Elevated FOXG1 in glioblastoma stem cells cooperates with Wnt/ß-catenin to induce exit from quiescence.

Glioblastoma (GBM) stem cells (GSCs) display phenotypic and molecular features reminiscent of normal neural stem cells and exhibit a spectrum of cell cycle states (dormant, quiescent, proliferative). However, mechanisms controlling the transition from quiescence to proliferation in both neural stem cells (NSCs) and GSCs are poorly understood. Elevated expression of the forebrain transcription factor FOXG1 is often observed in GBMs. Here, using small-molecule modulators and genetic perturbations, we identify a synergistic interaction between FOXG1 and Wnt/ß-catenin signaling. Increased FOXG1 enhances Wnt-driven transcriptional targets, enabling highly efficient cell cycle re-entry from quiescence; however, neither FOXG1 nor Wnt is essential in rapidly proliferating cells. We demonstrate that FOXG1 overexpression supports gliomagenesis in vivo and that additional ß-catenin induction drives accelerated tumor growth. These data indicate that elevated FOXG1 cooperates with Wnt signaling to support the transition from quiescence to proliferation in GSCs.

Lrig1 regulates the balance between proliferation and quiescence in glioblastoma stem cells.

Patients with glioblastoma (GBM) face a dismal prognosis. GBMs are driven by glioblastoma stem cells (GSCs) that display a neural stem cell (NSC)-like phenotype. These glioblastoma stem cells are often in a quiescent state that evades current therapies, namely debulking surgery and chemo/radiotherapy. Leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins have been implicated as regulators of growth factor signalling across many tissue stem cells. Lrig1 is highly expressed in gliomas and importantly, polymorphisms have been identified that are risk alleles for patients with GBM, which suggests some functional role in gliomagenesis. We previously reported that Lrig1 is a gatekeeper of quiescence exit in adult mouse neural stem cells, suppressing epidermal growth factor receptor signalling prior to cell cycle re-entry. Here, we perform gain- and loss-of-function studies to understand the function of Lrig1 in glioblastoma stem cells. Using a novel mouse glioblastoma stem cell model, we show that genetic ablation of Lrig1 in cultured GBM stem cells results in higher proliferation and loss of quiescence. In vivo, mice transplanted with glioblastoma stem cells lacking Lrig1 display lower survival compared to Lrig1 WT glioblastoma stem cells, with tumours displaying increased proportions of proliferative cells and reduced quiescent subpopulations. In contrast, Lrig1 overexpression in mouse glioblastoma stem cells results in enhanced quiescence and reduced proliferation, with impaired tumour formation upon orthotopic transplantation. Mechanistically, we find that Lrig1-null cells have a deficiency in BMP signalling responses that may underlie their lack of responsiveness to quiescence cues in vivo. These findings highlight important roles for Lrig1 in controlling responsiveness to both epidermal growth factor receptor and BMPR signalling, and hence the proportions of quiescent and proliferative subpopulations in GBMs.

Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.

Modelling glioblastoma tumour-host cell interactions using adult brain organotypic slice co-culture.

Glioblastoma multiforme (GBM) is an aggressive incurable brain cancer. The cells that fuel the growth of tumours resemble neural stem cells found in the developing and adult mammalian forebrain. These are referred to as glioma stem cells (GSCs). Similar to neural stem cells, GSCs exhibit a variety of phenotypic states: dormant, quiescent, proliferative and differentiating. How environmental cues within the brain influence these distinct states is not well understood. Laboratory models of GBM can be generated using either genetically engineered mouse models, or via intracranial transplantation of cultured tumour initiating cells (mouse or human). Unfortunately, these approaches are expensive, time-consuming, low-throughput and ill-suited for monitoring live cell behaviours. Here, we explored whole adult brain coronal organotypic slices as an alternative model. Mouse adult brain slices remain viable in a serum-free basal medium for several weeks. GSCs can be easily microinjected into specific anatomical sites ex vivo, and we demonstrate distinct responses of engrafted GSCs to diverse microenvironments in the brain tissue. Within the subependymal zone - one of the adult neural stem cell niches - injected tumour cells could effectively engraft and respond to endothelial niche signals. Tumour-transplanted slices were treated with the antimitotic drug temozolomide as proof of principle of the utility in modelling responses to existing treatments. Engraftment of mouse or human GSCs onto whole brain coronal organotypic brain slices therefore provides a simplified, yet flexible, experimental model. This will help to increase the precision and throughput of modelling GSC-host brain interactions and complements ongoing in vivo studies. This article has an associated First Person interview with the first author of the paper.

Elevated FOXG1 and SOX2 in glioblastoma enforces neural stem cell identity through transcriptional control of cell cycle and epigenetic regulators.

Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3, Plk1, Mycn, Dnmt1, Dnmt3b, and Tet3). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis-regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1-null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators.

Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells.

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.

Reduced connexin43 expression correlates with c-Src activation, proliferation, and glucose uptake in reactive astrocytes after an excitotoxic insult.

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.

Inhibition of ATP-sensitive potassium channels increases HSV-tk/GCV bystander effect in U373 human glioma cells by enhancing gap junctional intercellular communication.

It is well known that the efficiency of Herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) therapy is improved by the bystander effect, which mainly relies on gap junctional intercellular communication (GJIC). Malignant gliomas communicate poorly through gap junctions, consequently, agents with the ability to increase GJIC are good candidates to improve the efficiency of this therapy. Since we previously showed that the inhibition of ATP-sensitive potassium (KATP) channels promoted by tolbutamide increased GJIC in rat C6 glioma cells, we have investigated whether tolbutamide could increase the bystander effect in HSV-tk/GCV therapy against human glioma cells. We found that tolbutamide increased GJIC in U373 human glioma cells, an effect that was due to the up-regulation of connexin43, a protein that forms gap junctions channels. More interestingly, our results show that tolbutamide increased the efficiency of HSV-tk/GCV in co-cultures containing U373 cells and U373 cells transfected with HSV-tk. This effect was impaired in the presence of carbenoxolone, an inhibitor of GJIC. Furthermore, tolbutamide did not enhance the bystander effect in connexin43-silenced co-cultures. Together our results reveal that the inhibition of KATP channels promoted by tolbutamide enhances the bystander effect in HSV-tk/GCV therapy by increasing connexin43-mediated gap junctional intercellular communication in U373 human glioma cells.