Human Medulloblastoma Cell Research Articles

Enhanced inhibition of clonogenic survival of human medulloblastoma cells by multimodal treatment with ionizing irradiation, epigenetic modifiers, and differentiation-inducing drugs.

BackgroundMedulloblastoma (MB) is the most common pediatric brain tumor. Current treatment regimes consisting of primary surgery followed by radio- and chemotherapy, achieve 5-year overall survival rates of only about 60 %. Therapy-induced endocrine and neurocognitive deficits are common late adverse effects. Thus, improved antitumor strategies are urgently needed. In this study, we combined irradiation (IR) together with epigenetic modifiers and differentiation inducers in a multimodal approach to enhance the efficiency of tumor therapy in MB and also assessed possible late adverse effects on neurogenesis.MethodsIn three human MB cell lines (DAOY, MEB-Med8a, D283-Med) short-time survival (trypan blue exclusion assay), apoptosis, autophagy, cell cycle distribution, formation of gH2AX foci, and long-term reproductive survival (clonogenic assay) were analyzed after treatment with 5-aza-2′-deoxycytidine (5-azadC), valproic acid (VPA), suberanilohydroxamic acid (SAHA), abacavir (ABC), all-trans retinoic acid (ATRA) and resveratrol (RES) alone or combined with 5-aza-dC and/or IR. Effects of combinatorial treatments on neurogenesis were evaluated in cultured murine hippocampal slices from transgenic nestin-CFPnuc C57BL/J6 mice. Life imaging of nestin-positive neural stem cells was conducted at distinct time points for up to 28 days after treatment start.ResultsAll tested drugs showed a radiosynergistic action on overall clonogenic survival at least in two-outof-three MB cell lines. This effect was pronounced in multimodal treatments combining IR, 5-aza-dC and a second drug. Hereby, ABC and RES induced the strongest reduction of clongenic survival in all three MB cell lines and led to the induction of apoptosis (RES, ABC) and/or autophagy (ABC). Additionally, 5-aza-dC, RES, and ABC increased the S phase cell fraction and induced the formation of gH2AX foci at least in oneout-of-three cell lines. Thereby, the multimodal treatment with 5-aza-dC, IR, and RES or ABC did not change the number of normal neural progenitor cells in murine slice cultures.ConclusionIn conclusion, the radiosensitizing capacities of epigenetic and differentiation-inducing drugs presented here suggest that their adjuvant administration might improve MB therapy. Thereby, the combination of 5-aza-dC/IR with ABC and RES seemed to be the most promising to enhance tumor control without affecting the normal neural precursor cells.

MB-75EXPLOITING THE CELL CYCLE TO IMPROVE TREATMENT EFFICACY IN MEDULLOBLASTOMA

MB-75. EXPLOITING THE CELL CYCLE TO IMPROVE TREATMENT EFFICACY IN MEDULLOBLASTOMA Raelene Endersby1, Jacqueline Whitehouse1, Hilary Hii1, Mani Kuchibhotla1, Brett Patterson1, Brooke Strowger1, Tobias Schoep1, and Nick Gottardo1,2; Telethon Kids Institute and University of Western Australia, Perth, WA, Australia; Princess Margaret Hospital, Subiaco, WA, Australia Medulloblastoma is themost common malignantbraincancerof childhood. It arises in the cerebellum and has a predilection to spread throughout the central nervous system. The most effective treatment is a combination of surgery, followed by radiotherapy and intensive cytotoxic chemotherapy; however, devastating long-term sequelae, due to the collateral damage of healthy tissue by these treatments, including developmental defects, psychosocial deficits and secondary tumours are frequently encountered by survivors, and for patients with metastatic disease prognosis are dismal. Using high-throughput, cell-based assays human medulloblastoma cells (n 1⁄4 6) were screened against a library of approximately 3200 compounds. Fifty effective compounds were identified and further in vitro assessment identified several drugs that enhanced the cytotoxic activity of clinically-used chemotherapeutics: cyclophosphamide, cisplatin, and gemcitabine. Cell cycle checkpoint kinase (CHK1/2) inhibitors (iCHKs), were further assessed in vivo using mice bearing intracranial implants of human medulloblastoma cells. When combined with DNA-damaging chemotherapeutics, iCHK treatment reduced tumour burden as measured by bioluminescence imaging and significantly increased survival of animals with medulloblastoma up to three-fold, in several different PDX models. Immunohistochemical assessment of tumours showed the combination treatment significantly decreased tumour proliferation and significantly induced apoptosis compared with controls. These data demonstrate our experimental approach has robustly identified effective new therapies for paediatric medulloblastoma, and our findings strongly suggest CHK1/2 inhibitors have promising potential to improve treatments for this disease. Neuro-Oncology 18:iii97–iii122, 2016. doi:10.1093/neuonc/now076.71 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Thymoquinone inhibits growth of human medulloblastoma cells by inducing oxidative stress and caspase-dependent apoptosis while suppressing NF-κB signaling and IL-8 expression.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. The transcription factor NF-κB is overexpressed in human MB and is a critical factor for MB tumor growth. NF-κB is known to regulate the expression of interleukin-8 (IL-8), the chemokine that enhances cancer cell growth and resistance to chemotherapy. We have recently shown that thymoquinone (TQ) suppresses growth of hepatocellular carcinoma cells in part by inhibiting NF-κB signaling. Here we sought to extend these studies in MB cells and show that TQ suppresses growth of MB cells in a dose- and time-dependent manner, causes G2M cell cycle arrest, and induces apoptosis. TQ significantly increased generation of reactive oxygen species (ROS), while pretreatment of MB cells with the ROS scavenger N-acetylcysteine (NAC) abrogated TQ-induced cell death and apoptosis, suggesting that TQ-induced cell death and apoptosis are oxidative stress-mediated. TQ inhibitory effects were associated with inhibition of NF-κB and altered expression of its downstream effectors IL-8 and its receptors, the anti-apoptotic Bcl-2, Bcl-xL, X-IAP, and FLIP, as well as the pro-apoptotic TRAIL-R1, caspase-8, caspase-9, Bcl-xS, and cytochrome c. TQ-triggered apoptosis was substantiated by up-regulation of the executioner caspase-3 and caspase-7, as well as cleavage of the death substrate poly(ADP-ribose)polymerase. Interestingly, pretreatment of MB cells with NAC or the pan-caspase inhibitor zVAD-fmk abrogated TQ-induced apoptosis, loss of cyclin B1 and NF-κB activity, suggesting that these TQ-mediated effects are oxidative stress- and caspase-dependent. These findings reveal that TQ induces both extrinsic and intrinsic pathways of apoptosis in MB cells, and suggest its potential usefulness in the treatment of MB.

SMARCA4/Brg1 coordinates genetic and epigenetic networks underlying Shh-type medulloblastoma development.

Recent large-scale genomic studies have classified medulloblastoma into four subtypes: Wnt, Shh, Group 3 and Group 4. Each is characterized by specific mutations and distinct epigenetic states. Previously, we showed that a chromatin regulator SMARCA4/Brg1 is required for Gli-mediated transcription activation in Sonic hedgehog (Shh) signaling. We report here that Brg1 controls a transcriptional program that specifically regulates Shh-type medulloblastoma growth. Using a mouse model of Shh-type medulloblastoma, we deleted Brg1 in precancerous progenitors and primary or transplanted tumors. Brg1 deletion significantly inhibited tumor formation and progression. Genome-wide expression analyses and binding experiments indicate that Brg1 specifically coordinates with key transcription factors including Gli1, Atoh1 and REST to regulate the expression of both oncogenes and tumor suppressors that are required for medulloblastoma identity and proliferation. Shh-type medulloblastoma displays distinct H3K27me3 properties. We demonstrate that Brg1 modulates activities of H3K27me3 modifiers to regulate the expression of medulloblastoma genes. Brg1-regulated pathways are conserved in human Shh-type medulloblastoma, and Brg1 is important for the growth of a human medulloblastoma cell line. Thus, Brg1 coordinates a genetic and epigenetic network that regulates the transcriptional program underlying the Shh-type medulloblastoma development.

Abstract A51: Dianhydrogalactitol (VAL-083) offers potential therapeutic alternatives in the treatment of pediatric brain tumors

Abstract Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor, accounting for 15-30% of all childhood intracranial neoplasms. High grade gliomas (HGG) are much rarer in children than in adults, comprising only 5%-10% of childhood brain tumors. Although multidisciplinary treatment has improved the 5-year survival rates in children significantly, the prognosis for recurrent MB and HGG remains poor with median overall survival <1 year. Temozolomide (TMZ) is frequently employed in the treatment of MB and pediatric HGG; however, clinical evidence is lacking and poor outcomes due to high-expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is correlated with TMZ resistance, have been reported. Dianhydrogalactitol (VAL-083) is a structurally unique bi-functional alkylating agent causing DNA crosslinks at N7 position of guanine. VAL-083 readily crosses the blood brain barrier and has been shown to accumulate in brain tumor tissue. Furthermore, VAL-083 demonstrated clinical activity against MB and HGG in historical NCI-sponsored clinical studies. We have recently shown that VAL-083 demonstrates cytotoxic activity in GBM independent of MGMT expression in vitro and in vivo. We have further shown that VAL-083 is highly effective against GBM cancer stem cells (CSC) and non-CSC and that it acts as a radiosensitizer in GBM cell lines, in vitro. VAL-083 is currently in phase II clinical trials for recurrent GBM in adults. In the current adult GBM clinical trial VAL-083 displayed a favorable safety-profile and preliminary analysis supports a survival benefit at doses chosen for further investigation. Based on these recent results and data supporting VAL-083's clinical activity in historical MB and HGG studies, we sought to investigate the cytotoxic activity of VAL-083 as a potential therapeutic alternative for pediatric brain tumors by studying the drug against MB and pediatric HGG cell lines in vitro. Methods: Human MB cell lines DAOY, UW228, ONS-76 and UW426 and pediatric HGG cell line SF188 were treated with VAL-083 at concentrations of 0.1 100 μM for 72 h. Growth inhibition was measured by high content screening analysis. Neurosphere formation of DAOY and SF188 cells was determined by neurosphere colony assay. Results: VAL-083 inhibited growth of all cell lines (DAOY, UW228, ONS-76 UW426 and SF188) with IC50 at low micro-molar concentrations. DAOY, UW228 and SF188 cells were most sensitive to VAL-083. Primary neurosphere formation of DAOY cells was completely inhibited at 5 μM VAL-083 and VAL-083 was superior to TMZ against primary neurospheres formation of SF-188 cells; complete inhibition of SF-188 neurospheres was observed with the combination of VAL-083 and TMZ. Conclusion: These results suggest that VAL-083 is active against MB and pediatric HGG cells as well as CSC in vitro. Taken together with the results of historical NCI-sponsored clinical trials, these data warrant further investigations of VAL-083 as a possible therapeutic alternative for the treatment of recurrent pediatric MB and HGG. Citation Format: Jeffrey A. Bacha, Abbas Fotovati, Kaiji Hu, Anne Steino, Sarath Kanekal, Dennis M. Brown. Dianhydrogalactitol (VAL-083) offers potential therapeutic alternatives in the treatment of pediatric brain tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A51.

MB3W1 is an orthotopic xenograft model for anaplastic medulloblastoma displaying cancer stem cell- and Group 3-properties.

BackgroundMedulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup.MethodsWe established a robust orthotopic xenograft model with a cell line derived from the malignant pleural effusions of a child suffering from a Group 3 medulloblastoma.ResultsBesides classical characteristics of this tumor subgroup, the cells display cancer stem cell characteristics including neurosphere formation, multilineage differentiation, CD133/CD15 expression, high ALDH-activity and high tumorigenicity in immunocompromised mice with xenografts exactly recapitulating the original tumor architecture.ConclusionsThis model using unmanipulated, human medulloblastoma cells will enable translational research, specifically focused on Group 3 medulloblastoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2170-z) contains supplementary material, which is available to authorized users.

Studies on the Red Sea Sponge Haliclona sp. for its Chemical and Cytotoxic Properties.

Background:A great number of novel compounds with rich chemical diversity and significant bioactivity have been reported from Red Sea sponges.Objective:To isolate, identify, and evaluate the cytotoxic activity of the chemical constituents of a sponge belonging to genus Haliclona collected from the Eastern coast of the Red Sea.Materials and Methods:The total ethanolic extract of the titled sponge was subjected to intensive chromatographic fractionation and purification guided by cytotoxic bioassay toward various cancer cell lines. The structures of the isolated compounds were elucidated using spectroscopic techniques including one-dimension and two-dimension nuclear magnetic resonance, mass spectrometry, ultraviolet, and infrared data, as well as comparison with the reported spectral data for the known compounds. X-ray single-crystal structure determination was performed to determine the absolute configuration of compound 4. The screening of antiproliferative activity of the compounds was carried on three tumor cell lines, namely the human cervical cancer (HeLa), human hepatocellular carcinoma (HepG2), and human medulloblastoma (Daoy) cells using MTT assay.Results:This investigation resulted in the isolation of a new indole alkaloid, 1-(1H-indol-3-yloxy) propan-2-ol (1), with the previously synthesized pyrrolidine alkaloid, (2R, 3S, 4R, 5R) pyrrolidine-(1-hydroxyethyl)-3,4-diol hydrochloride (4), isolated here from a natural source for the first time. In addition, six known compounds tetillapyrone (2), nortetillapyrone (3), 2-methyl maleimide-5-oxime (5), maleimide-5-oxime (6), 5-(hydroxymethyl) dihydrofuran-2 (3H)-one (7), and ergosta-5,24 (28)-dien-3-ol (8) were also identified. Most of the isolated compounds exhibited weak cytotoxic activity against HepG-2, Daoy, and HeLa cancer cell lines.Conclusion:This is the first report of the occurrence of the indole and pyrrolidine alkaloids, 1-(1H-indol-2-yloxy) propan-2-ol (1), and the - (1-hydroxyethyl)-3,4-diol hydrochloride (4), in the Red Sea Haliclona sp.SUMMARY From the Red Sea Haliclona sp. two alkaloids with indole and pyrrolidine nuclei, 1-(1H-indol-2-yloxy) propan-2-ol-(1) and pyrrolidine-(1-hydroxyethyl)-3,4-diol hydrochloride (4) were isolated and fully characterized; in addition to six known compounds (2, 3, 5-8)The absolute configuration and the three-dimension stereo-molecular structure of compound 4 were determined by X-ray crystallographyThe different extracts and isolated compounds showed weak cytotoxic activity against HepG-2, Daoy, and HeLa cancer cell lines.

THE RATIO OF TrkA AND p75 RECEPTORS ON THE CELL NEUROEPITHELIAL TUMORS – A NEW TARGET EFFECT OF NERVE GROWTH FACTOR WITH CISPLATIN AND TEMOZOLOMIDE AND THE COMBINATIONS

Objective. To study the change of expression of TrkA, p75 receptors and their ratio on the human anaplastic astrocytoma, glioblastoma and medulloblastoma cells cultures exposed to nerve growth factor (NGF) and its combination with cisplatin or temozolomide, to establish participation of expression of the receptors in sensitivity tumor cells to NGF and its combinations with chemotherapeutical drugs. Materials and methods . In primary cell cultures of anaplastic astrocytoma, glioblastoma and medulloblastoma were studied: 1) cytotoxicity effects of nerve growth factor and its combination with cisplatin or temozolomide; 2) the expression of TrkA, p75 receptors and the its ratio by immunofluorescence; 3) correlation between expression of TrkA, p75 receptors, the its ratio and index of cytotoxicity of reagents. Results. It is found that the expression of the receptors and the relationship depends on the tumor type and reagents’ mechanism of action. Expression of TrkA and p75 receptors and their ratio correlated with sensitivity of anaplastic astrocytoma and glioblastoma cells to NGF and its combination with cisplatin or temozolomide. Conclusion. Relative expression of TrkA/p75 receptor, and its coefficient of correlation with index of cytotoxicity point to a mechanism (signaling pathway), on which there is cell death.

Abstract IA27: Functional genomics identifies drivers of medulloblastoma dissemination

Abstract The pediatric brain tumor medulloblastoma shows a characteristic pattern of metastasis, called leptomeningeal dissemination (LMD), in which tumor cells disseminate via the cerebrospinal fluid to the leptomeningeal surfaces of the brain and spinal cord. This pattern distinguishes medulloblastomas from other solid tumors, which metastasize through the bloodstream or lymphatic channels. The invasion-metastasis cascade, a widely accepted paradigm derived from studies of hematogenous spread of carcinomas, is envisioned as a multistep process, governed by three set of genes: initiation, progression, virulence genes. By contrast, our knowledge of the molecular mediators of medulloblastoma LMD is rudimentary. Initial insight into the genetic drivers of LMD came from a transposon insertion mutagenesis study (Wu et al, Nature 482: 529-33, 2012). Mobilizing the Sleeping Beauty transposon in cerebellar neural progenitor cells caused widespread dissemination of typically localized, nonmetastatic medulloblastomas in Patched+/- mice, in which Sonic Hedgehog (Shh) signaling is hyperactive. Candidate LMD genes were identified by sequencing the insertion sites and then mapping these sequences back to the mouse genome. To determine whether genes located at transposon insertion sites directly caused medulloblastomas to disseminate, we overexpressed candidate genes in Nestin+ neural progenitors in the cerebella of mice by retroviral transfer in combination with Shh and examined histological sections of brain and spinal cord during a 4-month observation period. Ectopic expression of the following Sleeping Beauty common insertion site genes shifted the in vivo growth characteristics of Shh-induced medulloblastomas from a localized pattern to a disseminated pattern: Eras (embryonic stem cell-expressed Ras), Lhx1 (LIM homeobox protein), Ccrk (cell cycle-related kinase), Akt (PI3K signaling protein), Arnt (hypoxia-inducible factor-1β), Gdi2 (Rab-GDP dissociation inhibitor), and Smad5 (TGF-β signal transducer). To elucidate the physiological traits conferred by these genes, we overexpressed them in a human medulloblastoma cell line (DAOY) and an immortalized, nontransformed cell line derived from mouse granule neuron precursors (SHH-NPD) and then quantified migration, invasiveness, and anchorage independence, growth characteristics that are closely associated with metastatic competence in carcinomas. Although the effects were quantitatively different in DAOY compared with SHH-NPD, all genes that induced LMD in vivo conferred these traits to cells in culture. We have used the set of genes discovered through Sleeping Beauty mutagenesis to begin constructing an LMD cascade, analogous to the invasion-metastasis cascade. Some LMD driver genes (Eras and Akt) enhanced the growth of the primary tumor, as indicated by shortened animal survival, while others did not. Therefore, we propose a two-category scheme for the LMD cascade, in which genes that increase primary tumor growth (Eras and Akt) are analogous to initiation/progression genes, whereas genes that only promote the growth and survival of disseminating cells (Lhx1, Ccrk, Arnt, Gdi2, and Smad5) are analogous to virulence genes. This simplified scheme will serve as a framework, upon which future research can build more sophisticated versions that better reflect the complexity of LMD. Citation Format: Daniel W. Fults. Functional genomics identifies drivers of medulloblastoma dissemination. [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 IA27.

PTPS-08HEMATOGENOUS DISSEMINATION OF MEDULLOBLASTOMA METASTASES TO THE LEPTOMENINGES

Metastatic medulloblastoma is a high-risk disease, because the majority of children dying of medulloblastoma expire from metastases rather than recurrence of the primary tumor. Current dogma dictates that medulloblastoma cells are shed from the primary tumor into the CSF, float through the spinal fluid and establish metastatic colonies on the leptomeningeal surface and spinal cord. Empirical evidence for this mechanism is lacking in the literature. Whole genome sequencing on matched trios of human primary tumor, metastases, and peripheral blood showed somatic mutations from the medulloblastoma at low clonal frequency in the blood samples — consistent with circulating tumor cells. In addition, we identified circulating tumor cells (CTC) in the blood of transgenic mice bearing metastatic medulloblastoma, and in mice intra-cranially implanted with patient derived MB. Surprisingly, when we implanted human or mouse medulloblastoma cells in the flank of immunocompromised mice, the MB cells colonized the leptomeningeal space causing death. As these MB xenografts are not in continuity with the CSF, they must have arisen through hematogenous dissemination. Importantly, flow cytometry shows that MB CTCs express CD34, the cell-surface marker used clinically in children to collect peripheral blood stem cells; suggesting that autologous bone marrow transplants from children with medulloblastoma are likely contaminated with CTCs. Finally we established a parabiosis model where two immunocompromised, sibling mice are surgically joined, and a common blood circulation is established. After parabiosis, one mouse is intra-cranially implanted with MB, and the mice are subsequently separated when the xenografted twin becomes symptomatic. The surviving twin goes on to die from leptomeningeal metastases. Our data challenge the existing paradigm of CSF mediated dissemination of medulloblastoma by demonstrating the capacity of medulloblastoma to spread through the blood system. Identification of a systemic route for MB metastases offers new avenues for the diagnosis and therapy of metastatic medulloblastoma.

PO11IN VITRO HUMAN, SPHEROID, CO-CULTURE MODEL FOR PRECLINICAL ASSESSMENT OF DIRECT APPLICATION THERAPEUTICS TO PAEDIATRIC BRAIN TUMOURS

INTRODUCTION: Spheroid co-cultures of brain tumours and human foetal brain tissue hold promise as physiologically-relevant in vitro models with high predictive potential. Apart from modelling tumour-host interactions, the human foetal tissue serves as a control comparing cytotoxicity and neurotoxicity. Previous co-culture models have suffered from spheroid-size variability, required manual sorting and offered limited quantitation capabilities. We describe a high-throughput platform yielding reproducible co-cultures amenable to automation, quantification and screening. METHOD: The human medulloblastoma cell line UW228-3 and foetal brain tissue were marked with two cytoplasmic, fluorescent dyes (CDCFDASE, Celltrace Violet) and seeded simultaneously in 96-well ultra-low-attachment plates. Tumour and normal cells formed reproducible (d = 600 µm, CV% = 10%), single co-culture spheroids in each well. After three days, the spheroids were exposed to the cytotoxic drug etoposide (0.3-100 µM) for 48h, followed by a 48h drug-free phase. The viability of each cell type was quantified using flow cytometry and multiphoton microscopy. RESULTS: Both dyes were retained in the mixed cultures facilitating normal and tumour tissue recognition and dose-response curve computation. Despite seeding as a mix, tumour and normal cells organised in two opposing poles in the co-cultures, with a main tumour mass and some tumour cells invading the normal tissue. Etoposide levels of 10 µM were found to maximise toxicity to tumours (6.5%viability), while normal tissue viability was 40%. CONCLUSION: The reproducible platform and universal compatibility of the marking procedure make this model transferable to other tumour types and primary tissues.

The p53 tumor suppressor protein protects against chemotherapeutic stress and apoptosis in human medulloblastoma cells.

Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB.

Ecto-5'-Nucleotidase Overexpression Reduces Tumor Growth in a Xenograph Medulloblastoma Model.

BackgroundEcto-5’-nucleotidase/CD73 (ecto-5’-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5’-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children.Materials and MethodsThe effects of ecto-5’-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified.ResultsThe human MB cell line D283, transfected with ecto-5’-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A1 adenosine receptor expression rates were detected in MB cells overexpressing ecto-5’-NT.ConclusionThis work suggests that ecto-5’-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A1 adenosine receptors. Therefore, ecto-5’-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy.

Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

Na,K-ATPase β1-subunit is a target of sonic hedgehog signaling and enhances medulloblastoma tumorigenicity.

BackgroundThe Sonic hedgehog (Shh) signaling pathway plays an important role in cerebellar development, and mutations leading to hyperactive Shh signaling have been associated with certain forms of medulloblastoma, a common form of pediatric brain cancer. While the fundamentals of this pathway are known, the molecular targets contributing to Shh-mediated proliferation and transformation are still poorly understood. Na,K-ATPase is a ubiquitous enzyme that maintains intracellular ion homeostasis and functions as a signaling scaffold and a cell adhesion molecule. Changes in Na,K-ATPase function and subunit expression have been reported in several cancers and loss of the β1-subunit has been associated with a poorly differentiated phenotype in carcinoma but its role in medulloblastoma progression is not known.MethodsHuman medulloblastoma cell lines and primary cultures of cerebellar granule cell precursors (CGP) were used to determine whether Shh regulates Na,K-ATPase expression. Smo/Smo medulloblastoma were used to assess the Na,K-ATPase levels in vivo. Na,K-ATPase β1-subunit was knocked down in DAOY cells to test its role in medulloblastoma cell proliferation and tumorigenicity.ResultsNa,K-ATPase β1-subunit levels increased with differentiation in normal CGP cells. Activation of Shh signaling resulted in reduced β1-subunit mRNA and protein levels and was mimicked by overexpression of Gli1and Bmi1, both members of the Shh signaling cascade; overexpression of Bmi1 reduced β1-subunit promoter activity. In human medulloblastoma cells, low β1-subunit levels were associated with increased cell proliferation and in vivo tumorigenesis.ConclusionsNa,K-ATPase β1-subunit is a target of the Shh signaling pathway and loss of β1-subunit expression may contribute to tumor development and progression not only in carcinoma but also in medulloblastoma, a tumor of neuronal origin.

MiR-367 promotes proliferation and stem-like traits in medulloblastoma cells.

In medulloblastoma, abnormal expression of pluripotency factors such as LIN28 and OCT4 has been correlated with poor patient survival. The miR-302/367 cluster has also been shown to control self-renewal and pluripotency in human embryonic stem cells and induced pluripotent stem cells, but there is limited, mostly correlational, information about these pluripotency-related miRNA in cancer. We evaluated whether aberrant expression of such miRNA could affect tumor cell behavior and stem-like traits, thereby contributing to the aggressiveness of medulloblastoma cells. Basal expression of primary and mature forms of miR-367 were detected in four human medulloblastoma cell lines and expression of the latter was found to be upregulated upon enforced expression of OCT4A. Transient overexpression of miR-367 significantly enhanced tumor features typically correlated with poor prognosis; namely, cell proliferation, 3-D tumor spheroid cell invasion and the ability to generate neurosphere-like structures enriched in CD133 expressing cells. A concurrent downregulation of the miR-367 cancer-related targets RYR3, ITGAV and RAB23, was also detected in miR-367-overexpressing cells. Overall, these findings support the pro-oncogenic activity of miR-367 in medulloblastoma and reveal a possible mechanism contributing to tumor aggressiveness, which could be further explored to improve patient stratification and treatment of this important type of pediatric brain cancer.

Characterization of cancer stem cells and primary cilia in medulloblastoma.

Medulloblastoma, a tumor of the cerebellum, is the most common pediatric central nervous system malignancy. These tumors are etiologically linked to mutations in the Sonic hedgehog (Shh) pathway, which signals through the primary, non-motile cilium. The growth of these aggressive tumors relies on self-renewal of tumor-propagating cells known as cancer stem cells (CSCs). Previous reports have implicated CD133-expressing cells as CSCs in brain tumors, while those expressing CD15 have been shown to propagate medulloblastoma. Here, we demonstrate that CD133+ and CD15+ cells are distinct medulloblastoma populations. CD15+ cells comprise approximately 0.5-1% of total human medulloblastoma cells, display CSC properties in culture and are detected in the Smoothened A1 transgenic mouse model of medulloblastoma. Additionally, we report on a medulloblastoma patient with enriched CD15+ cells in recurrent vs primary medulloblastoma. We also demonstrate that human medulloblastoma cells critically rely on establishment of primary cilia to drive Shh-mediated cell division. Primary cilia are found in external granule cells of human fetal cerebellum and in 12/14 medulloblastoma samples. Yet, CD15+ medulloblastoma cells lack primary cilia, suggesting that this CSC population signals independently of Shh. These results are important when considering the effects of current and prospective treatment modalities on medulloblastoma CSC populations.

PM-08 * A HUMANIZED CO-TRANSPLANT XENOGRAFT MOUSE MODEL, TO STUDY TOXICITY OF DRUGS TARGETED AGAINST PEDIATRIC BRAIN TUMORS

BACKGROUND: Side effects of current pediatric brain tumor therapeutics, usually resulting from neural cell toxicity, call for testing of novel therapeutic modalities on appropriate preclinical models, which recapitulates normal human neural environment with malignant brain tumors. Here, we present a new model to study toxicity of humanized antibodies against xenografted human normal neural cells sequentially co-transplanted with primary medulloblastoma (MB) cells. METHODS: Human neural progenitor cells (HuNPCs) derived from human fetal neural tissue, were infected with td-Tomato-luciferase expressing virus and injected into newborn pups (p1-p3 days old). HuNPC engraftment was confirmed with bioluminescence imaging and previously characterized for multi-potent differentiation into neurons, astrocytes and oligodendrocytes. HuNPC engrafted mice were then injected with unlabeled human MB cells at 1.5 months of age. MB cells used were previously confirmed to have 100% penetrance in multiple cohorts. After 2 weeks of tumor cell transplantation, mice were randomized and treated with either humanized anti-CD47 monoclonal antibody (HuCD47 mAb) or control. Human neural cells viability and proliferation in all mice were measured through subsequent bioluminescence imaging. RESULTS: Follow-up bioluminescence imaging revealed minimal change in flux values of treated mice with HuCD47 mAb, representing viability of engrafted normal neural cell, compared to control. Moreover, survival rates as well as subsequent H & E staining show HuCD47 mAb dramatically decreased tumor size and subsequently increased the survival of mice treated with HuCD47 mAb compared to control similar to experiments carried out in mice transplanted with human MB cells only. CONCLUSION: The presented in vivo model provides a new platform to test any novel potential therapeutic (here, HuCD47 mAb) in the context of xenografted human normal neural environment. This way we not only measure effect of the proposed drug on tumor regression but impact of developed agent on human normal neural tissue in the vicinity of the tumor.

MB-14 * DISCOVERING THE TREATMENT REFRACTORY BTIC POPULATION IN GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant pediatric brain tumour, and is categorized into four molecular subgroups: WNT, SHH, Group 3 and Group 4 of which Group 3 tumours are associated with increased chances of metastasis and poor patient outcome. Using a cell-surface marker, CD133, a subpopulation of cells with stem cell properties, termed brain tumour initiating cells (BTICs) was identified, and was further shown to drive medulloblastoma tumorigenesis in vitro and in vivo. Previous in silico analyses revealed enriched expression of many stem cell self-renewal regulatory genes in Group 3 MBs. In this work, we aim to identify and characterize the treatment-refractory BTIC population in Group 3 MBs by developing a mouse- adapted therapy treatment model that mimics the clinical treatment of MB. A mouse-adapted MB therapy model was developed using our human-mouse BTIC xenograft, in which MB cells tagged with GFP were intracranially transplanted into the frontal lobes of NOD-SCID mice. After tumour engraftment, mice assigned to receive treatment of craniospinal radiation, followed by intraperitoneal injections of chemotherapeutic drugs Vincristine, Cisplatin and Cyclophosphamide. Following enrichment of tumour cells from cultured brains, in vitro stem cell assays for self-renewal, flow characterization, and candidate gene expression profiling by NanoString were performed. Spinal cords were also harvested from mice in order to elucidate spinal dissemination of human MB cells. We found that the cells cultured from xenografts of treatment group showed an increase in self-renewal and despite evident tumour regression post-therapy, treated tumours showed an increase in expression of CD133, Sox2 and Bmi1 compared to untreated tumours. Profiling genomic changes in “treatment-responsive” tumors against those that fail therapy will generate a differential profile of the refractory BTIC, which may guide future therapeutic approaches targeting this cell, and will serve as a model for targeting such CSCs in other TIC-driven solid tumours.

Serpine2/PN-1 Is Required for Proliferative Expansion of Pre-Neoplastic Lesions and Malignant Progression to Medulloblastoma

BackgroundMedulloblastomas are malignant childhood brain tumors that arise due to the aberrant activity of developmental pathways during postnatal cerebellar development and in adult humans. Transcriptome analysis has identified four major medulloblastoma subgroups. One of them, the Sonic hedgehog (SHH) subgroup, is caused by aberrant Hedgehog signal transduction due to mutations in the Patched1 (PTCH1) receptor or downstream effectors. Mice carrying a Patched-1 null allele (Ptch1 ∆/+) are a good model to study the alterations underlying medulloblastoma development as a consequence of aberrant Hedgehog pathway activity.ResultsTranscriptome analysis of human medulloblastomas shows that SERPINE2, also called Protease Nexin-1 (PN-1) is overexpressed in most medulloblastomas, in particular in the SHH and WNT subgroups. As siRNA-mediated lowering of SERPINE2/PN-1 in human medulloblastoma DAOY cells reduces cell proliferation, we analyzed its potential involvement in medulloblastoma development using the Ptch1 ∆/+ mouse model. In Ptch1 ∆/+ mice, medulloblastomas arise as a consequence of aberrant Hedgehog pathway activity. Genetic reduction of Serpine2/Pn-1 interferes with medulloblastoma development in Ptch1 ∆/+ mice, as ~60% of the pre-neoplastic lesions (PNLs) fail to develop into medulloblastomas and remain as small cerebellar nodules. In particular the transcription factor Atoh1, whose expression is essential for development of SHH subgroup medulloblastomas is lost. Comparative molecular analysis reveals the distinct nature of the PNLs in young Ptch1 ∆/+ Pn-1 Δ/+ mice. The remaining wild-type Ptch1 allele escapes transcriptional silencing in most cases and the aberrant Hedgehog pathway activity is normalized. Furthermore, cell proliferation and the expression of the cell-cycle regulators Mycn and Cdk6 are significantly reduced in PNLs of Ptch1 ∆/+ Pn-1 Δ/+ mice.ConclusionsOur analysis provides genetic evidence that aberrant Serpine2/Pn-1 is required for proliferation of human and mouse medulloblastoma cells. In summary, our analysis shows that Serpine2/PN-1 boosts malignant progression of PNLs to medulloblastomas, in which the Hedgehog pathway is activated in a SHH ligand-independent manner.