Glioblastoma Tumorigenicity Research Articles

YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.

The role of YAP (Yes associated protein 1 gene) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G-protein coupled receptors) and RhoA, in growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor initiating cells derived from patient derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using shRNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor gene; TF), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (Heparin binding EGF-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

Targeting TWIST1 through loss of function inhibits tumorigenicity of human glioblastoma.

TWIST1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial‐to‐mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion, and self‐renewal in glioblastoma (GBM) cells. However, the potential therapeutic relevance of TW has not been established through loss‐of‐function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knockdown) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW‐associated mechanisms. Multiple bioinformatic tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases, and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time, we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting protumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM.

CDC7-dependent transcriptional regulation of RAD54L is essential for tumorigenicity and radio-resistance of glioblastoma

Accumulating evidence indicates that cell division cycle 7-related protein kinase(CDC7) plays an essential role in tumor cells and it could induces cell proliferation and could be related to prognosis in multiple types of cancer. However, the biological role and molecular mechanism of CDC7 in GBM still remains unclear. In this study, we identified that CDC7 expression was enriched in glioblastoma (GBM) tumors and was functionally required for tumor proliferation and its expression was associated to poor prognosis in GBM patients. Mechanically, CDC7 induced radio resistance in GBM cells and CDC7 knock down increased cell apoptosis when combined with radiotherapy. Moreover, CDC7 regulated The DNA repair/recombination protein 54L (RAD54L) expression via regulation of RAD54L promoter activity. Therapeutically, we found that CDC7 inhibitor attenuated tumor growth both in vitro and in vivo. Collectively, CDC7 promotes proliferation, induces radio resistance in GBM, and could become a potential therapeutic target for GBM.

New aspects of glioblastoma multiforme revealed by similarities between neural and glioblastoma stem cells.

Neural stem cells (NSCs) undergo self-renewal and generate neurons and glial cells under the influence of specific signals from surrounding environments. Glioblastoma multiforme (GBM) is a highly lethal brain tumor arising from NSCs or glial precursor cells owing to dysregulation of transcriptional and epigenetic networks that control self-renewal and differentiation of NSCs. Highly tumorigenic glioblastoma stem cells (GSCs) constitute a small subpopulation of GBM cells, which share several characteristic similarities with NSCs. GSCs exist atop a stem cell hierarchy and generate heterogeneous populations that participate in tumor propagation, drug resistance, and relapse. During multimodal treatment, GSCs de-differentiate and convert into cells with malignant characteristics, and thus play critical roles in tumor propagation. In contrast, differentiation therapy that induces GBM cells or GSCs to differentiate into a neuronal or glial lineage is expected to inhibit their proliferation. Since stem cell differentiation is specified by the cells' epigenetic status, understanding their stemness and the epigenomic situation in the ancestor, NSCs, is important and expected to be helpful for developing treatment modalities for GBM. Here, we review the current findings regarding the epigenetic regulatory mechanisms of NSC fate in the developing brain, as well as those of GBM and GSCs. Furthermore, considering the similarities between NSCs and GSCs, we also discuss potential new strategies for GBM treatment.

MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma

SummaryATG4B stimulates autophagy by promoting autophagosome formation through reversible modification of ATG8. We identify ATG4B as a substrate of mammalian sterile20-like kinase (STK) 26/MST4. MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities using genetic approaches or an inhibitor of ATG4B suppresses autophagy and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation, and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM xenograft markedly slows tumor growth and provides a significant survival benefit. Our work describes an MST4-ATG4B signaling axis that influences GBM autophagy and malignancy, and whose therapeutic targeting enhances the anti-tumor effects of radiotherapy.

The FOXM1/BUB1B signaling pathway is essential for the tumorigenicity and radioresistance of glioblastoma.

Accumulating evidence indicates that mitotic checkpoint serine/threonine kinase B (BUB1B) plays a critical role in multiple types of cancer. However, the biological function and molecular regulatory mechanism of BUB1B in glioblastoma (GBM) remain unclear. In the present study, we identified that BUB1B expression was enriched in GBM tumors and was functionally required for tumor proliferation both in vitro and in vivo. Clinically, BUB1B expression was associated with poor prognosis in GBM patients and BUB1B-dependent radioresistance in GBM was decreased by targeting BUB1B via shRNAs. Mechanistically, forkhead box protein M1 (FOXM1) transcriptionally regulated BUB1B expression by binding to and then activating the BUB1B promoter. Therapeutically, we found that FOXM1 inhibitor attenuated tumorigenesis and radioresistance of GBM both in vitro and in vivo. Altogether, BUB1B promotes tumor proliferation and induces radioresistance in GBM, indicating that BUB1B could be a potential therapeutic target for GBM.

Abstract LB-207: PI3Kγ inhibition suppresses glioblastoma tumorigenicity through disruption of an IL11-STAT3-MYC signaling axis between microglia and glioblastoma

Abstract While mutations in isocitrate dehydrogenase (IDH) gene are associated with favorable prognosis in glioblastoma patients, the biologic basis for favorable survival in patients with wild-type IDH (wtIDH) glioblastoma remains poorly understood. We identified an inflammatory gene signature whose expression inversely correlated with patient survival in three independent, wtIDH glioblastoma cohorts (The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and REMEBRANDT). Systematic analysis of the inflammatory infiltrates of the glioblastoma microenvironment revealed microglia as the predominant cell type driving this survival association. We show that the growth stimulatory effects of microglia can be recapitulated using conditioned media derived microglia. We cross-referenced proteomic profile of microglia conditioned media with the TCGA and the CGGA to identify constituent soluble factors with survival association and identified IL11. In both clinical datasets, increased IL11 expression was associated with shortened survival. Further, IL11 mRNA and protein expression are elevated in clinical glioblastoma specimen relative to the surrounding cerebrum. Analysis of samples secured from three unrelated patients revealed that IL11 level in microglia was significantly higher than in tumor cells. In vitro and in vivo experiments demonstrated that IL11 was necessary and sufficient for enhancement of glioblastoma tumorigenicity. IL11 induced activation of a STAT3-MYC signaling axis in tumor cells, which upregulates the genes required for glioblastoma tumorigenicity, including OLIG2, SOX2, and POU3F2. PI3Kγ activation in myeloid-derived cells, including microglia, is essential for its trafficking into the tumor microenvironment. We hypothesized that PI3Kγ inhibition or inactivation should impede microglia trafficking to the glioblastoma microenvironment and suppress tumorigenicity. Supporting our hypothesis, PI3Kγ inhibitor suppressed tumorigenicity in vivo by reducing microglia density and IL11 release in murine glioblastoma GL261 tumors. These effects were recapitulated when GL261 was implanted into PI3Kγ-/- mice. Importantly, ectopic expression IL11 reversed the tumor-suppressive effect of PI3Kγ inhibitor. The anti-glioblastoma effects of PI3Kγ inhibitor were enhanced by temozolomide, the standard of care chemotherapy for glioblastoma. These results suggest microglia is a mediator of clinical glioblastoma survival and disruption of microglia-glioblastoma interaction as potential therapeutic strategy. Citation Format: Jie Li, Megan M. Kaneda, Jiangfei Wang, Kunal Patel, Johnny Akers, Valya Ramakrishnan, Tao Jiang, Bob S. Carter, Judith A. Varner, Clark C. Chen. PI3Kγ inhibition suppresses glioblastoma tumorigenicity through disruption of an IL11-STAT3-MYC signaling axis between microglia and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-207. doi:10.1158/1538-7445.AM2017-LB-207

AdipoR1-mediated miR-3908 inhibits glioblastoma tumorigenicity through downregulation of STAT2 associated with the AMPK/SIRT1 pathway.

A prospective method of treatment for cancer is to inhibit oncogene signaling pathways with microRNA (miRNA or miR). In the present study, whether the expression of STAT2, AdipoR1/AMPK/SIRT1 pathway of glioma is regulated by miR-3908 was explored. To confirm whether the predicted miR-3908 is matched with STAT2 and AdipoR1, 3'UTR luciferase activity of STAT2 and AdipoR1 was assessed. In the presence of the mimics or inhibitors of miR-3908, cell function of glioma cells, such as proliferation, growth, migration, invasion and apoptosis were analyzed. The expression of AdipoR1 and its downstream AMPK/SIRT1 pathway proteins or STAT2 were examined. Luciferase reporter analysis showed that miR-3908 directly target STAT2 and AdipoR1. miR-3908 suppressed expression of STAT2 or AdipoR1 and downregulated AdipoR1 pathway genes, including AMPK, p-AMPK and SIRT1. miR-3908 inhibited tumorigenicity, migration, growth and invasion in glioma cell lines U251 and U87 as well as increased apoptosis of these cells. The pathways related to tumorigenicity and tumor progression, STAT2 and AdipoR1/AMPK/SIRT1 could be restrained by miR-3908. In conclusion, restoration of miR-3908 expression induced suppression of cancer progression and glioblastoma tumorigenicity. The present study discovered novel tumorigenesis associated with miR-3908, which may represent a new target in treatment for glioblastoma.

OS06.7 PI-3Kγ inhibition suppresses glioblastoma tumorigenicity through disruption of an IL11-STAT3-MYC signaling axis between microglia and glioblastoma

AbstractWhile mutations in isocitrate dehydrogenase (IDH) gene are associated with favorable prognosis in glioblastoma patients, the biologic basis for favorable survival in patients with wild-type IDH (wtIDH) glioblastoma remains poorly understood. We identified that an inflammatory gene signature whose expression inversely correlated with patient survival in three independent, wtIDH glioblastoma cohorts (The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and REMEBRANDT). Systematic analysis of the inflammatory infiltrates of the glioblastoma microenvironment revealed microglia as the predominant cell type driving this survival association. The pro-tumorgenic effects of microglia can be recapitulated using conditioned media derived from microglia. We cross-referenced proteomic profile of microglia conditioned media with the TCGA and the CGGA to identify constituent soluble factors with survival association and identified IL11. In both clinical datasets, increased IL11 expression was associated with shortened survival. Moreover, IL11 expression closely correlated with the survival-associated inflammatory signature in both TCGA and CGGA glioblastomas. IL11 mRNA and protein expression are elevated in clinical glioblastoma specimen relative to the surrounding cerebrum. Analysis of samples secured from three unrelated patients revealed that IL11 expression was largely found in the microglia fraction. and experiments demonstrate that IL11 was necessary and sufficient for enhancement of glioblastoma tumorigenicity. Microglia released IL11 induced activation of a STAT3-MYC signaling axis in glioblastoma cells, which ultimately lead to up-regulation of genes required for glioblastoma tumorigenicity, including OLIG2, SOX2, and POU3F2. PI3Kγ activation in myeloid-derived cells, including microglia, is essential for its trafficking into the tumor microenvironment. We hypothesized that PI3Kγ inhibition or inactivation should impede microglia trafficking to the glioblastoma microenvironment and suppress tumorigenicity. Supporting our hypothesis, treatment with the PI3Kγ, TG100-115, suppressed glioblastoma tumorigenicity by reduced microglia density and IL11 release in the glioblastoma microenvironment. These effects were recapitulated when murine glioblastoma GL261 were implanted into p110γ mice. Importantly, ectopic expression IL11 in GL261 reversed the tumor-suppressive effect of TG100-115. The anti-glioblastoma effects of TG100-115 were enhanced by temozolomide, the standard of care chemotherapy for glioblastoma. These results suggest microglia is a mediator of clinical glioblastoma survival and disruption of microglia-glioblastoma interaction as potential therapeutic strategy.

Glycolipid GD3 and GD3 synthase are key drivers for glioblastoma stem cells and tumorigenicity

The cancer stem cells (CSCs) of glioblastoma multiforme (GBM), a grade IV astrocytoma, have been enriched by the expressed marker CD133. However, recent studies have shown that CD133(-) cells also possess tumor-initiating potential. By analysis of gangliosides on various cells, we show that ganglioside D3 (GD3) is overexpressed on eight neurospheres and tumor cells; in combination with CD133, the sorted cells exhibit a higher expression of stemness genes and self-renewal potential; and as few as six cells will form neurospheres and 20-30 cells will grow tumor in mice. Furthermore, GD3 synthase (GD3S) is increased in neurospheres and human GBM tissues, but not in normal brain tissues, and suppression of GD3S results in decreased GBM stem cell (GSC)-associated properties. In addition, a GD3 antibody is shown to induce complement-dependent cytotoxicity against cells expressing GD3 and inhibition of GBM tumor growth in vivo. Our results demonstrate that GD3 and GD3S are highly expressed in GSCs, play a key role in glioblastoma tumorigenicity, and are potential therapeutic targets against GBM.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

The transcriptional modulator HMGA2 promotes stemness and tumorigenicity in glioblastoma

Glioblastoma (GBM) contains a population of stem-like cells that promote tumor invasion and resistance to therapy. Identifying and targeting stem cell factors in GBM may lead to the development of more effective therapies. High Mobility Group AT-hook 2 (HMGA2) is a transcriptional modulator that mediates motility and self-renewal in normal and cancer stem cells. We identified increased expression of HMGA2 in the majority of primary human GBM tumors and cell lines compared to normal brain. Additionally, HMGA2 expression was increased in CD133+ GBM neurosphere cells compared to CD133− cells. Targeting HMGA2 with lentiviral short hairpin RNA (shRNA) led to decreased GBM stemness, invasion, and tumorigenicity. Ectopic expression of HMGA2 in GBM cell lines promoted stemness, invasion, and tumorigenicity. Our data suggests that targeting HMGA2 in GBM may be therapeutically beneficial.

Downregulation of TLX induces TET3 expression and inhibits glioblastoma stem cell self-renewal and tumorigenesis.

Glioblastomas have been proposed to be maintained by highly tumorigenic glioblastoma stem cells (GSCs) that are resistant to current therapy. Therefore, targeting GSCs is critical for developing effective therapies for glioblastoma. In this study, we identify the regulatory cascade of the nuclear receptor TLX and the DNA hydroxylase Ten eleven translocation 3 (TET3) as a target for human GSCs. We show that knockdown of TLX expression inhibits human GSC tumorigenicity in mice. Treatment of human GSC-grafted mice with viral vector-delivered TLX shRNA or nanovector-delivered TLX siRNA inhibits tumour development and prolongs survival. Moreover, we identify TET3 as a potent tumour suppressor downstream of TLX to regulate the growth and self-renewal in GSCs. This study identifies the TLX-TET3 axis as a potential therapeutic target for glioblastoma.

Increased level of H19 long noncoding RNA promotes invasion, angiogenesis, and stemness of glioblastoma cells.

OBJECT Increased levels of H19 long noncoding RNA (lncRNA) have been observed in many cancers, suggesting that overexpression of H19 may be important in the development of carcinogenesis. However, the role of H19 in human glioblastoma is still unclear. The object of this study was to examine the level of H19 in glioblastoma samples and investigate the role of H19 in glioblastoma carcinogenesis. METHODS Glioblastoma and nontumor brain tissue specimens were obtained from tissue obtained during tumor resection in 30 patients with glioblastoma. The level of H19 lncRNA was detected by real-time quantitative reverse transcription polymerase chain reaction. The role of H19 in invasion, angiogenesis, and stemness of glioblastoma cells was then investigated using commercially produced cell lines (U87 and U373). The effects of H19 overexpression on glioblastoma cell invasion and angiogenesis were detected by in vitro Matrigel invasion and endothelial tube formation assay. The effects of H19 on glioblastoma cell stemness and tumorigenicity were investigated by neurosphere formation and an in vivo murine xenograft model. RESULTS The authors found that H19 is significantly overexpressed in glioblastoma tissues, and the level of expression was associated with patient survival. In the subsequent investigations, the authors found that overexpression of H19 promotes glioblastoma cell invasion and angiogenesis in vitro. Interestingly, H19 was also significantly overexpressed in CD133(+) glioblastoma cells, and overexpression of H19 was associated with increased neurosphere formation of glioblastoma cells. Finally, stable overexpression of H19 was associated with increased tumor growth in the murine xenograft model. CONCLUSIONS The results of this study suggest that increased expression of H19 lncRNA promotes invasion, angiogenesis, stemness, and tumorigenicity of glioblastoma cells. Taken together, these findings indicate that H19 plays an important role in tumorigenicity and stemness of glioblastoma and thus could be a therapeutic target for treatment of glioblastoma in the future.

EPIG-02DYNAMIC EPIGENETIC MODULATION OF GLIOBLASTOMA TUMORIGENICITY BY INHIBITION OF LYSINE-SPECIFIC DEMETHYLASE 1 (LSD1)

Glioblastoma is one of the most devastating of human cancers, with near-uniform fatality within two years of diagnosis. Therapeutic failure is thought to be related to small subpopulation of cells that exhibit the properties of self-renewal and tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells (“elite” model) or as a dynamically acquired property (“stochastic” model). We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary patient-derived glioblastoma (PDX) lines. In multiple PDX lines, glioblastoma subclones that are isogenic (within the limits of the current DNA sequencing technology) nevertheless display varying tumorigenicity. Moreover, the property can be gained or lost at low frequencies. The results indicated that while glioblastoma tumorigenicity largely conforms to the “elite” model, there is dynamic interplay between tumorgenic and non-tumorgenic cell states. Using independent Genetically Engineered Murine Models (GEMMs) of glioblastoma, PDX glioblastoma models, and single cell analysis of clinical glioblastoma specimens, we demonstrate that these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD1 depletion increases H3K4me3 accumulation at the MYC locus, which elevates MYC expression. MYC, in turn, regulates OLIG2, SOX2 and POU3F2, a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Altered tumorgenic potential was not accompanied by a change in the molecular subtype (as defined by the TCGA) and was therefore not related to subtype plasticity. Importantly, complete inhibition or silencing of LSD1 led to suppression of MYC expression and glioblastoma tumorigenicity in vitro and in vivo. However, partial inhibition of LSD1 lead to epigenetic reprogramming resulting in heritable increase MYC expression and glioblastoma tumorigenicity. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.

Abstract PR02: Dynamic epigenetic regulation of glioblastoma tumorigenicity through a LSD1-MYC-OLIG2 axis

Abstract Glioblastoma is one of the most devastating of human cancers, with near-uniform fatality within two years of diagnosis. Therapeutic failure is thought to be related to small subpopulation of cells that exhibit the properties of self-renewal and tumorigenicity. Understanding how such subpopulations attain and retain these properties remains a central question in oncology. One fundamental issue is whether tumorigenicity exists within a static population of elite cells or whether the capacity is stochastically acquired. To test these models, we assayed the tumorigenicity of single-cell subclones derived from long-terms passaged and primary patient-derived xenograft (PDX) glioblastoma lines. Our findings were best described by a hybrid model that is largely deterministic (elite) but with opportunities for dynamic (stochastic) interchange between non-tumorigenic and tumorigenic states. To identify molecular determinants of tumorigenicity, we performed gene expression profiling of the subclones. Analysis of the data suggested that tumorigenicity in glioblastoma is a dynamic property driven by variation in MYC expression, which in turn regulates Olig2 expression, a neural stem cell marker. Ectopic expression of MYC conferred tumorigenicty and MYC silencing abolished tumorigenicity in vitro and in vivo for multiple PDX and GEMM models. Transition between tumorigenic and non-tumorigenic cell states was associated with changes in histone modification at the MYC locus mediated by expression of lysine-specific demethylase 1 (LSD1). The model suggests a critical LSD1-MYC-OLIG2 axis that regulates the dynamic transition between glioblastoma cell states of differing tumorigenicity and unveils a novel framework for glioblastoma therapeutic development. Citation Format: Clark Chen, David Kozono, Jie Li, Masayuki Nitta, Oltea Sampetrean, Kimberly Ng, David Gonda, Deepa S. Kushwaha, Dmitry Merzon, Valya Ramakrishnan, Shan Zhu, Kaya Zhu, Hiroko Matsui, Olivier Harismendy, Wei Hua, Ying Mao, Chang-Hyuk Kwon, Keith L. Ligon, Hideyuki Saya, Bob S. Carter, Donald P. Pizzo, Scott R. VandenBerg, Frank Furnari, Webster Cavenee. Dynamic epigenetic regulation of glioblastoma tumorigenicity through a LSD1-MYC-OLIG2 axis. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR02.

Increased level of H19 long noncoding RNA promotes invasion, angiogenesis, and stemness of glioblastoma cells.

OBJECTIVE Increased levels of H19 long noncoding RNA (lncRNA) have been observed in many cancers, suggesting that overexpression of H19 may be important in the development of carcinogenesis. However, the role of H19 in human glioblastoma is still unclear. The object of this study was to examine the level of H19 in glioblastoma samples and investigate the role of H19 in glioblastoma carcinogenesis. METHODS Glioblastoma and nontumor brain tissue specimens were obtained from tissue obtained during tumor resection in 30 patients with glioblastoma. The level of H19 lncRNA was detected by real-time quantitative reverse transcription polymerase chain reaction. The role of H19 in invasion, angiogenesis, and stemness of glioblastoma cells was then investigated using commercially produced cell lines (U87 and U373). The effects of H19 overexpression on glioblastoma cell invasion and angiogenesis were detected by in vitro Matrigel invasion and endothelial tube formation assay. The effects of H19 on glioblastoma cell stemness and tumorigenicity were investigated by neurosphere formation and an in vivo murine xenograft model. RESULTS The authors found that H19 is significantly overexpressed in glioblastoma tissues, and the level of expression was associated with patient survival. In the subsequent investigations, the authors found that overexpression of H19 promotes glioblastoma cell invasion and angiogenesis in vitro. Interestingly, H19 was also significantly overexpressed in CD133+ glioblastoma cells, and overexpression of H19 was associated with increased neurosphere formation of glioblastoma cells. Finally, stable overexpression of H19 was associated with increased tumor growth in the murine xenograft model. CONCLUSIONS The results of this study suggest that increased expression of H19 lncRNA promotes invasion, angiogenesis, stemness, and tumorigenicity of glioblastoma cells. Taken together, these findings indicate that H19 plays an important role in tumorigenicity and stemness of glioblastoma and thus could be a therapeutic target for treatment of glioblastoma in the future.

Abstract 979: Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression

Abstract Glioblastoma is the most common form of primary brain cancer and remains one of the deadliest of human cancers with near-uniform fatality. Increasing evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells with self-renew ability and tumorigenicity, termed as tumor initiating cells. The mechanism how the tumor initiating cells maintain and gain tumorigenicity in glioblastoma still remains unclear. Here, we used sphere formation and tumor propagating potential to measure the tumorigenicity in established cell line and primary glioblastoma cells. The results indicated that glioblastoma tumorigenicity appears largely deterministic, though spontaneous gain and loss of this property occur at low frequency. Mechanically, this dynamic transition in tumorigenicity was governed by MYC level which was modulated epigenetically by the lysine-specific demethylase 1 (LSD1). Elevated MYC expression, in turn, regulates OLIG2, SOX2 and POU3F2, a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors facilitates transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development. Importantly, the effect of LSD1 on tumorigenity is “Janus”-like; partial depletion of LSD1 caused increased MYC expression and a pro-tumorigenic state. In contrast, complete suppression of LSD1 induced cell death. As such, therapeutic strategies targeting LSD1 and other targets manifesting similar “Janus” effect should be designed to prevent unintended induction of tumorigenesis during treatment. Citation Format: Jie Li, David Kozono, Masayuki Nitta, Oltea Sampetrean, David Gonda, Deepa S. Kushwaha, Dmitry Merzon, Valya Ramakrishnan, Shan Zhu, Kaya Zhu, Hiroko Matsui, Olivier Harismendy, Wei Hua, Ying Mao, Chang-Hyuk Kwon, Hideyuki Saya, Bob S. Carter, Donald P. Pizzo, Scott R. VandenBerg, Clark C. Chen. Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 979. doi:10.1158/1538-7445.AM2015-979

Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression

The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.

HG-12 * LIN28 AND MYC EXPRESSING PEDIATRIC GLIOBLASTOMAS ARE SENSITIVE TO INHIBITORS OF GLUTAMINE METABOLISM

We have previously shown that LIN28A protein is highly expressed in a subset of pediatric glioblastoma (GBM). Knockdown of LIN28A using lentiviral shRNA in GBM cell lines inhibits their invasion, growth and clonogenicity. LIN28A downregulates the let-7 family of tumor suppressing microRNAs, and these in turn inhibit multiple oncogenes including MYC. LIN28 is also known to regulate cellular metabolism in normal and transformed cells. We hypothesized that LIN28 would contribute to GBM tumorigenicity in part by modifying cellular metabolism. Pediatric GBM express MYC and NMYC, and these oncogenes also promote tumorigenicity through increasing proliferation, invasion and altering cellular metabolism. Addition of LIN28A to the JHH- GBM14 cell line leads to upregulation of MYC and glutaminase (GLS) as measured by western blot. The inhibitors of glutamine metabolism DON (6-Diazo-5-oxo-L-norleucine) and acivicin suppress the growth of LIN28A-transduced GBM14 cells to a greater extent than control empty-vector transduced cells in MTT assays (p = 0.008 for 5 uM DON, Student's t-test; p = 0.03, 5 uM acivicin, Student's t-test). We found that the pediatric high grade glioma cell lines SF188, UW479, SJ-GBM2, and KNS42 express varying levels of LIN28, MYC, and NMYC and have robust GLS expression by western blot. Acivicin treatment decreased the growth of SF188, KNS42 and UW479 in MTT Assays (P < .005 10 uM acivicin, Student's t-test). DON and acivicin have been tested in phase I clinical trials in children, but never extensively investigated in aggressive pediatric brain tumors. Our data suggest that LIN28 can alter GBM cellular metabolism in a way that confers sensitivity to inhibitors of glutamine metabolism and that these agents may be of therapeutic use in pediatric high grade gliomas.