Elevated fibroblast growth factor-inducible 14 expression transforms proneural-like gliomas into more aggressive and lethal brain cancer.

High-grade gliomas (HGGs) are aggressive, treatment-resistant, and often fatal human brain cancers. The TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) signaling axis is involved in tissue repair after injury and constitutive signaling has been implicated in the pathogenesis of numerous solid cancers. The Fn14 gene is expressed at low levels in the normal, uninjured brain but is highly expressed in primary isocitrate dehydrogenase wild-type and recurrent HGGs. Fn14 signaling is implicated in numerous aspects of glioma biology including brain invasion and chemotherapy resistance, but whether Fn14 overexpression can directly promote tumor malignancy has not been reported. Here, we used the replication-competent avian sarcoma-leukosis virus/tumor virus A system to examine the impact of Fn14 expression on glioma development and pathobiology. We found that the sole addition of Fn14 to an established oncogenic cocktail previously shown to generate proneural-like gliomas led to the development of highly invasive and lethal brain cancer with striking biological features including extensive pseudopalisading necrosis, constitutive canonical and noncanonical NF-κB pathway signaling, and high plasminogen activator inhibitor-1 (PAI-1) expression. Analyses of HGG patient datasets revealed that high human PAI-1 gene (SERPINE1) expression correlates with shorter patient survival, and that the SERPINE1 and Fn14 (TNFRSF12A) genes are frequently co-expressed in bulk tumor tissues, in tumor subregions, and in malignant cells residing in the tumor microenvironment. These findings provide new insights into the potential importance of Fn14 in human HGG pathobiology and designate both the NF-κB signaling node and PAI-1 as potential targets for therapeutic intervention. MAIN POINTS: This work demonstrates that elevated levels of the TWEAK receptor Fn14 in tumor-initiating, neural progenitor cells leads to the transformation of proneural-like gliomas into more aggressive and lethal tumors that exhibit constitutive NF-κB pathway activation and plasminogen activator inhibitor-1 overexpression.

Targeting BCL-xL improves the efficacy of bromodomain and extra-terminal protein inhibitors in triple-negative breast cancer by eliciting the death of senescent cells.

Inhibitors of bromodomain and extra-terminal proteins (BETi) suppress oncogenic gene expression and have been shown to be efficacious in many in vitro and murine models of cancer, including triple-negative breast cancer (TNBC), a highly aggressive disease. However, in most cancer models, responses to BETi can be highly variable. We previously reported that TNBC cells either undergo senescence or apoptosis in response to BETi, but the specific mechanisms dictating these two cell fates remain unknown. Using six human TNBC cell lines, we show that the terminal response of TNBC cells to BETi is dictated by the intrinsic expression levels of the anti-apoptotic protein B-cell lymphoma-extra large (BCL-xL). BCL-xL levels were higher in cell lines that senesce in response to BETi compared with lines that primarily die in response to these drugs. Moreover, BCL-xL expression was further reduced in cells that undergo BETi-mediated apoptosis. Forced BCL-xL overexpression in cells that normally undergo apoptosis following BETi treatment shifted them to senescence without affecting the reported mechanism of action of BETi in TNBC, that is, mitotic catastrophe. Most importantly, pharmacological or genetic inhibition of BCL-xL induced apoptosis in response to BETi, and inhibiting BCL-xL, even after BETi-induced senescence had already occurred, still induced cell death. These results indicate that BCL-xL provides a senescent cell death-inducing or senolytic target that may be exploited to improve therapeutic outcomes of TNBC in response to BETi. They also suggest that the basal levels of BCL-xL should be predictive of tumor responses to BETi in current clinical trials.

GPR68-ATF4 signaling is a novel prosurvival pathway in glioblastoma activated by acidic extracellular microenvironment.

BACKGROUND: Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of research, the survival rates for GBM remain effectively stagnant. A defining hallmark of GBM is a highly acidic tumor microenvironment, which is thought to activate pro-tumorigenic pathways. This acidification is the result of altered tumor metabolism favoring aerobic glycolysis, a phenomenon known as the Warburg effect. Low extracellular pH confers radioresistant tumors to glial cells. Notably GPR68, an acid sensing GPCR, is upregulated in radioresistant GBM. Usage of Lorazepam, which has off target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. However, the role of tumor microenvironment acidification in GPR68 activation has not been assessed in cancer. Here we interrogate the role of GPR68 specifically in GBM cells using a novel highly specific small molecule inhibitor of GPR68 named Ogremorphin (OGM) to induce the iron mediated cell death pathway: ferroptosis. METHOD: OGM was identified in a non-biased zebrafish embryonic development screen and validated with Morpholino and CRISPR based approaches. Next, A GPI-anchored pH reporter, pHluorin2, was stably expressed in U87 glioblastoma cells to probe extracellular acidification. Cell survival assays, via nuclei counting and cell titer glo, were used to demonstrate sensitivity to GPR68 inhibition in twelve immortalized and PDX GBM lines. To determine GPR68 inhibition's mechanism of cell death we use DAVID pathway analysis of RNAseq. Our major indication, ferroptosis, was then confirmed by western blotting and qRT-PCR of reporter genes including TFRC. This finding was further validated by transmission electron microscopy and liperfluo staining to assess lipid peroxidation. Lastly, we use siRNA and CRISPRi to demonstrate the critical role of ATF4 suppression via GPR68 for GBM survival. RESULTS: We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68. Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested, irrespective of genetic and phenotypic heterogeneity, or resistance to the mainstay GBM chemotherapeutic temozolomide. We use U87 and U138 glioblastoma cell lines to show how selective induction of ferroptosis occurs in an ATF4-dependent manner. Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells. CONCLUSION: These results indicate GPR68 emerges as a critical sensor for an autocrine pro-tumorigenic signaling cascade triggered by extracellular acidification in glioblastoma cells. In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death. These findings provide a promising therapeutic approach to selectively induce ferroptosis in glioblastoma cells while sparing healthy neural tissue.

DHODH inhibition impedes glioma stem cell proliferation, induces DNA damage, and prolongs survival in orthotopic glioblastoma xenografts.

Glioma stem cells (GSCs) promote tumor progression and therapeutic resistance and exhibit remarkable bioenergetic and metabolic plasticity, a phenomenon that has been linked to their ability to escape standard and targeted therapies. However, specific mechanisms that promote therapeutic resistance have been somewhat elusive. We hypothesized that because GSCs proliferate continuously, they may require the salvage and de novo nucleotide synthesis pathways to satisfy their bioenergetic needs. Here, we demonstrate that GSCs lacking EGFR (or EGFRvIII) amplification are exquisitely sensitive to de novo pyrimidine synthesis perturbations, while GSCs that amplify EGFR are utterly resistant. Furthermore, we show that EGFRvIII promotes BAY2402234 resistance in otherwise BAY2402234 responsive GSCs. Remarkably, a novel, orally bioavailable, blood-brain-barrier penetrating, dihydroorotate dehydrogenase (DHODH) inhibitor BAY2402234 was found to abrogate GSC proliferation, block cell-cycle progression, and induce DNA damage and apoptosis. When dosed daily by oral gavage, BAY2402234 significantly impaired the growth of two different intracranial human glioblastoma xenograft models in mice. Given this observed efficacy and the previously established safety profiles in preclinical animal models and human clinical trials, the clinical testing of BAY2402234 in patients with primary glioblastoma that lacks EGFR amplification is warranted.

Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres.

Hypoxia promotes the expansion of non-neoplastic stem and precursor cell populations in the normal brain, and is common in malignant brain tumors. We examined the effects of hypoxia on stem-like cells in glioblastoma (GBM). When GBM-derived neurosphere cultures are grown in 1% oxygen, hypoxia-inducible factor 1alpha (HIF1alpha) protein levels increase dramatically, and mRNA encoding other hypoxic response genes, such as those encoding hypoxia-inducible gene-2, lysyl oxidase, and vascular endothelial growth factor, are induced over 10-fold. Hypoxia increases the stem-like side population over fivefold, and the percentage of cells expressing CD133 threefold or more. Notch pathway ligands and targets are also induced. The rise in the stem-like fraction in GBM following hypoxia is paralleled by a twofold increase in clonogenicity. We believe HIF1alpha plays a causal role in these changes, as when oxygen-stable HIF1alpha is expressed in normoxic glioma cells CD133 is induced. We used digoxin, which has been shown to lower HIF protein levels in vitro and in vivo, to inhibit the hypoxic response. Digoxin suppressed HIF1alpha protein expression, HIF1alpha downstream targets, and slowed tumor growth in vivo. In addition, pretreatment with digoxin reduced GBM flank xenograft engraftment of hypoxic GBM cells, and daily intraperitoneal injections of digoxin were able to significantly inhibit the growth of established subcutaneous glioblastoma xenografts, and suppressed expression of vascular endothelial growth factor.

The Alternative Splicing Factor, MBNL1, Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-Induced Stemness.

Muscleblind-like proteins (MBNL) belong to a family of tissue-specific regulators of RNA metabolism that control premessenger RNA splicing. Inactivation of MBNL causes an adult-to-fetal alternative splicing transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer, glioblastoma (GBM), maintains stem-like features (glioma stem cell, GSC) through hypoxia-induced responses. Accordingly, we hypothesize here that hypoxia-induced responses in GBM might also include MBNL-based alternative splicing to promote tumor progression. When cultured in hypoxia condition, GSCs rapidly exported muscleblind-like-1 (MBNL1) out of the nucleus, resulting in significant inhibition of MBNL1 activity. Notably, hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression, resulting in significantly prolonged survival. This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, where hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may therefore, yield potent tumor suppressor activities, uncovering new therapeutic opportunities to counter this disease. SIGNIFICANCE: This study describes an unexpected mechanism by which RNA-binding protein, MBNL1, activity is inhibited in hypoxia by a simple isoform switch to regulate glioma stem cell self-renewal, tumorigenicity, and progression.

Histone deacetylase inhibitor MPT0B291 suppresses Glioma Growth in vitro and in vivo partially through acetylation of p53.

Background: Histone deacetylase (HDAC) inhibitors have emerged as a new class of anti-tumor agents for various types of tumors, including glioblastoma. Methods and results: We found that a novel HDAC inhibitor, MPT0B291, significantly reduced the cell viability and increased cell death of human and rat glioma cell lines, but not in normal astrocytes. We also demonstrated that MPT0B291 suppressed proliferation by inducing G1 phase cell cycle arrest and increased apoptosis in human and rat glioma cell lines by flow cytometry and immunocytochemistry. We further investigated the anti-tumor effects of MPT0B291 in xenograft (mouse) and allograft (rat) models. The IVIS200 images and histological analysis indicated MPT0B291 (25 mg/kg, p. o.) reduced tumor volume. Mechanistically, MPT0B291 increased phosphorylation and acetylation/activation of p53 and increased mRNA levels of the apoptosis related genes PUMA, Bax, and Apaf1 as well as increased protein level of PUMA, Apaf1 in C6 cell line. The expression of cell cycle related gene p21 was also increased and Cdk2, Cdk4 were decreased by MPT0B291. Conclusion: Our study highlights the anti-tumor efficacy of a novel compound MPT0B291 on glioma growth.

MCT4 regulates de novo pyrimidine biosynthesis in GBM in a lactate-independent manner.

BACKGROUND: Necrotic foci with surrounding hypoxic cellular pseudopalisades and microvascular hyperplasia are histological features found in glioblastoma (GBM). We have previously shown that monocarboxylate transporter 4 (MCT4) is highly expressed in necrotic/hypoxic regions in GBM and that increased levels of MCT4 are associated with worse clinical outcomes. METHODS: A combined transcriptomics and metabolomics analysis was performed to study the effects of MCT4 depletion in hypoxic GBM neurospheres. Stable and inducible MCT4-depletion systems were used to evaluate the effects of and underlining mechanisms associated with MCT4 depletion in vitro and in vivo, alone and in combination with radiation. RESULTS: This study establishes that conditional depletion of MCT4 profoundly impairs self-renewal and reduces the frequency and tumorigenicity of aggressive, therapy-resistant, glioblastoma stem cells. Mechanistically, we observed that MCT4 depletion induces anaplerotic glutaminolysis and abrogates de novo pyrimidine biosynthesis. The latter results in a dramatic increase in DNA damage and apoptotic cell death, phenotypes that were readily rescued by pyrimidine nucleosides supplementation. Consequently, we found that MCT4 depletion promoted a significant prolongation of survival of animals bearing established orthotopic xenografts, an effect that was extended by adjuvant treatment with focused radiation. CONCLUSIONS: Our findings establish a novel role for MCT4 as a critical regulator of cellular deoxyribonucleotide levels and provide a new therapeutic direction related to MCT4 depletion in GBM.

Dse1 may control cross talk between the pheromone and filamentation pathways in yeast.

The filamentous/invasive growth pathway is activated by nutrient limitation in the haploid form of the yeast Saccharomyces cerevisiae, whereas exposure to mating-pheromone causes cells to differentiate into gametes. Although these two pathways respond to very different stimuli and generate very different responses, they utilize many of the same signaling components. This implies the need for robust mechanisms to maintain signal fidelity. Dse1 was identified in an allele-specific suppressor screen for proteins that interact with the pheromone-responsive Gbetagamma, and found to bind both to a Gbetagamma-affinity column, and to the shared MEKK, Ste11. Although overexpression of Dse1 stimulated invasive growth and transcription of both filamentation and mating-specific transcriptional reporters, deletion of DSE1 had no effect on these outputs. In contrast, pheromone hyper-induced transcription of the filamentation reporter in cells lacking Dse1 and in cells expressing a mutant form of Gbeta that exhibits diminished interaction with Dse1. Thus, the interaction of Dse1 with both Gbeta and Ste11 may be designed to control cross talk between the pheromone and filamentation pathways.

Frequent gains at chromosome 7q34 involving BRAF in pilocytic astrocytoma.

Relatively little is known about the molecular changes that promote the formation or growth of pilocytic astrocytomas. We investigated genomic alterations in 25 pilocytic astrocytomas, including 5 supratentorial and 20 posterior fossa tumors, using oligonucleotide array comparative genomic hybridization. Large changes were identified in 7 tumors and included gains of chromosomes 5, 6, and 7 and losses of chromosomes 16, 17, 19, and 22. The most common alteration was a 1.9-MB region of low-level gain at chromosome 7q34 identified in 17 of 20 posterior fossa tumors. In most tumors, the region of gain ended within the BRAF locus and encompassed only exons that encode the BRAF kinase domain. We confirmed copy number increase at the 7q34 locus using quantitative polymerase chain reaction with primers adjacent to the HIPK2, RAB19B, and BRAF genes. Western blot analysis revealed that 3 of 6 pilocytic astrocytomas with 7q34 gain contained high levels of phosphorylated extracellular signal-related kinase (ERK) and nitrogen-activated protein kinase/ERK kinase (MEK), while 1 tumor lacking 7q34 gain and 2 normal brain specimens did not. Immunohistochemical stains of a tissue microarray containing 43 pilocytic astrocytoma identified ERK phosphorylation in 35 (81%). These data indicate that focal gains at chromosome 7q34 and increased BRAF-MEK-ERK signaling are common findings in sporadic pilocytic astrocytomas.

Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma.

Brain tumors can arise following deregulation of signaling pathways normally activated during brain development and may derive from neural stem cells. Given the requirement for Hedgehog in non-neoplastic stem cells, we investigated whether Hedgehog blockade could target the stem-like population in glioblastoma multiforme (GBM). We found that Gli1, a key Hedgehog pathway target, was highly expressed in 5 of 19 primary GBM and in 4 of 7 GBM cell lines. Shh ligand was expressed in some primary tumors, and in GBM-derived neurospheres, suggesting a potential mechanism for pathway activation. Hedgehog pathway blockade by cyclopamine caused a 40%-60% reduction in growth of adherent glioma lines highly expressing Gli1 but not in those lacking evidence of pathway activity. When GBM-derived neurospheres were treated with cyclopamine and then dissociated and seeded in media lacking the inhibitor, no new neurospheres formed, suggesting that the clonogenic cancer stem cells had been depleted. Consistent with this hypothesis, the stem-like fraction in gliomas marked by both aldehyde dehydrogenase activity and Hoechst dye excretion (side population) was significantly reduced or eliminated by cyclopamine. In contrast, we found that radiation treatment of our GBM neurospheres increased the percentage of these stem-like cells, suggesting that this standard therapy preferentially targets better-differentiated neoplastic cells. Most importantly, viable GBM cells injected intracranially following Hedgehog blockade were no longer able to form tumors in athymic mice, indicating that a cancer stem cell population critical for ongoing growth had been removed. Disclosure of potential conflicts of interest is found at the end of this article.

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

MMB triazole analogs are potent NF-κB inhibitors and anti-cancer agents against both hematological and solid tumor cells.

Triazole derivatives of melampomagnolide B (MMB) have been synthesized via click chemistry methodologies and screened against a panel of 60 human cancer cell lines. Several derivatives showed promising anti-cancer activity, affording growth inhibition (GI50) values in the nanomolar range (GI50 = 0.02-0.99 µM). Lead compound 7h exhibited EC50 values of 400 nM and 700 nM, respectively, against two AML clinical specimens. Compound 7h was significantly more potent than parthenolide as an inhibitor of p65 phosphorylation in both hematological and solid tumor cell lines, indicating its ability to inhibit the NF-κB pathway. In TMD-231 breast cancer cells, treatment with 7h reduced DNA binding activity of NF-κB through inhibition of IKK-ß mediated p65 phosphorylation and caused elevation of basal IκBα levels through inhibition of constitutive IκBα turnover and NF-κB activation. Molecular docking and dynamic modeling studies indicated that 7h interacts with the kinase domain of the monomeric IKKß subunit, leading to inhibition of IKKß activation, and compromising phosphorylation of downstream targets of the NF-κB pathway; dynamic modeling studies show that this interaction also causes unwinding of the α-helix of the NEMO binding site on IKKß. Molecular docking studies with 10, a water-soluble analog of 7h, demonstrate that this analog interacts with the dimerization/oligomerization domain of monomeric IKKß and may inhibit oligomer formation and subsequent autophosphorylation. Sesquiterpene lactones 7h and 10 are considered ideal candidates for potential clinical development.

Disruption of the monocarboxylate transporter-4-basigin interaction inhibits the hypoxic response, proliferation, and tumor progression.

We have previously shown that glioblastoma stem cells (GSCs) are enriched in the hypoxic tumor microenvironment, and that monocarboxylate transporter-4 (MCT4) is critical for mediating GSC signaling in hypoxia. Basigin is involved in many physiological functions during early stages of development and in cancer and is required for functional plasma membrane expression of MCT4. We sought to determine if disruption of the MCT-Basigin interaction may be achieved with a small molecule. Using a cell-based drug-screening assay, we identified Acriflavine (ACF), a small molecule that inhibits the binding between Basigin and MCT4. Surface plasmon resonance and cellular thermal-shift-assays confirmed ACF binding to basigin in vitro and in live glioblastoma cells, respectively. ACF significantly inhibited growth and self-renewal potential of several glioblastoma neurosphere lines in vitro, and this activity was further augmented by hypoxia. Finally, treatment of mice bearing GSC-derived xenografts resulted in significant inhibition of tumor progression in early and late-stage disease. ACF treatment inhibited intratumoral expression of VEGF and tumor vascularization. Our work serves as a proof-of-concept as it shows, for the first time, that disruption of MCT binding to their chaperon, Basigin, may be an effective approach to target GSC and to inhibit angiogenesis and tumor progression.

Targetable T-type Calcium Channels Drive Glioblastoma.

Glioblastoma (GBM) stem-like cells (GSC) promote tumor initiation, progression, and therapeutic resistance. Here, we show how GSCs can be targeted by the FDA-approved drug mibefradil, which inhibits the T-type calcium channel Cav3.2. This calcium channel was highly expressed in human GBM specimens and enriched in GSCs. Analyses of the The Cancer Genome Atlas and REMBRANDT databases confirmed upregulation of Cav3.2 in a subset of tumors and showed that overexpression associated with worse prognosis. Mibefradil treatment or RNAi-mediated attenuation of Cav3.2 was sufficient to inhibit the growth, survival, and stemness of GSCs and also sensitized them to temozolomide chemotherapy. Proteomic and transcriptomic analyses revealed that Cav3.2 inhibition altered cancer signaling pathways and gene transcription. Cav3.2 inhibition suppressed GSC growth in part by inhibiting prosurvival AKT/mTOR pathways and stimulating proapoptotic survivin and BAX pathways. Furthermore, Cav3.2 inhibition decreased expression of oncogenes (PDGFA, PDGFB, and TGFB1) and increased expression of tumor suppressor genes (TNFRSF14 and HSD17B14). Oral administration of mibefradil inhibited growth of GSC-derived GBM murine xenografts, prolonged host survival, and sensitized tumors to temozolomide treatment. Our results offer a comprehensive characterization of Cav3.2 in GBM tumors and GSCs and provide a preclinical proof of concept for repurposing mibefradil as a mechanism-based treatment strategy for GBM. Cancer Res; 77(13); 3479-90. ©2017 AACR.

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo.

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.

Atracurium Besylate and other neuromuscular blocking agents promote astroglial differentiation and deplete glioblastoma stem cells.

Glioblastoma multiforme (GBM) are the most common primary malignant brain tumor in adults, with a median survival of about one year. This poor prognosis is attributed primarily to therapeutic resistance and tumor recurrence after surgical removal, with the root cause suggested to be found in glioblastoma stem cells (GSCs). Using glial fibrillary acidic protein (GFAP) as a reporter of astrocytic differentiation, we isolated multiple clones from three independent GSC lines which express GFAP in a remarkably stable fashion. We next show that elevated expression of GFAP is associated with reduced clonogenicity in vitro and tumorigenicity in vivo. Utilizing this in vitro cell-based differentiation reporter system we screened chemical libraries and identified the non-depolarizing neuromuscular blocker (NNMB), Atracurium Besylate, as a small molecule which effectively induces astroglial but not neuronal differentiation of GSCs. Functionally, Atracurium Besylate treatment significantly inhibited the clonogenic capacity of several independent patient-derived GSC neurosphere lines, a phenomenon which was largely irreversible. A second NNMB, Vecuronium, also induced GSC astrocytic differentiation while Dimethylphenylpiperazinium (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist, significantly blocked Atracurium Besylate pro-differentiation activity. To investigate the clinical importance of nAChRs in gliomas, we examined clinical outcomes and found that glioma patients with tumors overexpressing CHRNA1 or CHRNA9 (encoding for the AChR-α1 or AChR-α9) exhibit significant shorter overall survival. Finally, we found that ex-vivo pre-treatment of GSCs, expressing CHRNA1 and CHRNA9, with Atracurium Besylate significantly increased the survival of mice xenotransplanted with these cells, therefore suggesting that tumor initiating subpopulations have been reduced.

ZEB1 Promotes Invasion in Human Fetal Neural Stem Cells and Hypoxic Glioma Neurospheres.

Diffuse spread through brain parenchyma and the presence of hypoxic foci rimmed by neoplastic cells are two cardinal features of glioblastoma, and low oxygen is thought to drive movement of malignant gliomas in the core of the lesions. Transcription factors associated with epithelial-to-mesenchymal transition (EMT) have been linked to this invasion, and we found that hypoxia increased in vitro invasion up to fourfold in glioblastoma neurosphere lines and induced the expression of ZEB1. Immunohistochemical assessment of 295 surgical specimens consisting of various types of pediatric and adult brain cancers showed that ZEB1 expression was significantly higher in infiltrative lesions than less invasive tumors such as pilocytic astrocytoma and ependymoma. ZEB1 protein was also present in human fetal periventricular stem and progenitor cells and ZEB1 inhibition impaired migration of in vitro propagated human neural stem cells. The induction of ZEB1 protein in hypoxic glioblastoma neurospheres could be partially blocked by the HIF1alpha inhibitor digoxin. Targeting ZEB1 blocked hypoxia-augmented invasion of glioblastoma cells in addition to slowing them in normoxia. These data support the role for ZEB1 in invasive and high-grade brain tumors and suggest its key role in promoting invasion in the hypoxic tumor core as well as in the periphery.

Lateral inhibition of Notch signaling in neoplastic cells.

During normal development, heterogeneous expression of Notch ligands can result in pathway suppression in the signal-sending cell, a process known as lateral inhibition. It is unclear if an analogous phenomenon occurs in malignant cells. We observed significant induction of Notch ligands in glioblastoma neurospheres and pancreatic carcinoma cells cultured in low oxygen, suggesting that this phenomenon could occur around hypoxic regions. To model lateral inhibition in these tumors, the ligand Jagged1 was overexpressed in glioblastoma and pancreatic carcinoma cells, resulting in overall induction of pathway targets. However, when ligand high and ligand low cells from a single line were co-cultured and then separated, we noted suppression of Notch pathway targets in the former and induction in the latter, suggesting that neoplastic lateral inhibition can occur. We also found that repression of Notch pathway targets in signal-sending cells may occur through the activity of a Notch ligand intracellular domain, which translocates into the nucleus. Understanding how this neoplastic lateral inhibition process functions in cancer cells may be important in targeting ligand driven Notch signaling in solid tumors.