Src: coordinating metabolism in cancer.

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.

Impairment of Autophagic Flux Participates in the Antitumor Effects of TAT-Cx43266-283 in Glioblastoma Stem Cells.

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.

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. 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).

Connexin43 peptide, TAT-Cx43266-283, selectively targets glioma cells, impairs malignant growth, and enhances survival in mouse models in vivo.

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.

Identification of potassium channel proteins Kv7.2/7.3 as common partners of the dopamine and glutamate transporters DAT and GLT-1.

Dopamine and glutamate transporters (DAT and GLT-1, respectively) share some biophysical characteristics, as both are secondary active carriers coupled to electrochemical ion gradients. In order to identify common or specific components of their respective proteomes, we performed a proximity labelling assay (BioID) in the hippocampal cell line HT22. While most of the identified proteins were specific for each transporter (and will be analyzed elsewhere), we detected two membrane proteins in the shared interactome of GLT-1 and DAT: the transmembrane protein 263 (Tmem263) and the potassium channel protein Kv7.3. However, only Kv7.3 formed immunoprecipitable complexes with GLT-1 and DAT in lysates of transfected HEK293 cells. Moreover, either DAT or GLT-1 co-clustered with Kv7.2/7.3 along the axonal tracts in co-transfected primary neurons, indicating a close spatial proximity between these proteins. Kv7.3, forming heterotetramers with the closely related subunit Kv7.2, underlies the M-currents that control the resting membrane potential and spiking activity in neurons. To investigate whether the presence of the potassium channel affected DAT or GLT-1 function, we performed uptake determinations using radioactive substrate and electrophysiological measurements. Uptake through both transporters was mildly stimulated by the presence of the channel, an effect that was reversed by the potassium channel blocker XE-991. Electrophysiological recording (in transfected HT22 and differentiated SH-SY5Y cells) indicated that the depolarizing effect induced by the presence of the neurotransmitter was reverted by the activity of the potassium channel. Altogether, these data suggest a tight spatial and functional relationship between the DAT/GLT-1 transporters and the Kv7.2/7.3 potassium channel that immediately readjusts the membrane potential of the neuron, probably to limit the neurotransmitter-mediated neuronal depolarization. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.