Medulloblastoma Cell Line DAOY Research Articles

Abstract B41: miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1

Abstract Purpose: Medulloblastoma, a most common pediatric malignant brain tumor consists of four molecular subgroups viz. WNT, SHH, Group 3 and Group 4. MiR-148a is over-expressed in the WNT subgroup tumors, which have the lowest incidence of metastasis and excellent survival among all medulloblastomas. Therefore, the role of miR-148a in medulloblastoma biology was investigated. Experimental Design: MiR-148a was expressed either in a transient manner using synthetic mimic or in a stable doxycycline inducible manner using a lentiviral vector. Effect of miR-148a expression on the growth and malignant behavior of medulloblastoma cell lines was investigated. CpG Methylation status of region upstream to pri-miR-148a was determined by performing bisulphite sequencing of this region in medulloblastoma cell lines and tissues. Results: Expression of miR-148a to the levels comparable to that in the WNT subgroup tumors was found to inhibit proliferation, clonogenic potential and in vitro invasion potential of medulloblastoma cells. In vivo tumorigenicity of medulloblastoma cells Daoy, D425 and D283, as seen by either generating subcutaneous xenografts or orthotopic xenografts in immunodeficient mouse, was found to be reduced significantly upon miR-148a expression. NRP1, ROCK1 and DNMT1 were identified as direct targets of miR-148a with NRP1 being novel target. Restoration of NRP1 expression in medulloblastoma cells was found to rescue the reduction in the invasion potential and tumorigenicity brought about by miR-148a expression. NRP1expression in medulloblastomas was found to be associated with poor survival, with little or no expression in majority of the WNT tumors. This observation is consistent with high miR-148a expression and low incidence of metastasis and excellent survival of the WNT subgroup tumors. miR-148a is known to be down-regulated as a result of promoter hypermethylation in colon, lung, breast, head and neck carcinomas and melanoma. Bisulphite sequencing of the region upstream to pri-miR-148a revealed presence of CpG methylation in four medulloblastoma cell lines Daoy, D283, D341 and D425 and non WNT medulloblastoma tissues, while it was found to be un-methylated in WNT medulloblastomas. Conclusions: The tumor suppressive effect of miR-148a expression in medulloblastoma cells accompanied by the down-regulation of NRP1, ROCK1 and DNMT1 makes miR-148a an attractive therapeutic agent for the treatment of medulloblastomas. Preliminary findings provided by bisulphite sequencing suggest that under-expression of miR-148a may be as a result of CpG methylation of upstream region to pri-miR-148a sequence. Citation Format: Kedar Narsinha Yogi, Epari Sridhar, Naina Goel, Rakesh Jalali, Atul Goel, Aliasgar Moiyadi, Rahul Thorat, Pooja Panwalkar, Atul Khire, Archya Dasgupta, Prakash Shetty, Neelam Shirsat. miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B41.

PO19TARGETING ASCT2 BLOCKS GROWTH AND MIGRATION IN A MEDULLOBLASTOMA CELL LINE

INTRODUCTION: Medulloblastoma is the most common malignant paediatric brain tumour. Current treatments include surgery and radio/chemotherapy, which are associated with significant side effects, such as neurological, intellectual and physical disabilities. Metabolic adaptation has emerged as a hallmark of cancer and as a promising therapeutic target. Rapidly proliferating cancer cells adapt their metabolism by increasing nutrient uptake and by reorganising metabolic flux to support cell growth, promoting macromolecule synthesis and ATP production. Glutamine is a conditionally essential amino acid in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production as well as for anaplerotic reactions fuelling the tricarboxylic acid cycle. METHOD: In this study, we used the DAOY medulloblastoma cell line. Amino acid starvation reduced cell proliferation but only glutamine starvation induced apoptosis. DAOY cells express high levels of ASCT2. Chemical inhibition (GPNA) or shRNA knockdown of ASCT2 in DAOY cells reduced cell growth and migration in the scratch assay. RESULTS: We demonstrate that the glutamine transporter ASCT2 is highly expressed in the DAOY medulloblastoma cell line. We show that chemical or shRNA-mediated inhibition of ASCT2 in vitro significantly inhibits cell growth, induce apoptosis and inhibit cell migration. CONCLUSION: Glutamine contributes to essentially every core metabolic task of proliferating tumour cells. ASCT2-mediated glutamine uptake is essential for multiple pathways regulating cell growth and cell migration in a medulloblastoma cell line, and it could therefore be a putative therapeutic target for medulloblastoma.

Inhibition of GPR137 suppresses proliferation of medulloblastoma cells in vitro.

Medulloblastoma is the most common malignant pediatric brain tumor in children. GPR137 is a ubiquitously expressed gene in the central nervous system. It has been reported that GPR137 modulates malignant proliferation of glioma cells. However, the relationship between GPR137 and medulloblastoma is still unknown. In this study, we knocked down GPR137 in the medulloblastoma cell line Daoy via a lentivirus-based RNA interference system to explore its role in medulloblastoma. Functional analyses showed that cell proliferation and colony formation were obviously restrained in Daoy cells after GPR137 knockdown. Furthermore, knockdown of GPR137 in Daoy cells led to a significant increase in cell percentage in the G0/G1 phase but a decrease in the S phase. Additionally, the cell population in the sub-G1 phase, which represents apoptotic cells, was remarkably increased in GPR137 knockdown cells. GPR137 inhibition induced a strong proapoptotic effect in Daoy cells, as confirmed by annexin V-APC/7-AAD double staining. In conclusion, GPR137 knockdown inhibited growth of Daoy medulloblastoma cells via disturbing cell cycle progression and inducing apoptosis. Our investigation suggested that GPR137 could be a potential oncogene in medulloblastoma cells and might serve as a target for the treatment of medulloblastoma.

Abstract 3282: Induction and characterization of cyclopamine resistance in SK-N-DZ neuroblastoma and DAOY medulloblastoma cell lines

Abstract Neuroblastoma and medulloblastoma are malignant solid tumors of childhood and early adolescence, that often reappear after clinical treatment due of an acquired chemo-resistance. These tumors present an increased expression of the Sonic Hedgehog signaling pathway. In the last years, inhibitors of this pathway, such as cyclopamine are being studied as a possible alternative to current treatment. In this research, we aim at analyzing the appearance and characteristics of resistance derived from the treatment with mutation-selective concentrations of cyclopamine. The aim of this study is the design of different ways to bypass the resistance generated by the tumor once its biology is completely understood. Cells were exposed to several treatment cycles. A 24 h cyclopamine exposure was performed when cell culture reached 60-70% confluence. Basal half-maximal inhibitory concentration (IC50) was 10 μM. Hence, SK-N-DZ cells received 5 treatment cycles of cyclopamine at 12 μM dissolved in DMSO; whereas Daoy cells received 5 treatment cycles of cyclopamine at 10 μM. The control group only received the diluent. Anchorage-independent growth assays (in soft agar), clonogenicity assays, cell migration scratch (wound-healing) assays, and relative mRNA expression were studied during cycles 1-5. During the first 5 treatment cycles, mRNA expression of SMO remained constant. CD15 expression decreased. MYCN, Gli and expression showed an increasing trend. Decrease in the pro-apoptotic status was also described. Soft agar showed an increase tendency in relative colony formation capacity. No statistical differences were found in the clonogenicity assays for the SK-ND-Z cells. The results highlighted the evidence that cells became resistant because they were able to grow in soft agar without attachment, to migrate and to overexpress genes related to cancer stem cells. Results shown are congruent with a resistance generation possibly owing to multidrug efflux pumps. However, more clear results would be seen with 48 h treatment. Further analysis based on this work could include 48 h treatment, water-dissolvable cyclopamine performance study, a more complete gene analysis, or studies with other neurological tumors. Note: This abstract was not presented at the meeting. Citation Format: Javier de la Rosa, Amaia Paredes, Javier Asensio-Salazar, Xing Fan, Bárbara Meléndez, Juan A. Rey, Javier S. Castresana. Induction and characterization of cyclopamine resistance in SK-N-DZ neuroblastoma and DAOY medulloblastoma cell lines. [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 3282. doi:10.1158/1538-7445.AM2015-3282

SU‐E‐J‐274: Responses of Medulloblastoma Cells to Radiation Dosimetric Parameters in Intensity‐Modulated Radiation Therapy

Purpose:To evaluate radiation responses of the medulloblastoma cell line Daoy in intensity‐modulated radiation therapy (IMRT), quantitative variations to variable radiation dosimetic parameters were tracked by bioluminescent images (BLIs).Methods:The luciferase and green fluorescent protein positive Daoy cells were cultured on dishes. The medulloblastoma cells irradiated to different dose rate, interval of fractionated doses, field margin and misalignment, and dose uniformity in IMRT were monitored using bioluminescent images. The cultured cells were placed into a dedicated acrylic phantom to deliver intensity‐modulated fluences and calculate accurate predicted dose distribution. The radiation with dose rate from 0.5 Gy/min to 15 Gy/min was irradiated by adjusting monitor unit per minute and source‐to‐surface distances. The intervals of fractionated dose delivery were changed considering the repair time of double strand breaks (DSB) revealed by straining of gamma‐H2AX.The effect of non‐uniform doses on the cells were visualized by registering dose distributions and BLIs. The viability according to dosimetric parameters was correlated with bioluminescent intensities for cross‐check of radiation responses.Results:The DSB and cell responses due to the first fractionated dose delivery significantly affected final tumor control rather than other parameters. The missing tumor volumes due to the smaller field margin than the tumor periphery or field misalignment caused relapse of cell responses on BLIs. The dose rate and gradient had effect on initial responses but could not bring out the distinguishable killing effect on cancer cells.Conclusion:Visualized and quantified bioluminescent images were useful to correlate the dose distributions with spatial radiation effects on cells. This would derive the effective combination of dose delivery parameters and fractionation. Radiation responses in particular IMRT configuration could be reflected to image based‐dose re‐optimization.

Folate receptor-targeted cytarabine for inhibiting the proliferation of medulloblastoma and promoting its apoptosis: an in vitro experimental study

Objective To investigate the folate receptor-targeted cytarabine (FR-Ara-C) for the proliferation inhibition and apoptosis promotion effect of the Daoy medulloblastoma cell lines. Methods The immunofluorescence and confocal laser scanning techniques were used to detect the expression of folate receptors-α (FRs-α) in Daoy cell lines. Methyl thiazolyl tetrazolium (MTT) colorimetric method was used to detect the proliferation inhibition rate after different concentrations (0.5-100.0×10-3 mmol/L FR-Ara-C and ordinary cytarabine [Ara-C]) acting on Daoy cells at different times (2-24 h) and their differences were compared. The group with the most obvious difference was selected for conducting AnnexinV-FITC/PI apoptosis detection. Results FRs-α expressed in both Daoy cell membrane and nucleus. When the same concentration of FR-Ara-C and Ara-C acted on Daoy cells at different times, the proliferation inhibition rate of FR-Ara-C was higher than that of Ara-C. There was significant difference (P<0.05); when the different concentrations of FR-Ara-C and Ara-C acted on Daoy cells at the same time, the proliferation inhibition rate of FR-Ara-C was higher than that of Ara-C. There was significant difference (P<0.05); when FR-Ara-C acted on for 8 h, the increased inhibition rate was most obvious compared with the Ara-C effect, 50% concentration of inhibition (IC50) at this time point was the optimal action concentration. Flow cytometry analysis showed that when 50×10-3 mmol/L concentration of FR-Ara-C and Ara-C acted on for 8 h, the apoptosis rates were 92.8±2.3% and 62.3±1.5%; when 10.0×10-3 mmol/L concentration of FR-Ara-C and Ara-C acted on for 8 h, the apoptosis rates were 87.1±2.4% and 44.7±1.7%; when the concentration was the same, the apoptosis rate was significantly higher than that of Ara-C after the action of FR-Ara-C. There was significant difference (P<0.05). Conclution FR-Ara-C has more significant proliferation inhibition and apoptosis promotion effect on medulloblastoma in vitro. Key words: Medulloblastoma; Folate receptor 1; Cytarabine; Inhibitory concentration 50

Overexpression of the ∆Np73 isoform is associated with centrosome amplification in brain tumor cell lines.

The p73 protein is a member of the p53 family, and this protein is known to be essential for the maintenance of genomic stability, DNA repair, and apoptosis regulation. Transcription from two promoters leads to two main N-terminal isoforms: the TAp73 isoform is reported to have tumor suppressor function, whereas the ΔNp73 isoform likely has oncogenic potential. The present study is focused on the investigation of a possible role of both these p73 N-terminal isoforms in the process of centrosome amplification. HGG-02 and GM7 glioblastoma cell lines and the Daoy medulloblastoma cell line were used in this study. The cells were analyzed using indirect immunofluorescence to determine TAp73 and ΔNp73 expression patterns and possible co-localization with the BubR1 protein, as well as the number of centrosomes. A transiently transfected GM7 cell line was used to verify the results concerning the N-terminal isoforms in relation to centrosome amplification. We found that increased immunoreactivity for the ΔNp73 isoform is associated with the occurrence of an abnormal number of centrosomes in particular cells. Using the transiently transfected GM7 cell line, we confirmed that centrosome amplification is present in cells with overexpression of the ΔNp73 isoform. In contrast, the immunoreactivity for the TAp73 isoform was weak or medium in most of the cells with an aberrant number of centrosomes. To determine the putative counterpart of the p73 N-terminal isoforms among spindle assembly checkpoint (SAC) proteins, we also evaluated possible interactions between the N-terminal isoforms and BubR1 protein, but no co-localization of these proteins was observed.

MB-12 * INDUCED CISPLATIN RESISTANCE IN MEDULLOBLASTOMAS

INTRODUCTION: Medulloblastoma is the most common malignant brain tumor in children. Treatment typically involves surgery followed by chemotherapy, incorporating cisplatin. Tumor recurrence is common after treatment, especially in type 3 (C-MYC-expressing) medulloblastomas, and less commonly in sonic-hedgehog-associated tumors (type 2). Though clinically devastating, little is known about recurrent medulloblastomas compared to primary tumors and almost nothing is known about the mechanisms of chemotherapeutic resistance in medulloblastoma. OBJECTIVES: Develop a cellular model for medulloblastoma recurrence and drug resistance by establishing a cisplatin-resistant medulloblastoma cell line, and evaluate sensitivity of this model to other treatments. METHODS: Medulloblastoma cell lines DAOY and D341-MED, representative of type 2 and type 3 medulloblastoma were pulsed with cisplatin or cultured in escalating concentrations of cisplatin, respectively. Resistance to cisplatin was assessed by cell proliferation assay (XTT). RESULTS: XTT analysis at 48 hours revealed an 8-fold increase in IC-50 between the wild-type D341 and cisplatin-resistant D341 cell line (termed CR4). DAOY cells evaluated similarly at 72 hours showed an ∼3 fold difference between wild type and cisplatin-pulsed DAOY (CR5P). Interestingly, CR4 cells displayed an increased sensitivity to doxorubicin relative to wild-type D341. DISCUSSION: Recurrent medulloblastoma show growth after repeated exposure to multiple chemotherapeutics. Our newly-created CR4 and CR5P cells represent this element of recurrent medulloblastoma, allowing analysis of biochemical and genetic differences between primary and recurrent medulloblastomas. We plan to further analyze hypersensitivity to other drugs with the aim of identifying candidates for combination chemotherapy. We also plan to evaluate the genetic, and phenotypic differences between wild type and resistant cells in response to cisplatin to better understand post-chemotherapy recurrence. CONCLUSION: We have created the first 2 medulloblastoma cell lines that show cisplatin resistance, serving as a novel model by which to study medulloblastoma recurrence.

Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization.

The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.

MXD3 regulation of DAOY cell proliferation dictated by time course of activation.

BackgroundMXD3 is a basic-helix-loop-helix-leucine-zipper transcription factor involved in cellular proliferation. In previous studies we demonstrated that knock-down of MXD3 in the human medulloblastoma cell line DAOY resulted in decreased proliferation. Surprisingly, overexpression of MXD3 in DAOY cells also decreased proliferation and increased cell death, suggesting that persistent expression of MXD3 triggers an apoptotic response, perhaps as a fail-safe mechanism. To investigate this apparent paradox in detail we developed a tamoxifen inducible system to analyze the temporal effects of MXD3 in the proliferation and transcriptional response of DAOY cells upon acute induction compared with long-term expression of MXD3.ResultsWe find that acute induction of MXD3 initially promotes cell cycle progression as assessed by a transient increase in bromodeoxyuridine incorporation. However, persistent induction of MXD3 ultimately results in decreased proliferation based on cell counts. Finally, with microarray expression profiling and gene ontology analysis we identify several major pathways enriched in response to acute (immune response, apoptosis, cell cycle) versus persistent (cell adhesion) MXD3 activation.ConclusionsIn this study, we demonstrate that acute MXD3 activation results in a transient increase in cell proliferation while persistent activation of MXD3 eventually results in an overall decrease in cell number, suggesting that the time course of MXD3 expression dictates the cellular outcome. Microarray expression profiling and gene ontology analysis indicate that MXD3 regulates distinct genes and pathways upon acute induction compared with persistent expression, suggesting that the cellular outcome is specified by changes in MXD3 transcriptional program in a time-dependent manner.

Celecoxib suppresses the phosphorylation of STAT3 protein and can enhance the radiosensitivity of medulloblastoma-derived cancer stem-like cells.

Medulloblastoma (MB) is a malignant primary brain tumor with poor prognosis. MB-derived CD133/Nestin double-positive cells (MB-DPs) exhibit cancer stem-like cell (CSC)-like properties that may contribute to chemoradioresistance, tumorigenesis and recurrence. In various tumors, signal transducer and activator of transcription 3 (STAT3) upregulation including MB which can regulate the expression of Nestin. Celecoxib, a selective COX-2 inhibitor, has been shown to potentially reduce STAT3 phosphorylation. The aim of the present study was to investigate the role of celecoxib in enhancing the effects of ionizing radiotherapy (IR) on MB-DP. MB-DPs and MB-derived CD133/Nestin double-negative cells (MB-DNs) were isolated from medulloblastoma cell line Daoy. Then, both of them were treated with celecoxib in different concentrations, and cell viability was assessed. The assays of cell survival, sphere formation, radiosensitivity, colony formation, apoptotic activity and mouse xenografting experiments in MB-DPs and MB-DNs treated with celecoxib alone, radiation alone, or celecoxib combined with radiation were further evaluated. We isolated MB-DPs from MB cell line Daoy, which exhibited typical CSC-like characteristics. Microarray analysis and Western blotting both indicated the upregulation of Janus kinase (JAK)-STAT cascade and STAT3 phosphorylation. Incubation with celecoxib dose-dependently suppressed the CSC-like properties and enhanced the IR effect on the induction of apoptosis, as detected by TUNEL assay and staining for Caspase 3 and Annexin V. Finally, celecoxib also enhanced the IR effect to suppress tumorigenesis and synergistically improve the recipient survival in orthotopic MB-derived CD133/Nestin double-positive cells (MB-DP cells) bearing mice.

Identification of vitamin d3-based hedgehog pathway inhibitors that incorporate an aromatic a-ring isostere.

Previous structure-activity relationship studies for vitamin D3 (VD3) inhibition of Hedgehog (Hh) signaling directed the design, synthesis, and evaluation of a series of VD3-based analogues that contain an aromatic A-ring mimic. Characterization of these compounds in a series of cellular assays demonstrated their ability to potently and selectively down-regulate Hh pathway signaling. The most active of these, 17, inhibited pathway signaling in Hh-dependent mouse fibroblasts (IC50 = 0.74 ± 0.1 μM) and cultured cancer cells (IC50 values 3.8 ± 0.1 to 5.2 ± 0.2 μM). In addition, 17 demonstrated reduced activation of the vitamin D receptor (VDR) compared to VD3 in these cellular models. These results suggest that VD3-based analogues with an aromatic A-ring are a valid scaffold for the development of more selective and potent Hh pathway inhibitors and identify 17 as an intriguing lead from this class of compounds for further development. In addition, our analysis of Hh pathway inhibitors in cancer cells suggests that the murine basal cell carcinoma cell line ASZ001 and the human medulloblastoma cell line DAOY are appropriate in vitro cancer models for early stage evaluation of pathway inhibition.

The SOX2-Interactome in Brain Cancer Cells Identifies the Requirement of MSI2 and USP9X for the Growth of Brain Tumor Cells

Medulloblastomas and glioblastomas, the most common primary brain tumors in children and adults, respectively, are extremely difficult to treat. Efforts to identify novel proteins essential for the growth of these tumors may help to further our understanding of the biology of these tumors, as well as, identify targets for future therapies. The recent identification of multiple transcription factor-centric protein interaction landscapes in embryonic stem cells has identified numerous understudied proteins that are essential for the self-renewal of these stem cells. To identify novel proteins essential for the fate of brain tumor cells, we examined the protein interaction network of the transcription factor, SOX2, in medulloblastoma cells. For this purpose, Multidimensional Protein Identification Technology (MudPIT) identified >280 SOX2-associated proteins in the medulloblastoma cell line DAOY. To begin to understand the roles of SOX2-associated proteins in brain cancer, we focused on two SOX2-associated proteins, Musashi 2 (MSI2) and Ubiquitin Specific Protease 9x (USP9X). Recent studies have implicated MSI2, a putative RNA binding protein, and USP9X, a deubiquitinating enzyme, in several cancers, but not brain tumors. We demonstrate that knockdown of MSI2 significantly reduces the growth of DAOY cells as well as U87 and U118 glioblastoma cells. We also demonstrate that the knockdown of USP9X in DAOY, U87 and U118 brain tumor cells strongly reduces their growth. Together, our studies identify a large set of SOX2-associated proteins in DAOY medulloblastoma cells and identify two proteins, MSI2 and USP9X, that warrant further investigation to determine whether they are potential therapeutic targets for brain cancer.

Abstract 3826: HoxA10 gene expression profile correlates with tumorigenic potential of medulloblastoma cell lines.

Abstract HOX genes are a family of homeodomain-containing transcription factors defined as master genes of development, altered in cancer cells and thus having implications for tumorigenesis. Developmental genes have been recognized as one of the keys to understanding the tumor progression in some type of cancers. This study aimed to correlate gene expression profile of some HOX genes with the tumorigenic potential of medulloblastoma cell lines (MCL). We used three human MCL, the UW473, UW472 and DAOY and two human cerebellum primary cultures (CPC). The MCL and CPC were characterized morphologically by light microscopy and immunophenotypically by flow cytometry. MCL were assessed by tumorigenic potential infusing 3x106 cells subcutaneously in NUDE mice. MCL and CPC were evaluated for gene expression profile of HOXA3, HOXA10, HOXB3, HOXB4 and HOXB6 genes by quantitative real time PCR. MCL are morphologically heterogeneous (polygonal and fibroblastoid morphology) and the CPC present fibroblastoid morphology. Immunophenotypically, MCL and CPC were similar for some CD markers and showed a high percentage (70-99%) for CD44, CD73, CD105, CD166 and CD29 and low or absence (0-5.3%) for CD144, CD31, CD34, CD45 and CD133. Some differences were observed for CD140b (0.28±0.11%; 6.2±8.3%; 0.78±1.1%; 0.44%), CD24 (52.5±1.7%; 64.6±6.4%; 20.5±6.4%; 1.9%), CD146 (60.4±8.8%; 90.6±3.8%; 34.6±12%; 98.4%), CD73 (77.2±6.9%; 81.4±8.98%; 52,97±12.4%; 99.1%), CD271 (3.3±3.7%; 26.9±16.22%; 0.6±0.8%; 0,26%) and CD90 (3,7±1%; 99.3±0.8%; 88.5±3.3%; 77%) in the UW472, UW473 and DAOY MCL, and CPC respectively. Regarding to tumorigenic potential, among the UW402, UW473 and DAOY MCL, only the DAOY cell line gave rise to tumor nodules that presented histology features similar to medulloblastoma. About gene expression, the HOXA3 gene was 4,027.7±430.9, 283.1±3.8 and 1.4±0.8 times higher expressed in DAOY, UW473 and UW402 MCL respectively when compared to CPC (p&amp;lt;0.0001, p&amp;lt;0.0001, p=0.32), the HOXA10 gene was 22,462.78±26.9, 0.89±0.6 and 1.18±0.7 times (p&amp;lt;0.0001, p=0.82, p=0.77), the HOXB3 gene was 2,867.2±1,318.6, 115.9±12.6 and 67.8±21.9 times (p=0.0074, p&amp;lt;0.0001, p=0.0022), the HOXB4 gene was 5,647.8±567.4, 53.6±24.1 and 60.6±34.6 times (p&amp;lt;0.0001, p=0.0021, p=0.0065), the HOXB6 gene was 2,422.4±579.6, 722.4±58.6 and 1.5±1.2 times (p&amp;lt;0.0001, p&amp;lt;0.0001, p=0.30). Taken together, this study demonstrates that MCL are morphologically heterogeneous and CPC present fibroblastoid morphology. MCL and CPC showed an immunophenotype somewhat different for some markers and DAOY cell line was the only one that gave rise to tumor nodules in NUDE mice. Correlating the tumorigenic potential with HOX gene expression level, the HOXA10 is strongly expressed in DAOY tumorigenic cell line and low expressed in UW402 and UW473 cell lines, suggesting that HOXA10 gene can be related to tumor development in medulloblastoma. Citation Format: Ricardo Bonfim-Silva, Thais Valeria A. C. Pimentel, Elvis T. Valera, Carlos Alberto Scrideli, Fernando S. Ramalho, Hélio Rubens Machado, Dimas Tadeu Covas, Gregory J. Riggins, Angelo A. Cardoso, Aparecida Maria Fontes. HoxA10 gene expression profile correlates with tumorigenic potential of medulloblastoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3826. doi:10.1158/1538-7445.AM2013-3826

Abstract 5053: Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line.

Abstract Medulloblastoma is one of the most frequent and aggressive tumors of childhood. The Sonic hedgehog (Shh) pathway, related to human development, is altered in most medulloblastomas: genes like Ptch, Smo, or Sufu suffer mutations in 15% to 25% of these tumors. We tested Shh inhibition in the Daoy medulloblastoma cell line by two methods: a molecular one (direct Gli1 siRNA inhibition); and a pharmacological inhibition of Smo, upstream of Gli1, by cyclopamine. Afterwards, a comparison of cellular and molecular responses was done. We proved that MTT cell viability, and cell migration assessed by the scratching assay decreased after Shh inhibition. Furthermore, colony formation assay in culture decreased by 70%, and colony formation assay in soft agar decreased up to 90% when Shh inhibition was applied. As a whole, Shh inhibition conferred a less in vitro tumorigenic status to Daoy cells. Moreover, we assessed the expression of different Gli1 target genes and other genes, before and after Shh inhibition, and found that Shh shows a crosstalk with oncogenes and tumor suppressor genes that have been described in numerous tumors. Therefore, we found downregulation of Ptch1, Cyclin D2, Plakoglobin, Nkx2.2, Bmi1, Smo and N-myc after Shh inhibition. Sufu and Gli3 showed parallel results, where Gli1 siRNA did neither decrease nor increase expression of both genes, whereas cyclopamine reduced them in 15-25%. Pax6 mRNA levels were upregulated by either Gli1 siRNA or cyclopamine. Finally, Notch1 was upregulated after inhibition of Shh, while Notch2 showed contrasting results, as siRNA inhibition decreased its expression, while cyclopamine increased it. All these experiments give an overview of the Shh pathway in medulloblastoma, its relationship with other genes, and the demonstration of the efficacy of cyclopamine and Gli 1 siRNA Shh inhibition in vitro. Citation Format: R Garcia-Lopez, B Vera-Cano, A Vacas-Oleas, J de la Rosa, G Gallo-Oller, M H. Shahi, X Fan, M M. Alonso, J A. Rey, Javier S. Castresana. Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5053. doi:10.1158/1538-7445.AM2013-5053

Abstract 1872: Targeting MYC-driven medulloblastoma using inhibitors of glutamine metabolism.

Abstract Medulloblastoma is the most common malignant brain tumor in children. Currently, treatment consists of surgical resection, chemotherapy, and whole brain and spine radiation, and is associated with high morbidity. Medulloblastoma is divided into four subgroups with different associated mutations and clinical prognoses. Group 3 medulloblastoma tumors are associated with elevated MYC levels and have the worst clinical prognosis of all four subtypes. Because MYC over-expression can lead to increased glutamine metabolism and glutamine addiction in other cancer types, we hypothesized that MYC-driven medulloblastoma would exhibit increased glutamine metabolism and be sensitive to inhibitors of glutamine metabolism. In medulloblastoma cell lines, MYC expression positively correlates with increased expression of enzymes involved in glutamine metabolism as measured by western blot and qPCR. Also, forced MYC expression in the non-MYC driven medulloblastoma cell lines UW228 and DAOY leads to corresponding increases in glutaminase expression as measured by western blot. To investigate the hypothesis that inhibition of glutamine metabolism would preferentially target MYC driven medulloblastoma, we tested the effects of two glutamine analogs—acivicin and 6-diazo-5-L-norleucine (DON)—that inhibit enzymes in the glutamine metabolism pathway. Acivicin decreased the growth of the high-MYC medulloblastoma cell lines D425 and D283 by 50% and 75% respectively compared to vehicle treated cells, but did not significantly affect UW228, a MYC-negative cell line. Acivicin treatment caused a 36% decrease in growth in UW228 cells forced to express MYC compared to a 5% decrease in growth in parental UW228 cells (p=.00029). We next tested the effect of acivicin on cell proliferation as measured by BrdU incorporation in neurosphere models of MYC and non-MYC driven medulloblastoma. MYC-expressing cells had a 30% decrease in proliferation following acivicin treatment, while MYC negative cells only showed a 7% decrease in proliferation (p=0.02). Treatment of MYC expressing cells with acivicin lead to increased apoptosis as measured by flow cytometry. MYC positive neurospheres treated with acivicin had a 65% increase in the percentage of cells in the sub2N population, while MYC-negative neurospheres did not show an increase in the sub2N population. Our laboratory is currently investigating the in vivo efficacy of glutamine metabolism inhibitors in orthotopic xenograft experiments using MYC-driven medulloblastoma cell lines. Taken together, our data suggests that glutamine metabolism may be a therapeutic target in MYC-driven medulloblastoma and that glutamine analogs may be useful therapeutic agents. Citation Format: Allison R. Hanaford, Charles G. Eberhart, Eric H. Raabe. Targeting MYC-driven medulloblastoma using inhibitors of glutamine metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1872. doi:10.1158/1538-7445.AM2013-1872

Comparison of Spheroids Formed by Rat Glioma Stem Cells and Neural Stem Cells Reveals Differences in Glucose Metabolism and Promising Therapeutic Applications

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

Carbon Monoxide Mediates the Anti-apoptotic Effects of Heme Oxygenase-1 in Medulloblastoma DAOY Cells via K+ Channel Inhibition

Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K(+) channels also play a crucial role in apoptosis, permitting K(+) efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K(+) channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K(+) currents in DAOY cells, and largely reversed the oxidant-induced increase in K(+) channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K(+) channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K(+) channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.

Abstract 3730: Cisplatin treatment enhancement with acetaminophen in a model of human medulloblastoma in nude rats

Abstract Purpose: To determine the efficacy of cisplatin treatment when enhanced by high dose acetaminophen (APAP) and followed by N-acetylcysteine (NAC) rescue in a nude rat model of medulloblastoma. Methods: Human medulloblastoma (DAOY) cells were inoculated into the cerebellum of female athymic nude rats (rnu/rnu). On day 10, rats were randomized to treatment groups (n = 6 per group): (1) saline controls; (2) IP cisplatin 2 mg/kg; (3) PO APAP 600 mg/kg + IP cisplatin; and (4) APAP + cisplatin + NAC 1 g/kg. All treatments were given twice a week for two weeks. Cisplatin treatment was given one hour after APAP, and NAC was given 4 hours following cisplatin. T2 and contrast-enhanced T1 magnetic resonance (MR) imaging sequences were performed prior to treatment and just prior to sacrifice at 14 days following initial treatment. Cerebrospinal fluid (CSF) was collected for polymerase chain reaction (PCR) assay of human DNA. In vitro cell sensitivity to a dose escalation of APAP and cisplatin was assessed in medulloblastoma cell lines DAOY and D238-MED. Results: In vitro viability assays showed that combination APAP plus cisplatin induced synergistic toxicity in human medulloblastoma cells. In saline control rats, the tumor xenograft localized within the cerebellum with spread to the meningeal surfaces and brainstem, and had a volume of 27.1 ±6.7mm3 (mean±SEM). All cisplatin therapy treatment groups showed a significant reduction in final tumor volume when compared to the control group (P &amp;lt; 0.0004). A trend for the cisplatin single therapy group to be less efficacious when compared to the combined cisplatin treatment groups was observed however, the difference was not significant. No reduction in efficacy was seen in the NAC rescue group, with a tumor volume of 4.42±1.36 mm3, compared to 3.30±0.99 mm3 in the APAP plus cisplatin group. Tumor response was not measurable by MR, however the presence of human DNA in the CSF, was correlated with tumor size. Conclusions: This treatment model for medulloblastoma may provide a mechanism to improve chemotherapy efficacy without increasing chemotherapy dose. In vivo and in vitro data correlate to show that the pretreatment with APAP sensitizes cells to cisplatin allowing efficacy at a lower doses, meanwhile delayed treatment with NAC staves off the systemic toxicity normally associated with cisplatin treatments. APAP enhancement with NAC rescue may provide a less toxic option to patients who might otherwise experience systemic toxicity from high doses of cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3730. doi:1538-7445.AM2012-3730

Novel cell lines established from pediatric brain tumors

The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat "fingerprints" matching that of the patient's tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors.