a-novel-high-resolution-vivo-imaging-technique-to-study-dynamic

A Novel High-resolution <em>In vivo</em> Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Glial fibrillary acidic protein (GFAP) is an intermediate filament expressed in glial cells that stabilizes and maintains the cytoskeleton of normal astrocytes. In glial tumors, GFAP expression is frequently lost with increasing grade of malignancy, suggesting that GFAP is important for maintaining glial cell morphology or regulating astrocytoma cell growth. Most permanent human glioma cell lines are GFAP negative by immunocytochemistry. Given that the GFAP gene is not mutated in human glioma specimens or glioma cell lines, we considered epigenetic mechanisms, such as promoter methylation, as a cause of silencing of GFAP in these tumors. In this study, we treated known GFAP-negative glioma cell lines with 5-aza-2'-deoxycytidine to examine GFAP promoter hypermethylation. Additionally, we performed bisulfite sequencing on primary glioma samples and glioma cell lines and showed an inverse relationship between GFAP promoter methylation status and GFAP expression. Using a gene reporter assay with the GFAP promoter cloned upstream of a luciferase gene, we showed that methylation of the GFAP promoter downregulates the expression of the luciferase gene. Our results suggest that epigenetic silencing of the GFAP gene through DNA methylation of its promoter region may be one mechanism by which GFAP is downregulated in human gliomas and glioma cell lines.

Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas.

Proliferating embryonic and cancer cells preferentially use aerobic glycolysis to support growth, a metabolic alteration commonly referred to as the "Warburg effect." Here, we show that the glycolytic enzyme hexokinase 2 (HK2) is crucial for the Warburg effect in human glioblastoma multiforme (GBM), the most common malignant brain tumor. In contrast to normal brain and low-grade gliomas, which express predominantly HK1, GBMs show increased HK2 expression. HK2 expression correlates with worse overall survival of GBM patients. Depletion of HK2, but neither HK1 nor pyruvate kinase M2, in GBM cells restored oxidative glucose metabolism and increased sensitivity to cell death inducers such as radiation and temozolomide. Intracranial xenografts of HK2-depleted GBM cells showed decreased proliferation and angiogenesis, but increased invasion, as well as diminished expression of hypoxia inducible factor 1Î± and vascular endothelial growth factor. In contrast, exogenous HK2 expression in GBM cells led to increased proliferation, therapeutic resistance, and intracranial growth. Growth was dependent on both glucose phosphorylation and mitochondrial translocation mediated by AKT signaling, which is often aberrantly activated in GBMs. Collectively, these findings suggest that therapeutic strategies to modulate the Warburg effect, such as targeting of HK2, may interfere with growth and therapeutic sensitivity of some GBMs.

Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme.

A key aberrant biological difference between tumor cells and normal differentiated cells is altered metabolism, whereby cancer cells acquire a number of stable genetic and epigenetic alterations to retain proliferation, survive under unfavorable microenvironments and invade into surrounding tissues. A classic biochemical adaptation is the metabolic shift to aerobic glycolysis rather than mitochondrial oxidative phosphorylation, regardless of oxygen availability, a phenomenon termed the "Warburg Effect". Aerobic glycolysis, characterized by high glucose uptake, low oxygen consumption and elevated production of lactate, is associated with a survival advantage as well as the generation of substrates such as fatty acids, amino acids and nucleotides necessary in rapidly proliferating cells. This review discusses the role of key metabolic enzymes and their association with aerobic glycolysis in Glioblastoma Multiforme (GBM), an aggressive, highly glycolytic and deadly brain tumor. Targeting key metabolic enzymes involved in modulating the "Warburg Effect" may provide a novel therapeutic approach either singularly or in combination with existing therapies in GBMs.

Targeting metabolic remodeling in glioblastoma multiforme.

Glioblastoma Multiforme (GBM), the most common and lethal primary human brain tumor, exhibits multiple molecular aberrations. We report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumor suppressor gene (TSG). Although GATA4 was expressed in normal brain, loss of GATA4 was observed in 94/163 GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor-like phenotype, formation of neurospheres, and the ability to differentiate into astrocytes, neurons, and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures, and brain tumor-initiating cells suppressed tumor growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21(CIP1), independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT (O-6-methylguanine-DNA-methyltransferase), the DNA repair enzyme which is often implicated in temozolomide resistance. Instead, GATA4 reduced expression of APNG (alkylpurine-DNA-N-glycosylase), a DNA repair enzyme which is poorly characterized in GBM-mediated temozolomide resistance. Identification and validation of GATA4 as a TSG and its downstream targets in GBM may yield promising novel therapeutic strategies.

A GATA4-regulated tumor suppressor network represses formation of malignant human astrocytomas.

Highly proliferating cells, normal or transformed, undergo aerobic glycolysis whereby glucose is metabolized to lactate rather than by oxidative metabolism, even in the presence of oxygen. This metabolic adaptation provides a survival advantage and facilitates synthesis of biosynthetic precursors required for continued cellular proliferation. An important mediator of aerobic glycolysis is our demonstration that in malignant gliomas there is over-expression of the glycolytic enzyme hexokinase 2 (HK2), phosphorylating glucose as the first step of the glycolytic pathway. In contrast, normal brain preferentially expresses HK1 and undergoes oxidative glucose metabolism. In this study, we examine whether this switch in HK isoform also occurs in the developing embryo and central nervous system (CNS). Bioinformatic analysis of available microarray data, including that of The Cancer Genome Atlas, demonstrated a ~17% overlap in metabolic-related genes in blastocyst stage embryo and human GBM tissue, including upregulation of HK2 and downregulation of HK1. Quantitative RT-PCR on mouse brains isolated at different embryonic and postnatal development time-points demonstrated HK2 expression was highest in the early embryo, while HK1 expression increased with CNS maturation. The downstream glycolytic enzymes PKM2 and LDHA had similar temporal profiles as HK2. Expression of the HK2 isoform was due in part to epigenetic regulation of HK2. In support, adult normal human brain and the few human GBM cell lines with low HK2 expression had methylation of CpG islands within intron 1 of HK2. In contrast, developing human fetal brain and GBM tissue expressing HK2 demonstrated significantly lower percent methylation. Furthermore, treatment of GBM cells lacking HK2 with 5-aza-2-deoxycytidine restored HK2 transcript expression. Overall, our results demonstrate that proliferative states including the developing embryo and malignant gliomas, which rely on aerobic glycolysis, preferentially express the HK2 isoform, found to be regulated in part epigenetically.

Developmental profile and regulation of the glycolytic enzyme hexokinase 2 in normal brain and glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). Oâ¶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, Oâ¶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions NÂ³-methyladenine and Nâ·-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

Alkylpurine-DNA-N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients.

Glioblastoma Multiforme (GBM) ist die h&auml;ufigsten und t&ouml;dliche alle Gliome. Der aktuelle Standard-Behandlung umfasst Operation gefolgt von begleitenden Strahlen- und Chemotherapie mit der DNA alkylierenden Agent Temozolomid (TMZ). O&#x2076;-Methylguanin-DNA-Methyltransferase (MGMT) repariert die meisten zytotoxischen L&auml;sionen von TMZ, O&#x2076;-Methylguanin generiert. Methylierung des Promotors MGMT in GBM korreliert mit therapeutischen empfindlicher auf Alkylantien Agent-Therapie. Jedoch sind einige Aspekte der TMZ Empfindlichkeit nicht durch MGMT-Promotor-Methylierung erkl&auml;rt. Hier untersuchten wir unsere Hypothese, dass die base Exzision Enzym Alkylpurine-DNA-N-Glycosylase (APNG), die repariert die zytotoxischen L&auml;sionen N&sup3;-Methyladenine und N&#x2077;-Methylguanin reparieren, kann dazu beitragen, TMZ Widerstand. Unterdr&uuml;ckender von APNG in gegr&uuml;ndet und prim&auml;re TMZ-resistenten GBM-Zelllinien, die endogen Ausdruck von MGMT und APNG abgeschw&auml;cht TMZ-induzierten DNA-Sch&auml;den zu reparieren und Apoptosis. Wiedereinf&uuml;hrung der Ausdruck der APNG in TMZ-Sensitive GBM Linien &uuml;bertragenen TMZ in-vitro-und in Orthotopic Xenograft-Mausmodelle. Dar&uuml;ber hinaus war Widerstand mit Coexpression Mgmt. Bewertung von APNG Protein erh&ouml;ht Ebenen in mehreren klinischen Datasets gezeigt, dass bei Patienten, hohe nukleare APNG-Ausdruck mit &auml;rmeren die Gesamt&uuml;berlebenszeit gegen&uuml;ber Patienten fehlt APNG Ausdruck korreliert. Verlust von APNG Ausdruck in einer Untergruppe von Patienten wurde auch erh&ouml;hte APNG-Promotor-Methylierung zugeordnet. Zusammenfassend zeigen unsere Daten, dass APNG zur TMZ-Resistenz bei GBM tr&auml;gt und kann n&uuml;tzlich in der Diagnose und Behandlung der Krankheit sein.

Alkylpurine-DNA-N-Glycosylase verleiht Resistenz gegen Temozolomid in Xenograft-Modelle von Glioblastoma Multiforme und Armen &Uuml;berleben bei Patienten zugeordnet ist.

Vestibular schwannomas (VS) are a common posterior fossa brain tumor, and though benign can cause significant morbidity, particularly loss of hearing, tinnitus, vertigo and facial paralysis. The current treatment options for VS include microsurgical resection, stereotactic radiosurgery or close surveillance monitoring, with each treatment option carrying associated complications and morbidities. Most importantly, none of these options can definitively reverse hearing loss or tinnitus. Identification of a novel medical therapy, through the use of targeted molecular inhibition, is therefore a highly desirable treatment strategy that may minimize complications arising from both tumor and treatment and more importantly be suitable for patients whose options are limited with respect to surgical or radiosurgical interventions. In this study we chose to examine the effect of Nilotinib on VS. Nilotinib (TasignaÂ®) is a second-generation receptor tyrosine kinase (RTK) inhibitor with a target profile similar to that of imatinib (GleevecÂ®), but increased potency, decreased toxicity and greater cellular and tissue penetration. Nilotinib targets not only the BCR-ABL oncoprotein, but also platelet-derived growth factor (PDGF) receptor signalling. In this preclinical study, the human NF2-null schwannoma cell line HEI-193 subjected to nilotinib inhibition demonstrated decreased viability, proliferation and anchorage-independent growth, and increased apoptosis. A daily dose of nilotinib for 5 days inhibited HEI-I93 proliferation at a clinically-relevant concentration in a dose-dependent manner (IC(50) 3-5 Âµmol/L) in PDGF-stimulated cells. These anti-tumorigenic effects of nilotinib were correlated to inhibited activation of PDGFR-Î± and PDGFR-Î² and major downstream signalling pathways. These experiments support a therapeutic potential for Nilotinib in VS.

Investigation of the in vitro therapeutic efficacy of nilotinib in immortalized human NF2-null vestibular schwannoma cells.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Very little is known about aggressive forms of this disease, such as metastatic or recurrent MBs. In order to identify pathways involved in aggressive MB pathophysiology, we performed unbiased, whole genome microarrays on MB tumors at both the human and murine levels. Primary human MBs were compared, transcriptomically, to their patient-matched recurrent or metastatic tumors. Expression profiling was also performed on murine tumors from two spontaneously developing MB mouse models (Ptch+/- and Smo/Smo) that present with differing clinical severities. At both the human and murine levels we identified transforming growth factor-beta (TGF-Î²) as a potential contributor to MB progression/metastasis. Smad3, a major downstream component of the TGF-Î² pathway, was also evaluated using immunohistochemistry in malignant human tissues and was shown to correlate with MB metastasis and survival. Similarly, Smad3 expression during development identified a subset of cerebellar neuronal precursors as putative cells of origin for the Smad3-positive MBs. To our knowledge, this is the first study that links TGF-Î² to MB pathogenesis. Our research suggests that canonical activation of this pathway leads to better prognosis for patients.

Canonical TGF-Î² pathway activity is a predictor of SHH-driven medulloblastoma survival and delineates putative precursors in cerebellar development.

Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite current advances in therapy consisting of surgery followed by chemotherapy and radiation, the overall survival rate still remains poor. Therapeutic failures are partly attributable to the highly infiltrative nature of tumor adjacent to normal brain parenchyma. Recently, evidence is mounting to suggest that actin cytoskeleton dynamics are critical components of the cell invasion process. Drebrin is an actin-binding protein involved in the regulation of actin filament organization, and plays a significant role in cell motility; however, the role of drebrin in glioma cell invasiveness has not yet been fully elucidated. Therefore, this study was aimed to clarify the role of drebrin in glioma cell morphology and cell motility. Here we show that drebrin is expressed in glioma cell lines and in operative specimens of GBM. We demonstrate that stable overexpression of drebrin in U87 cells leads to alterations in cell morphology, and induces increased invasiveness in vitro while knockdown of drebrin in U87 cells by small interfering RNA (siRNA) decreases invasion and migration. In addition, we show that depletion of drebrin by siRNA alters glioma cell morphology in A172 GBM cell line. Our results suggest that drebrin contributes to the maintenance of cell shape, and may play an important role in glioma cell motility.

The role of drebrin in glioma migration and invasion.

Glioblastoma (GBM) is the most common and lethal primary brain tumor. Over the past few years tremendous genomic and proteomic characterization along with robust animal models of GBM have provided invaluable data that show that "GBM", although histologically indistinguishable from one another, are comprised of molecularly heterogenous diseases. In addition, robust pre-clinical models and a better understanding of the core pathways disrupted in GBM are providing a renewed optimism for novel strategies targeting these devastating tumors. Here, we summarize a brief history of the disease, our current molecular knowledge, lessons from animal models and emerging concepts of angiogenesis, invasion, and metabolism in GBM that may lend themselves to therapeutic targeting.

Glioblastoma, a brief review of history, molecular genetics, animal models and novel therapeutic strategies.

The malignant glioma is the most common primary human brain tumor, and its migration and invasiveness away from the primary tumor mass are considered a leading cause of tumor recurrence and treatment failure. Recently, gene expression profiling revealed that the transmembrane glycoprotein CD99 is more highly expressed in malignant glioma than in normal brain. Although its function is not completely understood, CD99 is implicated in cell adhesion and migration in a variety of different cell types. CD99 has wild-type and splice variant isoforms. Previous studies have shown that wild-type CD99 may be an oncosuppressor in some tumors, distinct from the role of the splice variant isoform. In this study, our data reveal that only wild-type CD99 is expressed in human glioma cells and tissues. Using a tissue microarray, we validated that gliomas demonstrate higher expression of CD99 compared with nonneoplastic brain. To assess the role of CD99 in glioma migration and invasion, we inhibited CD99 expression by siRNA and demonstrated decreased glioma migration and invasion. In contrast, when CD99 was overexpressed in glioma cells, we observed enhancement of cell migration and invasiveness. An orthotopic brain tumor model demonstrates that CD99 overexpression significantly increases invasiveness and decreases survival rate. Interestingly, Rac activity was decreased and Rho activity was increased in CD99 overexpressing glioma cells, and the proportion of amoeboid cells to mesenchymal cells was significantly increased. Taken together, our findings suggest that CD99 may play an important role in the migration and invasion of human gliomas independent of Akt, ERK, or JNK signaling pathways. Moreover, CD99 might be involved in amoeboid-mesenchymal transition in glioma migration. CD99 may be an important future target to inhibit migration and invasion, especially in CD99-expressing gliomas.

Overexpression of CD99 Increases the Migration and Invasiveness of Human Malignant Glioma Cells.

Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. InÂ vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C.Â elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:

Article	Year
Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas. Neuro-oncology\| PubMed ID: 21075782	2011
Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme. The Journal of experimental medicine\| PubMed ID: 21242296	2011
Targeting metabolic remodeling in glioblastoma multiforme. Oncotarget\| PubMed ID: 21317451	2010
A GATA4-regulated tumor suppressor network represses formation of malignant human astrocytomas. The Journal of experimental medicine\| PubMed ID: 21464220	2011
Developmental profile and regulation of the glycolytic enzyme hexokinase 2 in normal brain and glioblastoma multiforme. Neurobiology of disease\| PubMed ID: 21726646	2011
Alkylpurine-DNA-N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients. The Journal of clinical investigation\| PubMed ID: 22156195	2012
Investigation of the in vitro therapeutic efficacy of nilotinib in immortalized human NF2-null vestibular schwannoma cells. PloS one\| PubMed ID: 22745749	2012
Canonical TGF-Î² pathway activity is a predictor of SHH-driven medulloblastoma survival and delineates putative precursors in cerebellar development. Brain pathology (Zurich, Switzerland)\| PubMed ID: 22966790	2013
The role of drebrin in glioma migration and invasion. Experimental cell research\| PubMed ID: 23201135	2013
Glioblastoma, a brief review of history, molecular genetics, animal models and novel therapeutic strategies. Archivum immunologiae et therapiae experimentalis\| PubMed ID: 23224339	2013
Overexpression of CD99 Increases the Migration and Invasiveness of Human Malignant Glioma Cells. Genes & cancer\| PubMed ID: 23486730	2012
The eEF2 Kinase Confers Resistance to Nutrient Deprivation by Blocking Translation Elongation. Cell\| PubMed ID: 23706743	2013

Sameer Agnihotri

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