cyclins D1 and D3 were affected by the levels of connexin43. In conclusion, our results indicate that connexin43 participates in the effects of endothelin-1 on glucose uptake and proliferation in astrocytes. Interestingly, although the rate of growth in connexin43 KO astrocytes has been reported to be reduced, we observed that an acute reduction in connexin43 by siRNA increased proliferation and glucose uptake.
Asunto(s)
Astrocitos/metabolismo , Proliferación Celular , Conexina 43/metabolismo , Endotelina-1/metabolismo , Uniones Comunicantes/metabolismo , Glucosa/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Comunicación Celular/fisiología , Células Cultivadas , Conexina 43/genética , Ciclinas/metabolismo , Regulación hacia Abajo/fisiología , Endotelina-1/farmacología , Metabolismo Energético/fisiología , Uniones Comunicantes/efectos de los fármacos , Transportador de Glucosa de Tipo 1/metabolismo , Hexoquinasa/metabolismo , Antígeno Ki-67/metabolismo , Ratones , Ratones Noqueados , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Proteína de Retinoblastoma/metabolismoRESUMEN
The synthesis and release of the neurotrophic factor oleic acid requires internalization of albumin into the astrocyte, which is mediated by megalin. In this study, we show that the binding and internalization of albumin involve its interaction with megalin, caveolin-1, caveolin-2 and cavin, but not with clathrin in astrocytes from primary culture. Electron microscopy analyses revealed albumin-gold complexes localized in caveolae, but not in clathrin-coated vesicles. Neither chlorpromazine nor silencing clathrin expression modified albumin uptake. Silencing caveolin-1 strongly reduced the binding and internalization of albumin and the distribution of megalin in the plasma membrane. However, silencing caveolin-2 only decreased albumin internalization, suggesting that caveolin-1 is responsible for megalin recruitment to the caveolae and that caveolin-2 participates in caveolae internalization. In most tissues, the cytosolic adaptor protein disabled (Dab)-2 connects megalin to clathrin, astrocytes lack Dab-2; instead, they express Dab-1, which interacts with caveolin-1 and megalin and is required for albumin internalization. The transcytosis of albumin in astrocytes, including the passage through the endoplasmic reticulum, which is a compulsory step for oleic acid synthesis, was confirmed by electron microscopy analyses. Thus, whereas silencing clathrin did not modify the synthesis and release of oleic acid, the knock-down of caveolin-1, caveolin-2 and Dab-1 strongly reduced the synthesis and release of this neurotrophic factor. In conclusion, caveola-mediated endocytosis of albumin requires megalin and the adaptor protein Dab-1 in cultured astrocytes. Albumin endocytosis may be a key step in brain development because it stimulates the synthesis of oleic acid, which in turn promotes neuronal differentiation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Albúminas/metabolismo , Astrocitos/metabolismo , Caveolas/metabolismo , Endocitosis/fisiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ácido Oléico/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Análisis de Varianza , Animales , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Células Cultivadas , Clorpromazina/farmacología , Cromatografía Líquida de Alta Presión/métodos , Antagonistas de Dopamina/farmacología , Endocitosis/efectos de los fármacos , Inmunoprecipitación , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Microscopía Electrónica de Transmisión/métodos , Proteínas del Tejido Nervioso/genética , Prosencéfalo/citología , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Transfección/métodos , Transferrina/metabolismoRESUMEN
Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential.
Asunto(s)
Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Neoplasias/metabolismo , Animales , Carcinogénesis , Comunicación Celular , Diferenciación Celular , Membrana Celular/metabolismo , Proliferación Celular , Citosol/metabolismo , Regulación de la Expresión Génica , Homeostasis , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Células Madre Neoplásicas/citología , Pronóstico , Dominios Proteicos , Isoformas de Proteínas , Investigación Biomédica Traslacional , Resultado del Tratamiento , Microambiente TumoralRESUMEN
We have previously shown that the uptake and transcytosis of albumin in astrocytes promote the synthesis of the neurotrophic factor oleic acid. Although the mechanism by which albumin induces oleic acid synthesis is well known, the mechanism of albumin uptake in astrocytes remains unknown. In this work, we found that astrocytes express megalin, an endocytic receptor for multiple ligands including albumin. In addition, when the activity of megalin is blocked by specific antibodies or by silencing megalin with specific siRNA, albumin binding and internalization is strongly reduced indicating that megalin is required for albumin binding and internalization in the astrocyte. Since the uptake of albumin in astrocytes aims at synthesizing the neurotrophic factor oleic acid, we tested the ability of megalin-silenced astrocytes to synthesize and release oleic acid in the presence of albumin. Our results showed that the amount of oleic acid found in the extracellular medium of megalin-silenced astrocytes was strongly reduced as compared with their controls. Together, the results of this work indicate that megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid. Consequently, the possible involvement of albumin in the holoprosencephalic syndrome observed in megalin-deficient mice is suggested.
Asunto(s)
Astrocitos/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Factores de Crecimiento Nervioso/biosíntesis , Ácido Oléico/biosíntesis , Receptores de Albúmina/metabolismo , Albúmina Sérica Bovina/farmacología , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Bovinos , Células Cultivadas , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Factores de Crecimiento Nervioso/genética , Ácido Oléico/genética , Ratas , Ratas Wistar , Receptores de Albúmina/genética , Receptores de Albúmina/fisiologíaRESUMEN
Amyloid-ß (Aß) peptides, Aß40, Aß42, and recently Aß25 - 35, have been directly implicated in the pathogenesis of Alzheimer's disease (AD). We have previously shown that all three peptides decrease neuronal viability, but Aß40 also promotes synaptic disassembling. In this work, we have studied the effects of these peptides on astrocytes in primary culture and found that the three Aß peptides were internalized by astrocytes and significantly decreased astrocyte viability, while increasing ROS production. Aß peptide internalization is temperature-dependent, a fact that supports the idea that Aß peptides are actively endocytosed by astrocytes. However, inhibiting caveolae formation by methyl-beta-cyclodextrin or by silencing caveolin-1 with RNA interference did not prevent Aß endocytosis, which suggests that Aß peptides do not use caveolae to enter astrocytes. Conversely, inhibition of clathrin-coated vesicle formation by chlorpromazine or by silencing clathrin with RNA interference significantly decreased Aß internalization and partially reverted the decrease of astrocyte viability caused by the presence of Aß. These results suggest that Aß is endocytosed by clathrin-coated vesicles in astrocytes. Aß-loaded astrocytes, when co-incubated with non-treated astrocytes in separate wells but with the same incubation medium, promoted cell death in non-treated astrocytes; a fact that was associated with the presence of Aß inside previously unloaded astrocytes. This phenomenon was inhibited by the presence of chlorpromazine in the co-incubation medium. These results suggest that astrocyte may perform Aß transcytosis, a process that could play a role in the clearance of Aß peptides from the brain to cerebrospinal fluid.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Astrocitos/efectos de los fármacos , Endocitosis/efectos de los fármacos , Transcitosis/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Animales , Animales Recién Nacidos , Antipsicóticos/farmacología , Encéfalo/citología , Caveolina 1/genética , Caveolina 1/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Clorpromazina/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Temperatura , Transfección , beta-Ciclodextrinas/farmacologíaRESUMEN
Osteoarthritis (OA), a chronic disease characterized by articular cartilage degeneration, is a leading cause of disability and pain worldwide. In OA, chondrocytes in cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favouring disease progression. Similar to other wound-healing disorders, chondrocytes from OA patients show a chronic increase in the gap junction channel protein connexin43 (Cx43), which regulates signal transduction through the exchange of elements or recruitment/release of signalling factors. Although immature or stem-like cells are present in cartilage from OA patients, their origin and role in disease progression are unknown. In this study, we found that Cx43 acts as a positive regulator of chondrocyte-mesenchymal transition. Overactive Cx43 largely maintains the immature phenotype by increasing nuclear translocation of Twist-1 and tissue remodelling and proinflammatory agents, such as MMPs and IL-1ß, which in turn cause cellular senescence through upregulation of p53, p16INK4a and NF-κB, contributing to the senescence-associated secretory phenotype (SASP). Downregulation of either Cx43 by CRISPR/Cas9 or Cx43-mediated gap junctional intercellular communication (GJIC) by carbenoxolone treatment triggered rediferentiation of osteoarthritic chondrocytes into a more differentiated state, associated with decreased synthesis of MMPs and proinflammatory factors, and reduced senescence. We have identified causal Cx43-sensitive circuit in chondrocytes that regulates dedifferentiation, redifferentiation and senescence. We propose that chondrocytes undergo chondrocyte-mesenchymal transition where increased Cx43-mediated GJIC during OA facilitates Twist-1 nuclear translocation as a novel mechanism involved in OA progression. These findings support the use of Cx43 as an appropriate therapeutic target to halt OA progression and to promote cartilage regeneration.
Asunto(s)
Cartílago Articular/inmunología , Comunicación Celular/genética , Senescencia Celular/genética , Condrocitos/inmunología , Conexina 43/genética , Osteoartritis/genética , Adipocitos/efectos de los fármacos , Adipocitos/inmunología , Adipocitos/patología , Antígenos CD/genética , Antígenos CD/inmunología , Carbenoxolona/farmacología , Cartílago Articular/patología , Estudios de Casos y Controles , Comunicación Celular/inmunología , Diferenciación Celular , Senescencia Celular/inmunología , Condrocitos/efectos de los fármacos , Condrocitos/patología , Conexina 43/inmunología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/inmunología , Regulación de la Expresión Génica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/inmunología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/patología , FN-kappa B/genética , FN-kappa B/inmunología , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Osteoartritis/inmunología , Osteoartritis/patología , Cultivo Primario de Células , Índice de Severidad de la Enfermedad , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunología , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/inmunologíaRESUMEN
Amyloid-ß (Aß), Aß40, Aß42, and, recently, Aß25-35 have been directly implicated in the pathogenesis of Alzheimer's disease. We have studied the effects of Aß on neuronal death, reactive oxygen species (ROS) production, and synaptic assembling in neurons in primary culture. Aß25-35, Aß40, and Aß42 significantly decreased neuronal viability, although Aß25-35 showed a higher effect. Aß25-35 showed a more penetrating ability to reach mitochondria while Aß40 did not enter the neuronal cytosol and Aß42 was scarcely internalized. We did not observe a direct correlation between ROS production and cell death because both Aß40 and Aß42 decreased neuronal viability but Aß40 did not change ROS production. Rather, ROS production seems to correlate with the penetrating ability of each Aß. No significant differences were found between Aß40 and Aß42 regarding the extent of the deleterious effects of both peptides on neuronal viability or synaptophysin expression. However, Aß40 elicited a clear delocalization of PSD-95 and synaptotagmin from prospective synapsis to the neuronal soma, suggesting the occurrence of a crucial effect of Aß40 on synaptic disassembling. The formation of Aß40- or Aß42-serum albumin complexes avoided the effects of these peptides on neuronal viability, synaptophysin expression, and PSD-95/synaptotagmin disarrangement suggesting that sequestration of Aß by albumin prevents deleterious effects of these peptides. We can conclude that Aß borne by albumin can be safely transported through body fluids, a fact that may be compulsory for Aß disposal by peripheral tissues.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Albúmina Sérica Humana/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Citosol/metabolismo , Citosol/patología , Homólogo 4 de la Proteína Discs Large/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas/patología , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Sinaptofisina/metabolismo , Sinaptotagminas/metabolismoRESUMEN
Connexin43 (CX43), a protein that forms gap junction channels and hemichannels in astrocytes, is downregulated in high-grade gliomas. Its relevance for glioma therapy has been thoroughly explored; however, its positive effects on proliferation are counterbalanced by its effects on migration and invasion. Here, we show that a cell-penetrating peptide based on CX43 (TAT-Cx43266-283) inhibited c-Src and focal adhesion kinase (FAK) and upregulated phosphatase and tensin homolog in glioma stem cells (GSCs) derived from patients. Consequently, TAT-Cx43266-283 reduced GSC motility, as analyzed by time-lapse microscopy, and strongly reduced their invasive ability. Interestingly, we investigated the effects of TAT-Cx43266-283 on freshly removed surgical specimens as undissociated glioblastoma blocks, which revealed a dramatic reduction in the growth, migration, and survival of these cells. In conclusion, a region of CX43 (amino acids 266-283) exerts an important anti-tumor effect in patient-derived glioblastoma models that includes impairment of GSC migration and invasion.
Asunto(s)
Conexina 43/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Glioma/metabolismo , Células Madre Neoplásicas/metabolismo , Fosfohidrolasa PTEN/metabolismo , Familia-src Quinasas/metabolismo , Proteína Tirosina Quinasa CSK , 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 , Conexina 43/química , Conexina 43/genética , Conexina 43/farmacología , Glioma/genética , Humanos , Modelos Biológicos , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.