Novel MYC-driven medulloblastoma models from multiple embryonic cerebellar cells.

Group3 medulloblastoma (MBG3) that predominantly occur in young children are usually associated with MYC amplification and/or overexpression, frequent metastasis and a dismal prognosis. Physiologically relevant MBG3 models are currently lacking, making inferences related to their cellular origin thus far limited. Using in utero electroporation, we here report that MBG3 mouse models can be developed in situ from different multipotent embryonic cerebellar progenitor cells via conditional expression of Myc and loss of Trp53 function in several Cre driver mouse lines. The Blbp-Cre driver that targets embryonic neural progenitors induced tumors exhibiting a large-cell/anaplastic histopathology adjacent to the fourth ventricle, recapitulating human MBG3. Enforced co-expression of luciferase together with Myc and a dominant-negative form of Trp53 revealed that GABAergic neuronal progenitors as well as cerebellar granule cells give rise to MBG3 with their distinct growth kinetics. Cross-species gene expression analysis revealed that these novel MBG3 models shared molecular characteristics with human MBG3, irrespective of their cellular origin. We here developed MBG3 mouse models in their physiological environment and we show that oncogenic insults drive this MB subgroup in different cerebellar lineages rather than in a specific cell of origin.

The WIP1 oncogene promotes progression and invasion of aggressive medulloblastoma variants.

Recent studies suggest that medulloblastoma, the most common malignant brain tumor of childhood, is comprised of four disease variants. The WIP1 oncogene is overexpressed in Group 3 and 4 tumors, which contain medulloblastomas with the most aggressive clinical behavior. Our data demonstrate increased WIP1 expression in metastatic medulloblastomas, and inferior progression-free and overall survival of patients with WIP1 high-expressing medulloblastoma. Microarray analysis identified upregulation of genes involved in tumor metastasis, including the G protein-coupled receptor CXCR4, in medulloblastoma cells with high WIP1 expression. Stimulation with the CXCR4 ligand SDF1α activated PI-3 kinase signaling, and promoted growth and invasion of WIP1 high-expressing medulloblastoma cells in a p53-dependent manner. When xenografted into the cerebellum of immunodeficient mice, medulloblastoma cells with stable or endogenous high WIP1 expression exhibited strong expression of CXCR4 and activated AKT in primary and invasive tumor cells. WIP1 or CXCR4 knockdown inhibited medulloblastoma growth and invasion. WIP1 knockdown also improved the survival of mice xenografted with WIP1 high-expressing medulloblastoma cells. WIP1 knockdown inhibited cell surface localization of CXCR4 by suppressing expression of the G protein receptor kinase 5, GRK5. Restoration of wild-type GRK5 promoted Ser339 phosphorylation of CXCR4 and inhibited the growth of WIP1-stable medulloblastoma cells. Conversely, GRK5 knockdown inhibited Ser339 phosphorylation of CXCR4, increased cell surface localization of CXCR4 and promoted the growth of medulloblastoma cells with low WIP1 expression. These results demonstrate crosstalk among WIP1, CXCR4 and GRK5, which may be important for the aggressive phenotype of a subclass of medulloblastomas in children.

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Transitioning from genotypes to epigenotypes: why the time has come for medulloblastoma epigenomics.

Recent advances in genomic technologies have allowed for tremendous progress in our understanding of the biology underlying medulloblastoma, a malignant childhood brain tumor. Consensus molecular subgroups have been put forth by the pediatric neuro-oncology community and next-generation genomic studies have led to an improved description of driver genes and pathways somatically altered in these subgroups. In contrast to the impressive pace at which advances have been made at the level of the medulloblastoma genome, comparable studies of the epigenome have lagged behind. Complementary data yielded from genomic sequencing and copy number profiling have verified frequent targeting of chromatin modifiers in medulloblastoma, highly suggestive of prominent epigenetic deregulation in the disease. Past studies of DNA methylation-dependent gene silencing and microRNA expression analyses further support the concept of medulloblastoma as an epigenetic disease. In this Review, we aim to summarize the key findings of past reports pertaining to medulloblastoma epigenetics as well as recent and ongoing genomic efforts linking somatic alterations of the genome with inferred deregulation of the epigenome. In addition, we predict what is on the horizon for medulloblastoma epigenetics and how aberrant changes in the medulloblastoma epigenome might serve as an attractive target for future therapies.

Oncogenic YAP promotes radioresistance and genomic instability in medulloblastoma through IGF2-mediated Akt activation.

Radiation therapy remains the standard of care for many cancers, including the malignant pediatric brain tumor medulloblastoma. Radiation leads to long-term side effects, whereas radioresistance contributes to tumor recurrence. Radio-resistant medulloblastoma cells occupy the perivascular niche. They express Yes-associated protein (YAP), a Sonic hedgehog (Shh) target markedly elevated in Shh-driven medulloblastomas. Here we report that YAP accelerates tumor growth and confers radioresistance, promoting ongoing proliferation after radiation. YAP activity enables cells to enter mitosis with un-repaired DNA through driving insulin-like growth factor 2 (IGF2) expression and Akt activation, resulting in ATM/Chk2 inactivation and abrogation of cell cycle checkpoints. Our results establish a central role for YAP in counteracting radiation-based therapies and driving genomic instability, and indicate the YAP/IGF2/Akt axis as a therapeutic target in medulloblastoma.

Sonic hedgehog regulates Bmi1 in human medulloblastoma brain tumor-initiating cells.

Bmi1 is a key stem cell regulatory gene implicated in the pathogenesis of many aggressive cancers, including medulloblastoma. Overexpression of Bmi1 promotes cell proliferation and is required for hedgehog (Hh) pathway-driven tumorigenesis. This study aimed to determine if Sonic hedgehog (Shh) modulates the key stem cell regulatory gene Bmi1 in childhood medulloblastoma brain tumor-initiating cells (BTICs). Although current literature suggests that there is a correlation between Shh pathway genes and Bmi1 expression, it is unclear whether there is indeed a direct regulatory mechanism. To address whether Shh induces expression of Bmi1, stem cell-enriched populations from medulloblastoma cell lines and primary samples were treated with Shh ligand and KAAD-cyclopamine (Shh antagonist). Our data indicate that Bmi1 expression positively correlates with increasing Shh ligand concentrations. Chromatin immunoprecipitation reveals that Gli1 preferentially binds to the Bmi1 promoter, and Bmi1 transcript levels are increased and decreased by Gli1 overexpression and downregulation, respectively. Knockdown experiments of Bmi1 in vitro and in vivo demonstrate that Hh signaling not only drives Bmi1 expression, but a feedback mechanism exists wherein downstream effectors of Bmi1 may, in turn, activate Hh pathway genes. These findings implicate Bmi1 and Hh as mutually indispensable pathways in medulloblastoma BTIC maintenance. Recent molecular characterization of medulloblastoma also reveals that Bmi1 is overexpressed across all subgroups of medulloblastoma, particularly in the most aggressive subtypes. Lastly, despite recent identification of BTIC markers, the molecular characterization of these cell populations remains unclear. In this work, we propose that the BTIC marker CD133 may segregate a cell population with a Hh-receptor phenotype, thus demonstrating a cell-cell interaction between the CD133+ Hh receptor cells and the CD133- Hh-secreting cells.

The SFRP family of WNT inhibitors function as novel tumor suppressor genes epigenetically silenced in medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Dysregulation of WNT signaling occurs in up to 20% of cases. Using a genome-wide approach, we identified the secreted frizzled-related protein 1, 2 and 3 (SFRP1, SFRP2 and SFRP3) family of WNT inhibitors as putative tumor suppressor genes silenced by promoter region methylation in MB. SFRP1, SFRP2 and SFRP3 expression increased after 5-aza-2'-deoxycytidine treatment. SFRP1, SFRP2 and SFRP3 methylation was identified in 23.5, 3.9 and 15.7% of primary MB specimens, respectively, by methylation-specific PCR. Stable SFRP1, SFRP2 and SFRP3 expression reduced phospho-DVL2 levels and hindered MB cell proliferation and colony formation in soft agar in vitro. In 60% of primary tumors, SFRP1 was expressed at levels twofold lower than that in normal cerebellum. SFRP1 expression impaired tumor formation in vivo in flank and orthotopic intracerebellar xenograft models and conferred a significant survival advantage (P<0.0001). We identify for the first time tumor suppressor gene function of SFRP genes in MB, and suggest that loss of WNT pathway inhibition due to SFRP gene silencing is an additional mechanism that may contribute to excessive WNT signaling in this disease.

Mechanism of cycling of migrating myoelectric complexes: effect of morphine.

Morphine was injected intravenously at various phases of the migrating myoelectric complex (MMC) cycle to study the oscillatory characteristics of MMCs by the premature initiation of phase IIIs. All injection timings were represented as a percentage of the normal MMC period at the most proximal duodenal electrode. During the initial 20% of the MMC cycle, the mechanism of initiation of MMCs was in an absolutely refractory state in the sense that a supramaximal dose of morphine (200-300 micrograms/kg) did not initiate a premature phase III. During the remainder of the MMC cycle, the control mechanism was in a relatively refractory state. As this state progressed, premature phase III activity was initiated with diminishing doses of morphine. This was called the relatively refractory state. The initiation of a premature phase III by morphine did not affect the phase III already in progress, except that its propagation velocity was increased. Truncal vagotomy did not affect the refractory characteristics of MMCs or the action of morphine. Only large doses of naloxone (2 mg/kg) blocked the above action of morphine. The study shows that the MMC cyclic phenomenon has the characteristics of relaxation oscillators that may result from enteric neural biological clocks. The period of these oscillators can be altered by stimulants such as morphine.

Pharmacists' tasks, skills and attributes. Their professional importance and desirable teaching environment in the Australian state of Tasmania.

The professional importance of tasks, skills and attributes of pharmacists in an Australian state have been evaluated by various groups of pharmacists and allied health professionals. The most desirable teaching environment (academic or workplace) for the teaching of these tasks and skills has also been determined. Essential tasks, skills and attributes include those on drug knowledge, prescription handling and bench skills, verbal skills and professional responsibility. Most pharmacists' tasks and skills should be taught in both the academic environment and the workplace.