CLIC1 regulation of cancer stem cells in glioblastoma.

Chloride intracellular channel 1 (CLIC1) has emerged as a therapeutic target in various cancers. CLIC1 promotes cell cycle progression and cancer stem cell (CSC) self-renewal. Furthermore, CLIC1 is shown to play diverse roles in proliferation, cell volume regulation, tumour invasion, migration, and angiogenesis. In glioblastoma (GB), CLIC1 facilitates the G1/S phase transition and tightly regulates glioma stem-like cells (GSCs), a rare population of self-renewing CSCs with central roles in tumour resistance to therapy and tumour recurrence. CLIC1 is found as either a monomeric soluble protein or as a non-covalent dimeric protein that can form an ion channel. The ratio of dimeric to monomeric protein is altered in GSCs and depends on the cell redox state. Elucidating the mechanisms underlying the alterations in CLIC1 expression and structural transitions will further our understanding of its role in GSC biology. This review will highlight the role of CLIC1 in GSCs and its significance in facilitating different hallmarks of cancer.

Transcriptional reprogramming of skeletal muscle stem cells by the niche environment.

Adult stem cells are indispensable for tissue regeneration, but their function declines with age. The niche environment in which the stem cells reside plays a critical role in their function. However, quantification of the niche effect on stem cell function is lacking. Using muscle stem cells (MuSC) as a model, we show that aging leads to a significant transcriptomic shift in their subpopulations accompanied by locus-specific gain and loss of chromatin accessibility and DNA methylation. By combining in vivo MuSC transplantation and computational methods, we show that the expression of approximately half of all age-altered genes in MuSCs from aged male mice can be restored by exposure to a young niche environment. While there is a correlation between gene reversibility and epigenetic alterations, restoration of gene expression occurs primarily at the level of transcription. The stem cell niche environment therefore represents an important therapeutic target to enhance tissue regeneration in aging.

In situ detection of protein-protein interaction by proximity ligation assay in patient derived brain tumor stem cells.

Improper or aberrant protein-protein interactions can lead to severe human diseases including cancer. Here, we describe an adapted proximity ligation assay (PLA) protocol for the assessment of galectin-1-HOXA5 interaction in brain tumor stem cells (BTSCs). We detail the steps for culturing and preparation of BTSCs followed by PLA and detection of protein interactions in situ using fluorescent microscopy. This PLA protocol is optimized specifically for BTSCs and includes key controls for effective result analysis. For complete details on the use and execution of this protocol, please refer to Sharanek et al. (2021).

Isolation of Adult Mouse Neural Stem Cells and Assessment of Self-Renewal by ELDA.

The generation of new neurons in the adult brain throughout life is integral to brain plasticity and repair. Adult neural stem cells (aNSCs), present in the subventricular zone (SVZ) of the lateral ventricle wall and the subgranular zone (SGZ) of the hippocampal dentate gyrus, divide symmetrically or asymmetrically to maintain the stem cell pool or become committed progenitors and differentiate into various cell lineages. Depletion or dysregulation of aNSCs impairs proper brain connectivity and function and can contribute to several brain diseases including cognitive and neurodegenerative disorders and brain cancer. In this chapter, we present our optimized method to obtain and maintain reproducible neurosphere cultures from the adult mouse brain followed by evaluation of self-renewal using the extreme limiting dilution assay (ELDA) software. We use this assay routinely on aNSCs obtained from patient mouse models to generate log fraction plots and provide confidence intervals for all limiting dilution assay (LDA) data. At the same time, given the low number of NSCs required for the completion of the ELDA experiment, it is feasible to employ this approach to conduct high-content compound screening for therapeutic interventions aimed at enhancing the stem cell pool or combating a cohort of genetic and epigenetic disorders.

Subcellular fractionation of brain tumor stem cells.

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.

Transcriptional control of brain tumor stem cells by a carbohydrate binding protein.

Brain tumor stem cells (BTSCs) and intratumoral heterogeneity represent major challenges in glioblastoma therapy. Here, we report that the LGALS1 gene, encoding the carbohydrate binding protein, galectin1, is a key regulator of BTSCs and glioblastoma resistance to therapy. Genetic deletion of LGALS1 alters BTSC gene expression profiles and results in downregulation of gene sets associated with the mesenchymal subtype of glioblastoma. Using a combination of pharmacological and genetic approaches, we establish that inhibition of LGALS1 signaling in BTSCs impairs self-renewal, suppresses tumorigenesis, prolongs lifespan, and improves glioblastoma response to ionizing radiation in preclinical animal models. Mechanistically, we show that LGALS1 is a direct transcriptional target of STAT3 with its expression robustly regulated by the ligand OSM. Importantly, we establish that galectin1 forms a complex with the transcription factor HOXA5 to reprogram the BTSC transcriptional landscape. Our data unravel an oncogenic signaling pathway by which the galectin1/HOXA5 complex maintains BTSCs and promotes glioblastoma.

OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation.

Glioblastoma contains a rare population of self-renewing brain tumor stem cells (BTSCs) which are endowed with properties to proliferate, spur the growth of new tumors, and at the same time, evade ionizing radiation (IR) and chemotherapy. However, the drivers of BTSC resistance to therapy remain unknown. The cytokine receptor for oncostatin M (OSMR) regulates BTSC proliferation and glioblastoma tumorigenesis. Here, we report our discovery of a mitochondrial OSMR that confers resistance to IR via regulation of oxidative phosphorylation, independent of its role in cell proliferation. Mechanistically, OSMR is targeted to the mitochondrial matrix via the presequence translocase-associated motor complex components, mtHSP70 and TIM44. OSMR interacts with NADH ubiquinone oxidoreductase 1/2 (NDUFS1/2) of complex I and promotes mitochondrial respiration. Deletion of OSMR impairs spare respiratory capacity, increases reactive oxygen species, and sensitizes BTSCs to IR-induced cell death. Importantly, suppression of OSMR improves glioblastoma response to IR and prolongs lifespan.

Characterization of a Mouse Model of Börjeson-Forssman-Lehmann Syndrome.

Mutations of the transcriptional regulator PHF6 cause the X-linked intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS), but the pathogenesis of BFLS remains poorly understood. Here, we report a mouse model of BFLS, generated using a CRISPR-Cas9 approach, in which cysteine 99 within the PHD domain of PHF6 is replaced with phenylalanine (C99F). Mice harboring the patient-specific C99F mutation display deficits in cognitive functions, emotionality, and social behavior, as well as reduced threshold to seizures. Electrophysiological studies reveal that the intrinsic excitability of entorhinal cortical stellate neurons is increased in PHF6 C99F mice. Transcriptomic analysis of the cerebral cortex in C99F knockin mice and PHF6 knockout mice show that PHF6 promotes the expression of neurogenic genes and represses synaptic genes. PHF6-regulated genes are also overrepresented in gene signatures and modules that are deregulated in neurodevelopmental disorders of cognition. Our findings advance our understanding of the mechanisms underlying BFLS pathogenesis.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

A focused compound screen highlights the significance of epidermal growth factor receptor signalling in chordoma pathogenesis.

Chordoma is a rare primary bone cancer with limited treatment options. Surgical resection followed by radiotherapy has proven effective; however, when, in 30-40% of patients, tumours recur and metastasize, a high level of resistance to chemotherapies leaves these patients with a dearth of treatment options. Recent work published in the Journal of Pathology by Scheipl et al describing a focused compound drug screen highlights the significance of epidermal growth factor receptor (EGFR) signalling in chordoma, and shows potential for EGFR inhibitors as a way forward for developing an effective treatment for chordoma. Importantly, combining EGFR inhibitors with a MET inhibitor induces a synergistic effect on growth inhibition of resistant chordoma cells, highlighting the significance of combined EGFR and MET inhibitors as a potential avenue to defeat chemoresistance in chordoma patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Control of glioblastoma tumorigenesis by feed-forward cytokine signaling.

EGFRvIII-STAT3 signaling is important in glioblastoma pathogenesis. Here, we identified the cytokine receptor OSMR as a direct target gene of the transcription factor STAT3 in mouse astrocytes and human brain tumor stem cells (BTSCs). We found that OSMR functioned as an essential co-receptor for EGFRvIII. OSMR formed a physical complex with EGFRvIII, and depletion of OSMR impaired EGFRvIII-STAT3 signaling. Conversely, pharmacological inhibition of EGFRvIII phosphorylation inhibited the EGFRvIII-OSMR interaction and activation of STAT3. EGFRvIII-OSMR signaling in tumors operated constitutively, whereas EGFR-OSMR signaling in nontumor cells was synergistically activated by the ligands EGF and OSM. Finally, knockdown of OSMR strongly suppressed cell proliferation and tumor growth of mouse glioblastoma cells and human BTSC xenografts in mice, and prolonged the lifespan of these mice. Our findings identify OSMR as a critical regulator of glioblastoma tumor growth that orchestrates a feed-forward signaling mechanism with EGFRvIII and STAT3 to drive tumorigenesis.

STAT3-iNOS Signaling Mediates EGFRvIII-Induced Glial Proliferation and Transformation.

Malignant gliomas, including glioblastoma multiforme, constitute the most common and aggressive primary brain tumors in adults. The transcription factor signal transducer and activator of transcription 3 (STAT3) plays an essential role in glioblastoma pathogenesis downstream of the major oncogenic protein epidermal growth factor receptor variant III (EGFRvIII). However, the critical gene targets of STAT3 that mediate EGFRvIII-induced glial transformation have remained unknown. Here, we identify inducible nitric oxide synthase (iNOS) as a novel target gene of STAT3 in EGFRvIII-expressing mouse astrocytes. Endogenous STAT3 occupies the endogenous iNOS promoter and stimulates iNOS transcription in EGFRvIII-expressing astrocytes. STAT3 does not appear to control iNOS transcription in astrocytes deficient in the major glioblastoma tumor suppressor protein phosphatase and tensin homolog (PTEN), suggesting that STAT3 regulates iNOS transcription specifically in EGFRvIII-expressing astrocytes. Importantly, inhibition of iNOS by distinct approaches, including knockdown by RNA interference, reduces cell population growth and invasiveness of EGFRvIII-expressing astrocytes. In addition, upon iNOS knockdown or administration of a small-molecule inhibitor of iNOS, EGFRvIII-expressing astrocytes form smaller tumors in vivo. These findings suggest that inhibition of iNOS may have potential therapeutic value for EGFRvIII-activated brain tumors.

Mcl-1 is a key regulator of apoptosis during CNS development and after DNA damage.

Despite the importance of Mcl-1, an anti-apoptotic Bcl-2 family member, in the regulation of apoptosis, little is known regarding its role in nervous system development and injury-induced neuronal cell death. Because germline deletion of Mcl-1 results in peri-implantation lethality, we address the function of Mcl-1 in the nervous system using two different conditional Mcl-1 mouse mutants in the developing nervous system. Here, we show for the first time that Mcl-1 is required for neuronal development. Neural precursors within the ventricular zone and newly committed neurons in the cortical plate express high levels of Mcl-1 throughout cortical neurogenesis. Loss of Mcl-1 in neuronal progenitors results in widespread apoptosis. Double labeling with active caspase 3 and Tuj1 reveals that newly committed Mcl1 deficient neurons undergo apoptosis as they commence migration away from the ventricular zone. Examination of neural progenitor differentiation in vitro demonstrated that cell death in the absence of Mcl1 is cell autonomous. Although conditional deletion of Mcl-1 in cultured neurons does not trigger apoptosis, loss of Mcl-1 sensitizes neurons to an acute DNA damaging insult. Indeed, the rapid reduction of Mcl-1 mRNA and protein levels are early events after DNA damage in neurons, and maintaining high Mcl-1 levels can protect neurons against death. Together, our results are the first to demonstrate the requirement of Mcl-1, an anti-apoptotic Bcl-2 family protein, for cortical neurogenesis and the survival of neurons after DNA damage.

Caspase-3-mediated cleavage of Akt: involvement of non-consensus sites and influence of phosphorylation.

Here, we show for the first time that Akt1 is cleaved in vitro at the caspase-3 consensus site DQDD(456) downward arrow SM. Our data suggest QEEE(116) downward arrow E(117) downward arrow MD, EEMD(119) downward arrow, TPPD(453) downward arrow QD and DAKE(398) downward arrow IM as novel non-consensus caspase-3 cleavage sites. More importantly, we demonstrate that phosphorylation of Akt1 modulates its cleavage in a site-specific manner: Resistance to cleavage at site DAKE(398) (within the kinase domain) in response to phosphorylation suggests a possible mechanism by which the anti-apoptotic role of Akt1 is regulated. Our result is important in biological models which rely on Akt1 for cell survival.

Chemoresistance in human ovarian cancer: the role of apoptotic regulators.

Ovarian cancer is among the most lethal of all malignancies in women. While chemotherapy is the preferred treatment modality, chemoresistance severely limits treatment success. Recent evidence suggests that deregulation of key pro- and anti-apoptotic pathways is a key factor in the onset and maintenance of chemoresistance. Furthermore, the discovery of novel interactions between these pathways suggests that chemoresistance may be multi-factorial. Ultimately, the decision of the cancer cell to live or die in response to a chemotherapeutic agent is a consequence of the overall apoptotic capacity of that cell. In this review, we discuss the biochemical pathways believed to promote cell survival and how they modulate chemosensitivity. We then conclude with some new research directions by which the fundamental mechanisms of chemoresistance can be elucidated.