RAPID resistance to BET inhibitors is mediated by FGFR1 in glioblastoma.

Bromodomain and extra-terminal domain (BET) proteins are therapeutic targets in several cancers including the most common malignant adult brain tumor glioblastoma (GBM). Multiple small molecule inhibitors of BET proteins have been utilized in preclinical and clinical studies. Unfortunately, BET inhibitors have not shown efficacy in clinical trials enrolling GBM patients. One possible reason for this may stem from resistance mechanisms that arise after prolonged treatment within a clinical setting. However, the mechanisms and timeframe of resistance to BET inhibitors in GBM is not known. To identify the temporal order of resistance mechanisms in GBM we performed quantitative proteomics using multiplex-inhibitor bead mass spectrometry and demonstrated that intrinsic resistance to BET inhibitors in GBM treatment occurs rapidly within hours and involves the fibroblast growth factor receptor 1 (FGFR1) protein. Additionally, small molecule inhibition of BET proteins and FGFR1 simultaneously induces synergy in reducing GBM tumor growth in vitro and in vivo. Further, FGFR1 knockdown synergizes with BET inhibitor mediated reduction of GBM cell proliferation. Collectively, our studies suggest that co-targeting BET and FGFR1 may dampen resistance mechanisms to yield a clinical response in GBM.

Dual role of the adhesion G-protein coupled receptor ADRGE5/CD97 in glioblastoma invasion and proliferation.

CD97, an adhesion G-protein coupled receptor highly expressed in glioblastoma (GBM), consists of two noncovalently bound domains: the N-terminal fragment (NTF) and C-terminal fragment. The C-terminal fragment contains a GPCR domain that couples to Gα12/13, while the NTF interacts with extracellular matrix components and other receptors. We investigated the effects of changing CD97 levels and its function on primary patient-derived GBM stem cells (pdGSCs) in vitro and in vivo. We created two functional mutants: a constitutively active ΔNTF and the noncleavable dominant-negative H436A mutant. The CD97 knockdown in pdGSCs decreased, while overexpression of CD97 increased tumor size. Unlike other constructs, the ΔNTF mutant promoted tumor cell proliferation, but the tumors were comparable in size to those with CD97 overexpression. As expected, the GBM tumors overexpressing CD97 were very invasive, but surprisingly, the knockdown did not inhibit invasiveness and even induced it in noninvasive U87 tumors. Importantly, our results indicate that NTF was present in the tumor core cells but absent in the pdGSCs invading the brain. Furthermore, the expression of noncleavable H436A mutant led to large tumors that invade by sending massive protrusions, but the invasion of individual tumor cells was substantially reduced. These data suggest that NTF association with CD97 GPCR domain inhibits individual cell dissemination but not overall tumor invasion. However, NTF dissociation facilitates pdGSCs brain infiltration and may promote tumor proliferation. Thus, the interplay between two functional domains regulates CD97 activity resulting in either enhanced cell adhesion or stimulation of tumor cell invasion and proliferation.

NAMPT Inhibition Induces Neuroblastoma Cell Death and Blocks Tumor Growth.

High-risk neuroblastoma (NB) portends very poor prognoses in children. Targeting tumor metabolism has emerged as a novel therapeutic strategy. High levels of nicotinamide-adenine-dinucleotide (NAD+) are required for rapid cell proliferation. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme for NAD+ salvage and is overexpressed in several cancers. Here, we determine the potential of NAMPT as a therapeutic target for NB treatment. NAMPT inhibition cytotoxicity was determined by trypan blue exclusion and LDH assays. Neuroblastoma stem cell self-renewal was evaluated by neurosphere assay. Protein expression was evaluated via Western blot. The effect of targeting NAMPT in vivo was determined using an NB1691-xenografted mouse model. Robust NAMPT expression was demonstrated in multiple N-MYC amplified, high-risk neuroblastoma cell lines. NAMPT inhibition with STF-118804 (STF) decreased ATP, induced apoptosis, and reduced NB stem cell neurosphere formation. STF treatment down-regulated N-MYC levels and abrogated AKT activation. AKT and glycolytic pathway inhibitors in combination with NAMPT inhibition induced robust, greater-than-additive neuroblastoma cell death. Lastly, STF treatment blocked neuroblastoma tumor growth in mouse xenograft models. NAMPT is a valid therapeutic target as inhibition promoted neuroblastoma cell death in vitro and prevented tumor growth in vivo. Further investigation is warranted to establish this therapy's role as an adjunctive modality.

Chalcones as Anti-Glioblastoma Stem Cell Agent Alone or as Nanoparticle Formulation Using Carbon Dots as Nanocarrier.

The current prognosis for glioblastoma is dismal. Treatment-resistant glioblastoma stem cells (GSCs) and the failure of most drugs to reach therapeutic levels within the tumor remain formidable obstacles to successful treatment. Chalcones are aromatic ketones demonstrated to reduce malignant properties in cancers including glioblastoma. Nanomedicines can increase drug accumulation and tumor cell death. Carbon-dots are promising nanocarriers that can be easily functionalized with tumor-targeting ligands and anti-cancer drugs. Therefore, we synthesized a series of 4'-amino chalcones with the rationale that the amino group would serve as a "handle" to facilitate covalent attachment to carbon-dots and tested their cytotoxicity toward GSCs. We generated 31 chalcones (22 4'-amino and 9 4' derivatives) including 5 novel chalcones, and found that 13 had an IC50 below 10 µM in all GSC lines. After confirming that the 4-amino group was not part of the active pharmacophore, chalcones were attached to transferrin-conjugated carbon-dots. These conjugates were significantly more cytotoxic than the free chalcones, with the C-dot-transferrin-2,5, dimethoxy chalcone conjugate inducing up to 100-fold more GSC death. Several of the tested chalcones represent promising lead compounds for the development of novel anti-GSC drugs. Furthermore, designing amino chalcones for carbon-dot mediated drug delivery is a rational and effective methodology.

The contribution of ketone bodies to glycolytic inhibition for the treatment of adult and pediatric glioblastoma.

PURPOSE: Glioblastoma (GBM) remains one of the most lethal primary brain tumors in children and adults. Targeting tumor metabolism has emerged as a promising-targeted therapeutic strategy for GBM and characteristically resistant GBM stem-like cells (GSCs). METHODS: Gene expression data was obtained from the online patient-histology database, GlioVis. GSC mitochondria morphology was examined by TEM. Cell viability and effect on GSC self-renewal was determined via MTS assay and neurosphere assay, respectively. Proteins were evaluated by Western Blot. RESULTS: Enzymes necessary for ketone catabolism (BDH1, OXCT1 and ACAT1) are significantly downregulated in adult and pediatric GBM. GSC mitochondrial ultrastructure suggested defects in oxidative phosphorylation. Treatment of both GBM and GSC cell lines resulted in dose-dependent decreases in viability in response to glycolytic inhibitor 2-deoxy-D-glucose (2-DG), and ketone body Acetoacetate (AA), but not ß-hydroxybutyrate (ßHB). AA induced apoptosis was confirmed by western blot analysis, indicating robust caspase activation and PARP cleavage. AA reduced neurosphere formation at concentrations as low as 1 mM. Combined treatment of low dose 2-DG (50 µM) with AA resulted in more cell death than either treatment alone. The effect was greater than additive at low concentrations of AA, reducing viability approximately 50% at 1 mM AA. AA was found to directly upregulate mitochondrial uncoupling protein 2 (UCP2), which may explain this potential drug synergism via multi-faceted inhibition of the glycolytic pathway. CONCLUSION: Targeting the metabolic pathway of GBM via glycolytic inhibition in conjunction with ketogenic diet or exogenous ketone body supplementation warrants further investigation as a promising adjunctive treatment to conventional therapy.

Novel Curcumin Inspired Bis-Chalcone Promotes Endoplasmic Reticulum Stress and Glioblastoma Neurosphere Cell Death.

Glioblastoma (GBM) has a dismal prognosis and successful elimination of GBM stem cells (GSCs) is a high-priority as these cells are responsible for tumor regrowth following therapy and ultimately patient relapse. Natural products and their derivatives continue to be a source for the development of effective anticancer drugs and have been shown to effectively target pathways necessary for cancer stem cell self-renewal and proliferation. We generated a series of curcumin inspired bis-chalcones and examined their effect in multiple patient-derived GSC lines. Of the 19 compounds synthesized, four analogs robustly induced GSC death in six separate GSC lines, with a half maximal inhibitory concentration (IC50) ranging from 2.7⁻5.8 µM and significantly reduced GSC neurosphere formation at sub-cytotoxic levels. Structural analysis indicated that the presence of a methoxy group at position 3 of the lateral phenylic appendages was important for activity. Pathway and drug connectivity analysis of gene expression changes in response to treatment with the most active bis-chalcone 4j (the 3,4,5 trimethoxy substituted analog) suggested that the mechanism of action was the induction of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) mediated cell death. This was confirmed by Western blot analysis in which 4j induced robust increases in CHOP, p-jun and caspase 12. The UPR is believed to play a significant role in GBM pathogenesis and resistance to therapy and as such represents a promising therapeutic target.

Targeting Glioblastoma Stem Cells with 2-Deoxy-D-Glucose (2-DG) Potentiates Radiation-Induced Unfolded Protein Response (UPR).

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, and despite optimized treatment options, median survival remains dismal. Contemporary evidence suggests disease recurrence results from expansion of a robustly radioresistant subset of GBM progenitor cells, termed GBM stem cells (GSCs). In this study, we utilized transmission electron microscopy to uncover ultrastructural effects on patient-derived GSC lines exposed to supratherapeutic radiotherapy levels. Elevated autophagosome formation and increased endoplasmic reticulum (ER) internal diameter, a surrogate for ER stress and activation of unfolded protein response (UPR), was uncovered. These observations were confirmed via protein expression through Western blot. Upon interrogating genomic data from an open-access GBM patient database, overexpression of UPR-related chaperone protein genes was inversely correlated with patient survival. This indicated controlled UPR may play a role in promoting radioresistance. To determine if potentiating UPR further can induce apoptosis, we exposed GSCs to radiation with an ER stress-inducing drug, 2-deoxy-D-glucose (2-DG), and found dose-dependent decreases in viability and increased apoptotic marker expression. Taken together, our results indicate GSC radioresistance is, in part, achieved by overexpression and overactivation of ER stress-related pathways, and this effect can be overcome via potentiation of UPR, leading to loss of GSC viability.

Drug and disease signature integration identifies synergistic combinations in glioblastoma.

Glioblastoma (GBM) is the most common primary adult brain tumor. Despite extensive efforts, the median survival for GBM patients is approximately 14 months. GBM therapy could benefit greatly from patient-specific targeted therapies that maximize treatment efficacy. Here we report a platform termed SynergySeq to identify drug combinations for the treatment of GBM by integrating information from The Cancer Genome Atlas (TCGA) and the Library of Integrated Network-Based Cellular Signatures (LINCS). We identify differentially expressed genes in GBM samples and devise a consensus gene expression signature for each compound using LINCS L1000 transcriptional profiling data. The SynergySeq platform computes disease discordance and drug concordance to identify combinations of FDA-approved drugs that induce a synergistic response in GBM. Collectively, our studies demonstrate that combining disease-specific gene expression signatures with LINCS small molecule perturbagen-response signatures can identify preclinical combinations for GBM, which can potentially be tested in humans.

Curcumin decreases malignant characteristics of glioblastoma stem cells via induction of reactive oxygen species.

BACKGROUND: Glioblastoma Multiforme (GBM) is the most common and lethal form of primary brain tumor in adults. Following standard treatment of surgery, radiation and chemotherapy, patients are expected to survive 12-14 months. Theorized cause of disease recurrence in these patients is tumor cell repopulation through the proliferation of treatment-resistant cancer stem cells. Current research has revealed curcumin, the principal ingredient in turmeric, can modulate multiple signaling pathways important for cancer stem cell self-renewal and survival. METHODS: Following resection, tumor specimens were dissociated and glioblastoma stem cells (GSCs) were propagated in neurosphere media and characterized via immunocytochemistry. Cell viability was determined with MTS assay. GSC proliferation, sphere forming and colony forming assays were conducted through standard counting methods. Reactive oxygen species (ROS) production was examined using the fluorescent molecular probe CM-H2DCFA. Effects on cell signaling pathways were elucidated by western blot. RESULTS: We evaluate the effects of curcumin on patient-derived GSC lines. We demonstrate a curcumin-induced dose-dependent decrease in GSC viability with an approximate IC50 of 25 µM. Treatment with sub-toxic levels (2.5 µM) of curcumin significantly decreased GSC proliferation, sphere forming ability and colony forming potential. Curcumin induced ROS, promoted MAPK pathway activation, downregulated STAT3 activity and IAP family members. Inhibition of ROS with the antioxidant N-acetylcysteine reversed these effects indicating a ROS dependent mechanism. CONCLUSIONS: Discoveries made in this investigation may lead to a non-toxic intervention designed to prevent recurrence in glioblastoma by targeting glioblastoma stem cells.

Neuropathic pain-induced depressive-like behavior and hippocampal neurogenesis and plasticity are dependent on TNFR1 signaling.

Patients suffering from neuropathic pain have a higher incidence of mood disorders such as depression. Increased expression of tumor necrosis factor (TNF) has been reported in neuropathic pain and depressive-like conditions and most of the pro-inflammatory effects of TNF are mediated by the TNF receptor 1 (TNFR1). Here we sought to investigate: (1) the occurrence of depressive-like behavior in chronic neuropathic pain and the associated forms of hippocampal plasticity, and (2) the involvement of TNFR1-mediated TNF signaling as a possible regulator of such events. Neuropathic pain was induced by chronic constriction injury of the sciatic nerve in wild-type and TNFR1(-/-) mice. Anhedonia, weight loss and physical state were measured as symptoms of depression. Hippocampal neurogenesis, neuroplasticity, myelin remodeling and TNF/TNFRs expression were analyzed by immunohistochemical analysis and western blot assay. We found that neuropathic pain resulted in the development of depressive symptoms in a time dependent manner and was associated with profound hippocampal alterations such as impaired neurogenesis, reduced expression of neuroplasticity markers and myelin proteins. The onset of depressive-like behavior also coincided with increased hippocampal levels of TNF, and decreased expression of TNF receptor 2 (TNFR2), which were all fully restored after mice spontaneously recovered from pain. Notably, TNFR1(-/-) mice did not develop depressive-like symptoms after injury, nor were there changes in hippocampal neurogenesis and plasticity. Our data show that neuropathic pain induces a cluster of depressive-like symptoms and profound hippocampal plasticity that are dependent on TNF signaling through TNFR1.

Activation of NF-κB in Schwann cells is dispensable for myelination in vivo.

Peripheral myelination is a dynamic process orchestrated by axons and Schwann cells. Although the signaling mechanisms governing myelination are not fully understood, NF-κB activation in Schwann cells has been implicated as a key regulator in vitro. Using a mouse model, we show that nuclear factor κB activation in Schwann cells is not required for myelination in vivo.

NIBP, a novel NIK and IKK(beta)-binding protein that enhances NF-(kappa)B activation.

The transcription factor NF-kappaB plays an important role in both physiological and pathological events in the central nervous system. Nevertheless, the mechanisms of NF-kappaB-mediated regulation of gene expression, and the signaling molecules participating in the NF-kappaB pathway in the central nervous system are, to date, poorly understood. To identify such molecules, we conducted a yeast two-hybrid screen of a human brain cDNA library using NIK as bait. As a result, we identified a novel NIK and IKK(beta) binding protein designated NIBP that is mainly expressed in brain, muscle, heart, and kidney. Interestingly, low levels of expression were detected in immune tissues such as spleen, thymus, and peripheral blood leukocytes, where NF-kappaB is known to modulate immune function. We demonstrated by immunohistochemistry that NIBP expression in the brain is localized to neurons. NIBP physically interacts with NIK, IKK(beta), but not IKK(alpha) or IKK(gamma). NIBP overexpression potentiates tumor necrosis factor-alpha-induced NF-kappaB activation through increased phosphorylation of the IKK complex and its downstream I(kappa)B(alpha) and p65 substrates. Finally, knockdown of NIBP expression by small interfering RNA reduces tumor necrosis factor-alpha-induced NF-kappaB activation, prevents nerve growth factor-induced neuronal differentiation, and decreases Bcl-xL gene expression in PC12 cells. Our data demonstrate that NIBP, by interacting with NIK and IKK(beta), is a new enhancer of the cytokine-induced NF-(kappa)B signaling pathway. Because of its neuronal expression, we propose that NIBP may be a potential target for modulating the NF-(kappa)B signaling cascade in neuronal pathologies dependent upon abnormal activation of this pathway.

TNAP, a novel repressor of NF-kappaB-inducing kinase, suppresses NF-kappaB activation.

NF-kappaB-inducing kinase (NIK) has been implicated as an essential component of NF-kappaB activation. However, the regulatory mechanism of NIK signaling remains elusive. We have identified a novel NIK interacting protein, TNAP (for TRAFs and NIK-associated protein). In mammalian cells, TNAP physically interacts with NIK, TRAF2, and TRAF3 but not IKK1 or IKK2. TNAP specifically inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, TNF receptor 1, TRADD, RIP, TRAF2, and NIK but does not affect IKK1- and IKK2-mediated NF-kappaB activation. Knockdown of TNAP by lentiviral-mediated small interference RNA potentiates TNF-alpha-induced NF-kappaB activation. TNAP suppresses NIK kinase activity and subsequently reduces p100 processing, p65 phosphorylation, and IkappaBalpha degradation. These data suggest that TNAP is a repressor of NIK activity and regulates both the classical and alternative NF-kappaB signaling pathways.

Neuronal glutamate transporter EAAT4 is expressed in astrocytes.

High-affinity excitatory amino acid transporters (EAATs) are essential to terminate glutamatergic neurotransmission and to prevent excitotoxicity. To date, five distinct EAATs have been cloned from animal and human tissues: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5. EAAT1 and EAAT2 are commonly known as glial glutamate transporters, whereas EAAT3, EAAT4, and EAAT5 are neuronal. EAAT4 is largely expressed in cerebellar Purkinje cells. In this study, using immunohistochemistry and Western blotting, we found that EAAT4-like immunoreactivity (ir) is enriched in the spinal cord and forebrain. Double-labeled fluorescent immunostaining and confocal image analysis indicated that EAAT4-like ir colocalizes with an astrocytic marker, glial fibrillary acidic protein (GFAP). The astrocytic localization of EAAT4 was further confirmed in astrocyte cultures by double-labeled fluorescent immunocytochemistry and Western blotting. Reverse transcriptase-polymerase chain reaction analysis demonstrated mRNA expression of EAAT4 in astrocyte cultures. Sequencing confirmed the specificity of the amplified fragment. These results demonstrate that EAAT4 is expressed in astrocytes. This astrocytic localization of neuronal EAAT4 may reveal a new function of EAAT4 in the central nervous system.

Identification and characterization of a novel Nogo-interacting mitochondrial protein (NIMP).

Nogo is a potent inhibitor of regeneration following spinal cord injury. To develop a better understanding of the mechanisms responsible for regenerative failure we used a yeast two-hybrid approach to try and identify proteins that interact with Nogo. We identified a novel mitochondrial protein designated Nogo-interacting mitochondrial protein (NIMP) in a screen of an adult human brain cDNA library. This interaction was confirmed by co-immunoprecipitation in both brain tissue (endogenous) and transfected HEK293T cells (overexpressed). In support of these studies we demonstrate that Nogo interacts with the UQCRC1 and UQCRC2 components of complex III, within the mitochondrial respiratory chain. The mitochondrial localization of NIMP was evidenced by confocal image analysis and western blot analysis of isolated mitochondria. NIMP is highly conserved and ubiquitously expressed in mitochondria-enriched tissues. Within the CNS, NIMP-like immunoreactivity is present in neurons and astrocytes. These data suggest that NIMP is a novel mitochondrial protein that interacts with Nogo. The interaction of Nogo with mitochondrial proteins may provide insight into the mechanisms for Nogo-induced inhibition of neurite growth.