Heparan Sulfate Synthesized by Ext1 Regulates Receptor Tyrosine Kinase Signaling and Promotes Resistance to EGFR Inhibitors in GBM.

Signaling from multiple receptor tyrosine kinases (RTK) contributes to therapeutic resistance in glioblastoma (GBM). Heparan sulfate (HS), present on cell surfaces and in the extracellular matrix, regulates cell signaling via several mechanisms. To investigate the role for HS in promoting RTK signaling in GBM, we generated neural progenitor cells deficient for HS by knockout of the essential HS-biosynthetic enzyme Ext1, and studied tumor initiation and progression. HS-null cells had decreased proliferation, invasion, and reduced activation of multiple RTKs compared with control. In vivo tumor establishment was significantly decreased, and rate of tumor growth reduced with HS-deficient cells implanted in an HS-poor microenvironment. To investigate if HS regulates RTK activation through platelet-derived growth factor receptor α (PDGFRα) signaling, we removed cell surface HS in patient-derived GBM lines and identified reduced cell surface PDGF-BB ligand. Reduced ligand levels were associated with decreased phosphorylation of PDGFRα, suggesting HS promotes ligand-receptor interaction. Using human GBM tumorspheres and a murine GBM model, we show that ligand-mediated signaling can partially rescue cells from targeted RTK inhibition and that this effect is regulated by HS. Indeed, tumor cells deficient for HS had increased sensitivity to EGFR inhibition in vitro and in vivo. IMPLICATIONS: Our study shows that HS expressed on tumor cells and in the tumor microenvironment regulates ligand-mediated signaling, promoting tumor cell proliferation and invasion, and these factors contribute to decreased tumor cell response to targeted RTK inhibition.

Mechanisms of Resistance to EGFR Inhibition Reveal Metabolic Vulnerabilities in Human GBM.

Amplification of the epidermal growth factor receptor gene (EGFR) represents one of the most commonly observed genetic lesions in glioblastoma (GBM); however, therapies targeting this signaling pathway have failed clinically. Here, using human tumors, primary patient-derived xenografts (PDX), and a murine model for GBM, we demonstrate that EGFR inhibition leads to increased invasion of tumor cells. Further, EGFR inhibitor-treated GBM demonstrates altered oxidative stress, with increased lipid peroxidation, and generation of toxic lipid peroxidation products. A tumor cell subpopulation with elevated aldehyde dehydrogenase (ALDH) levels was determined to comprise a significant proportion of the invasive cells observed in EGFR inhibitor-treated GBM. Our analysis of the ALDH1A1 protein in newly diagnosed GBM revealed detectable ALDH1A1 expression in 69% (35/51) of the cases, but in relatively low percentages of tumor cells. Analysis of paired human GBM before and after EGFR inhibitor therapy showed an increase in ALDH1A1 expression in EGFR-amplified tumors (P < 0.05, n = 13 tumor pairs), and in murine GBM ALDH1A1-high clones were more resistant to EGFR inhibition than ALDH1A1-low clones. Our data identify ALDH levels as a biomarker of GBM cells with high invasive potential, altered oxidative stress, and resistance to EGFR inhibition, and reveal a therapeutic target whose inhibition should limit GBM invasion.

A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment.

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

Antisecretory Factor-Mediated Inhibition of Cell Volume Dynamics Produces Antitumor Activity in Glioblastoma.

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AF's ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777-90. ©2018 AACR.

Glioma-induced SIRT1-dependent activation of hMOF histone H4 lysine 16 acetyltransferase in microglia promotes a tumor supporting phenotype.

High-grade gliomas are malignant aggressive primary brain tumors with limited therapeutic options, and dismal prognosis for patients. Microglia, the resident immune cells of the brain, are recruited and reprogrammed into tumor-supporting cells by glioma cells, which in turn positively influence tumor expansion and infiltration into surrounding brain tissues. Here, we report that glioma-induced microglia conversion is coupled to an increase of histone H4 lysine 16 (H4K16) acetylation level in microglia, through increased nuclear localization of the deacetylase SIRT1, which in turn results in deacetylation of the H4K16 acetyltransferase hMOF and its recruitment to the chromatin at promoter regions of microglial target genes. Furthermore, we demonstrate that manipulation of the microglial H4K16 acetylation level, taking advantage of the intrinsic H4K16 deacetylase or acetyltransferase activities of SIRT1 and hMOF, respectively, modulated the tumor-supporting function of microglia. This study provides evidence that post-translational modifications of histones and the histone-modifying enzymes controlling them, such as H4K16 acetylation regulated by hMOF and SIRT1, are part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells and represent potentially novel therapeutic targets.

Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion.

Glioblastoma (GBM) is the most common primary malignant brain tumor of adults and confers a poor prognosis due, in part, to diffuse invasion of tumor cells. Heparan sulfate (HS) glycosaminoglycans, present on the cell surface and in the extracellular matrix, regulate cell signaling pathways and cell-microenvironment interactions. In GBM, the expression of HS glycosaminoglycans and the enzymes that regulate their function are altered, but the actual HS content and structure are unknown. However, inhibition of HS glycosaminoglycan function is emerging as a promising therapeutic strategy for some cancers. In this study, we use liquid chromatography-mass spectrometry analysis to demonstrate differences in HS disaccharide content and structure across four patient-derived tumorsphere lines (GBM1, 5, 6, 43) and between two murine tumorsphere lines derived from murine GBM with enrichment of mesenchymal and proneural gene expression (mMES and mPN, respectively) markers. In GBM, the heterogeneous HS content and structure across patient-derived tumorsphere lines suggested diverse functions in the GBM tumor microenvironment. In GBM5 and mPN, elevated expression of sulfatase 2 (SULF2), an extracellular enzyme that alters ligand binding to HS, was associated with low trisulfated HS disaccharides, a substrate of SULF2. In contrast, other primary tumorsphere lines had elevated expression of the HS-modifying enzyme heparanase (HPSE). Using gene editing strategies to inhibit HPSE, a role for HPSE in promoting tumor cell adhesion and invasion was identified. These studies characterize the heterogeneity in HS glycosaminoglycan content and structure across GBM and reveal their role in tumor cell invasion.Implications: HS-interacting factors promote GBM invasion and are potential therapeutic targets. Mol Cancer Res; 15(11); 1623-33. ©2017 AACR.

GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity.

Abnormal activation of the epidermal growth factor receptor (EGFR) due to a deletion of exons 2-7 of EGFR (EGFRvIII) is a common alteration in glioblastoma (GBM). While this alteration can drive gliomagenesis, tumors harboring EGFRvIII are heterogeneous. To investigate the role for EGFRvIII activation in tumor phenotype we used a neural progenitor cell-based murine model of GBM driven by EGFR signaling and generated tumor progenitor cells with high and low EGFRvIII activation, pEGFRHi and pEGFRLo. In vivo, ex vivo, and in vitro studies suggested a direct association between EGFRvIII activity and increased tumor cell proliferation, decreased tumor cell adhesion to the extracellular matrix, and altered progenitor cell phenotype. Time-lapse confocal imaging of tumor cells in brain slice cultures demonstrated blood vessel co-option by tumor cells and highlighted differences in invasive pattern. Inhibition of EGFR signaling in pEGFRHi promoted cell differentiation and increased cell-matrix adhesion. Conversely, increased EGFRvIII activation in pEGFRLo reduced cell-matrix adhesion. Our study using a murine model for GBM driven by a single genetic driver, suggests differences in EGFR activation contribute to tumor heterogeneity and aggressiveness.

Epidermal growth factor treatment of the adult brain subventricular zone leads to focal microglia/macrophage accumulation and angiogenesis.

One of the major components of the subventricular zone (SVZ) neurogenic niche is the specialized vasculature. The SVZ vasculature is thought to be important in regulating progenitor cell proliferation and migration. Epidermal growth factor (EGF) is a mitogen with a wide range of effects. When stem and progenitor cells in the rat SVZ are treated with EGF, using intracerebroventricular infusion, dysplastic polyps are formed. Upon extended infusion, blood vessels are recruited into the polyps. In the current study we demonstrate how polyps develop through distinct stages leading up to angiogenesis. As polyps progress, microglia/macrophages accumulate in the polyp core concurrent with increasing cell death. Both microglia/macrophage accumulation and cell death peak during angiogenesis and subsequently decline following polyp vascularization. This model of inducible angiogenesis in the SVZ neurogenic niche suggests involvement of microglia/macrophages in acquired angiogenesis and can be used in detail to study angiogenesis in the adult brain.

Radixin inhibition decreases adult neural progenitor cell migration and proliferation in vitro and in vivo.

Neuronal progenitors capable of long distance migration are produced throughout life in the subventricular zone (SVZ). Migration from the SVZ is carried out along a well-defined pathway called the rostral migratory stream (RMS). Our recent finding of the specific expression of the cytoskeleton linker protein radixin in neuroblasts suggests a functional role for radixin in RMS migration. The ezrin-radixin-moesin (ERM) family of proteins is capable of regulating migration through interaction with the actin cytoskeleton and transmembrane proteins. The ERM proteins are differentially expressed in the RMS with radixin and moesin localized to neuroblasts, and ezrin expression confined to astrocytes of the glial tubes. Here, we inhibited radixin function using the quinocarmycin analog DX52-1 which resulted in reduced neuroblast migration in vitro, while glial migration remained unaltered. Furthermore, the morphology of neuroblasts was distorted resulting in a rounded shape with no or short polysialylated neural cell adhesion molecule positive processes. Intracerebroventricular infusion of the radixin inhibitor resulted in accumulation of neuroblasts in the anterior SVZ. Neuroblast chains were short and intermittently interrupted in the SVZ and considerably disorganized in the RMS. Moreover, we studied the proliferation activity in the RMS after radixin inhibition, since concentrated radixin expression has been demonstrated in the cleavage furrow of dividing cells, which indicates a role of radixin in cell division. Radixin inhibition decreased neuroblast proliferation, whereas the proliferation of other cells in the RMS was not affected. Our results demonstrate a significant role for radixin in neuroblast proliferation and migration.

Time-of-flight secondary ion mass spectrometry based molecular histology of human spinal cord tissue and motor neurons.

Secondary ion mass spectrometry is a powerful method for imaging biological samples with high spatial resolution. Whole section time-of-flight-secondary ion mass spectrometry (TOF-SIMS) scans and multivariate data analysis have been performed on the human spinal cord in order to delineate anatomical regions of interest based on their chemical distribution pattern. TOF-SIMS analysis of thoracic spinal cord sections was performed at 5 µm resolution within 2 h. Multivariate image analysis by means of principal component analysis and maximum auto correlation factor analysis resulted in detection of more than 400 m/z peaks that were found to be significantly changed. Here, the results show characteristic biochemical distributions that are well in line with major histological regions, including gray and white matter. As an approach for iterative segmentation, we further evaluated previously outlined regions of interest as identified by multivariate image analysis. Here, further discrimination of the gray matter into ventral, lateral, and dorsal neuroanatomical regions was observed. TOF-SIMS imaging has been carried out at submicrometer resolution obtaining localization and characterization of spinal motor neurons based on their chemical fingerprint, including neurotransmitter precursors that serve as molecular indicators for motor neuron integrity. Thus, TOF-SIMS can be used as an approach for chemical histology and pathology. TOF-SIMS holds immense potential for investigating the subcellular mechanisms underlying spinal cord related diseases including chronic pain and amyotrophic lateral sclerosis.

EGF-induced expansion of migratory cells in the rostral migratory stream.

The presence of neural stem cells in the adult brain is currently widely accepted and efforts are made to harness the regenerative potential of these cells. The dentate gyrus of the hippocampal formation, and the subventricular zone (SVZ) of the anterior lateral ventricles, are considered the main loci of adult neurogenesis. The rostral migratory stream (RMS) is the structure funneling SVZ progenitor cells through the forebrain to their final destination in the olfactory bulb. Moreover, extensive proliferation occurs in the RMS. Some evidence suggest the presence of stem cells in the RMS, but these cells are few and possibly of limited differentiation potential. We have recently demonstrated the specific expression of the cytoskeleton linker protein radixin in neuroblasts in the RMS and in oligodendrocyte progenitors throughout the brain. These cell populations are greatly altered after intracerebroventricular infusion of epidermal growth factor (EGF). In the current study we investigate the effect of EGF infusion on the rat RMS. We describe a specific increase of radixin(+)/Olig2(+) cells in the RMS. Negative for NG2 and CNPase, these radixin(+)/Olig2(+) cells are distinct from typical oligodendrocyte progenitors. The expanded Olig2(+) population responds rapidly to EGF and proliferates after only 24 hours along the entire RMS, suggesting local activation by EGF throughout the RMS rather than migration from the SVZ. In addition, the radixin(+)/Olig2(+) progenitors assemble in chains in vivo and migrate in chains in explant cultures, suggesting that they possess migratory properties within the RMS. In summary, these results provide insight into the adaptive capacity of the RMS and point to an additional stem cell source for future brain repair strategies.

Characterization of epidermal growth factor-induced dysplasia in the adult rat subventricular zone.

Epidermal growth factor (EGF) is a mitogen widely used when culturing adult neural stem cells in vitro. Although proliferative effects can also be observed in vivo, intracerebroventricular infusion of EGF has been found to counteract neuronal determination and promote glial differentiation instead. However, EGF receptor activation has different effects on the subventricular zone (SVZ) in mice and rats, possibly because of species differences in SVZ cell composition. Specifically in the rat, EGF stimulation of the SVZ induces the formation of hyperplastic polyps. The present study aims at molecular and morphological characterization of these subventricular polyps. Using immunohistochemistry, electron microscopy, and gene expression analysis, we demonstrate in hyperplastic EGF-induced polyps an upregulation in protein expression of Sox2, Olig2, GFAP, nestin, and vimentin. We found polyp-specific dysplastic changes in the form of coexpression of Sox2 and Olig2. This highly proliferative, Sox2/Olig2 coexpressing dysplastic cell type is >10-fold enriched in the hyperplastic polyps compared with control SVZ and most likely causes the polyp formation. Unique ultrastructural features of the polyps include a lack of ependymal cell lining as well as a large number of cells with large, light, ovoid nuclei and a cytoplasm with abundant ribosomes, whereas other polyp cells contain invaginated nuclei but fewer ribosomes. EGF also induced changes in the expression of Id genes Id1, Id2, and Id4 in the SVZ. Taken together, we here demonstrate dysplastic, structural, and phenotypical changes in the rat SVZ following EGF stimulation, which are specific to hyperplastic polyps.

Unique gene expression patterns indicate microglial contribution to neural stem cell recovery following irradiation.

Ionizing radiation results in damage to neural stem cells and reduced neurogenesis. The aim of the present study was to determine intrinsic and extrinsic factors that influence neural stem cell survival following irradiation, using qPCR. Gene expression of hippocampal and SVZ neurospheres were analyzed following irradiation, and results demonstrated that irradiated hippocampal and SVZ stem cells displayed similar gene expression profiles for intrinsic genes. Irradiated microglia (extrinsic factor) isolated from the SVZ exhibited increased gene expression of growth factors involved in stem cell maintenance, proliferation, and survival. However, microglial genes in the irradiated hippocampus responded less favorably with respect to stem cell recovery. This might explain the superior recovery of SVZ compared to hippocampal stem cells following in vivo irradiation. In addition, our results show that a combination of growth factors, which were upregulated in SVZ microglia, increased the proliferation and decreased cell death of irradiated neurospheres in vitro.