Metabolic remodeling in astrocytes: paving the path to brain tumor development

The brain is a highly metabolic organ, composed of multiple cell classes, that controls crucial functions of the body. Although neurons have traditionally been the main protagonist, astrocytes have gained significant attention over the last decade. In this regard, astrocytes are a type of glial cells that have recently emerged as critical regulators of central nervous system (CNS) function and play a significant role in maintaining brain energy metabolism. However, in certain scenarios, astrocyte behavior can go awry, which poses a significant threat to brain integrity and function. This is definitively the case for mutations that turn normal astrocytes and astrocytic precursors into gliomas, an aggressive type of brain tumor. In addition, healthy astrocytes can interact with tumor cells, becoming part of the tumor microenvironment and influencing disease progression. In this review, we discuss the recent evidence suggesting that disturbed metabolism in astrocytes can contribute to the development and progression of fatal human diseases such as cancer. Emphasis is placed on detailing the molecular bases and metabolic pathways of this disease and highlighting unique metabolic vulnerabilities that can potentially be exploited to develop successful therapeutic opportunitiesThis work was supported by grants to Dr. Balsa from the Spanish Government, Ministerio de Ciencia e Innovacion ´ (PID2019-110766GAI00), European Union’s Horizon 2020 research and innovation program (ERC-2020-STG-948478) and Fundacion ´ CRIS contra el cancer (PR_EX_2022-01

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

[EN] 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

Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes

[EN] Connexin43 (Cx43), the major protein forming gap junctions in astrocytes, is reduced in high-grade gliomas, where its ectopic expression exerts important effects, including the inhibition of the proto-oncogene tyrosine-protein kinase Src (c-Src). In this work we aimed to investigate the mechanism responsible for this effect. The inhibition of c-Src requires phosphorylation at tyrosine 527 mediated by C-terminal Src kinase (Csk) and dephosphorylation at tyrosine 416 mediated by phosphatases, such as phosphatase and tensin homolog (PTEN). Our results showed that the antiproliferative effect of Cx43 is reduced when Csk and PTEN are silenced in glioma cells, suggesting the involvement of both enzymes. Confocal microscopy and immunoprecipitation assays confirmed that Cx43, in addition to c-Src, binds to PTEN and Csk in glioma cells transfected with Cx43 and in astrocytes. Pull-down assays showed that region 266–283 in Cx43 is sufficient to recruit c-Src, PTEN and Csk and to inhibit the oncogenic activity of c-Src. As a result of c-Src inhibition, PTEN was increased with subsequent inactivation of Akt and reduction of proliferation of human glioblastoma stem cells. We conclude that the recruitment of Csk and PTEN to the region between residues 266 and 283 within the C-terminus of Cx43 leads to c-Src inhibition

A Short Region of Connexin43 Reduces Human Glioma Stem Cell Migration, Invasion, and Survival through Src, PTEN, and FAK

[EN] 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 onmigration and invasion. Here,weshowthat a cell-penetrating peptide based onCX43(TAT-Cx43266-283) inhibited c-Src and focal adhesion kinase (FAK) and upregulated phosphatase and tensinhomolog inglioma 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

Targeting metabolic plasticity in glioma stem cells in vitro and in vivo through specific inhibition of c-Src by TAT-Cx43266-283

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.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)

Efectos antitumorales del péptido inhibidor de c-Src, TAT-Cx43 266-283 en células madre de glioma humano

Tesis por compendio de publicaciones[EN] Glioma stem cells (GSCs) constitute one of the challenges in treating glioblastoma, the most severe and frequent brain tumour. They have self-renewal capacity, multilineage differentiation properties, high oncogenic potential, resistance to conventional therapies and aggressive infiltration into the brain parenchyma. All these features make difficult the whole tumour resection and facilitate tumour recurrence. GSCs express very low levels of connexin43 (Cx43), the main gap-junction channel-forming protein in astrocytes. One important channel-independent function of Cx43 is to inhibit the oncogene c-Src, involved in cell proliferation, migration and survival. In fact, restoring Cx43 inhibits the strong activity of this oncogene in glioma cells, including GSCs. Interestingly, a cell-penetrating peptide based on the region of Cx43 that inhibits c-Src, TAT-Cx43266-283, reverses GSC phenotype and reduces neurosphere formation. In this thesis, we analysed the anti-tumour effects of TAT-Cx43266-283 on human G166 GSCs and more importantly, we set up a protocol for the culture of primary patient-derived GSCs that allowed us to broaden our study to these cells and to gain insight into the anti-tumour mechanism of TAT-Cx43266-283. First, we showed that TAT-Cx43266-283 inhibits G166 and patient-derived GSCs proliferation. To corroborate the proposed mechanism for Cx43-c-Src interaction previously described in rat C6 glioma cells and astrocytes, we used a biotinylated TAT-Cx43266-283 cell-penetrating peptide as a bait to identify its interacting partners. This confirmed that TAT-Cx43266-283 serves as a docking platform that favours the proximity of active c-Src with its endogenous inhibitors, PTEN and Csk, in GSCs. In addition to c-Src inhibition, TAT-Cx43266-283 upregulated PTEN contributing to the reduction of GSC proliferation by the downregulation of AKT activity in human GSCs. Since the inhibition of GSC proliferation by TAT-Cx43266-283 is lost when PTEN is silenced, it could be proposed that this phosphatase is required for the anti-proliferative effect of TAT-Cx43266-283 on GSCs. Because TAT-Cx43266-283 targets c-Src and PTEN, two regulators of the focal adhesion kinase (FAK), which in turn controls migration and invasion, we addressed the effects of TAT-Cx43266-283 on FAK activity. FAK active levels decreased after 15 h (Y576 and Y577 FAK) and 24 h (Y397 FAK) of treatment with TAT-Cx43266-283 on patient-derived GSCs. The inhibition of this c-Src downstream motility cascade was patent when individual G166 and patient-derived GSC trajectories were tracked because they showed a significant decrease in their lengths. Invasive properties through Matrigel-inserts were also reduced in G166 and patient-derived GSCs when these cells were exposed to TAT-Cx43266-283. Furthermore, we performed a time-lapse microscopy study on fresh tumour explants from the same surgical specimens used to obtain GSCs. Importantly, these movies showed a strong decrease in cell proliferation, migration and survival of those tumour explants treated with TAT-Cx43266-283. Finally, another region of Cx43 (amino acids 274-291) was used to confirm the specificity of TAT-Cx43266-283. We showed that TAT-Cx43274-291 did not affect significantly migration or proliferation in G166 and patient-derived GSCs, thus verifying TAT-Cx43266-283 specificity. This PhD thesis advances the understanding of the molecular mechanisms triggered by Cx43-Src interaction that includes the PTEN-FAK axis. As a consequence of this signalling pathway, this study shows the anti-tumour effects of TAT-Cx43266-283 on the reduction of cell proliferation, migration and survival in two highly relevant glioma models, such as patient-derived GSCs and freshly removed surgical specimens of malignant gliomas

Connexin43-Src interacting sequence as a cell-penetrating peptide to study human primary glioma stem cells invasion

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015.Connexin43 (Cx43) is the main gap junction channel-forming protein in astrocytes, the most abundant glial cells in central nervous system. This protein is downregulated in brain tumours called gliomas. Tumour initiation, relapse, and therapeutic resistance in gliomas is attributed to Glioma Stem Cells (GSCs). Interestingly, several cell-penetrating peptides (CPPs) containing different regions of Cx43 involved in c-Src interaction reverse Glioma Stem Cells (GSCs) phenotype and reduce the rate of cell growth. Considering the controversial Cx43 migration properties and the infi ltrative nature of these tumours, we have investigated the role of these CPPs in human primary GSC migration and invasion. Human primary GSCs were obtained from fresh tumour biopsies and were treated with CPPs. Human GSCs G166 were treated with CPPs. Migration was studied using tiny-tumour cultures, Time-Lapse live-cell Imaging and Immunocytochemistry. Invasion was studied using 8.0 μM pore transwell inserts with or without Matrigel. The mechanism involved in migration was studied by Western blot, evaluating the activity of Focal Adhesion Kinase (FAK). Our findings indicate that our CPPs reduced the rate of human primary GSCs and G166 GSCs migration and invasion. In addition, CPPs inhibited c-Src activity in these cells and consequently decreased FAK phosphorylation necessary to establish adequate focal adhesions in order to migrate. It should be mentioned that FAK is activated by Src-mediated phosphorylation. In conclusion, our results show that c-Src plays an essential role in the effects of Cx43 on migration and suggest these CPPs by inhibiting migration and invasion could be the basis for promising therapies.Peer reviewe

The invasion of glioblastoma stem cells is reduced by the sequence of the connexin43 that interacts with c-Src

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Glioblastoma stem cells (GSC) constitute a niche of cells with self-renewal ability and resistance to conventional therapies. Thus, they are responsible for relapse in these aggressive brain tumors when their resection is not complete. The expression of connexin43 (Cx43), the main gap junction channel-forming protein, and c-Src, a non receptor tyrosine kinase, is inversely related in glioblastoma cells. Interestingly, restoring Cx43 in GSC reverses GSC phenotype. The sequence responsible for this effect is the region that interacts with c-Src (amino acids 266-283). The Focal Adhesion Kinase (FAK) requires the c-Src-mediated phosphorylation in tyrosines 576 and 577 in order to be fully active and promote cell migration. In this study, fresh human tumour biopsies were disaggregated to obtain human primary GSCs. These cells were treated with a cell-penetrating peptide containing the Cx43-Src interacting sequence (Tat-Cx43266-283). The migration was analyzed by tracking individual cell trajectories in cultures recorded by Time-Lapse Live-cell Imaging. Matrigel-treated transwell inserts were used to analyze the invasion and the levels of phosphorylation of FAK tyrosines 576 and 577 were evaluated by western Blot. Our results showed that Tat-Cx43266-283 reduced the migration and invasion of human primary GSC. The analysis of c-Src and FAK proteins suggests that the mechanism by which this reduction takes place includes the inhibition of c-Src/FAK axis. In conclusion, Tat-Cx43266-283 inhibits c-Src with the subsequent reduction in FAK phosphorylation required to establish appropriate focal adhesions for migration. All together, these results indicate an important antivasive role of the sequence of Cx43 that interacts with c-Src in glioma stem cells.Peer reviewe

A short region of connexin43 reduces human glioma stem cell migration, invasion, and survival through Src, PTEN, and FAK

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-Cx43) inhibited c-Src and focal adhesion kinase (FAK) and upregulated phosphatase and tensin homolog in glioma stem cells (GSCs) derived from patients. Consequently, TAT-Cx43 reduced GSC motility, as analyzed by time-lapse microscopy, and strongly reduced their invasive ability. Interestingly, we investigated the effects of TAT-Cx43 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.This work was supported by the Ministerio de Economía y Competitividad, Spain (FEDER BFU2015-70040-R), Junta de Castilla y León, Spain (FEDER SA026U16), and Fundación Ramón Areces. M.J.-R. was a fellowship recipient from the Junta de Castilla y León and the European Social Fund.Peer Reviewe

A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity

The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43266–283, inhibits Src activity, we investigated the effect of TAT-Cx43266–283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43266–283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43266–283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43266–283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43266–283. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43266–283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43266–283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay. In fact, TAT-Cx43266–283 and dasatinib, a potent c-Src inhibitor, strongly reduced the activation of astrocyte hemichannels. In conclusion, our results suggest that TAT-Cx43266–283 exerts a neuroprotective effect through the reduction of hemichannel activity likely mediated by c-Src in astrocytes. These data unveil a new role of c-Src in the regulation of Cx43-hemichannel activity that could be part of the mechanism by which astroglial c-Src participates in neuroinflammation