Medulloblastoma is the most common malignant paediatric brain tumour. Similar to other tumours, medulloblastoma pathogenesis involves abnormal regulation of several developmental growth pathways. As my thesis project, I studied the effects of aberrant HGF/MET signalling on medulloblastoma formation in two ways. In my first objective, I investigated the role that mutations play in activated HGF/MET signalling in medulloblastoma by searching for mutations in HGF/MET pathway genes, SPINT1, SPINT2, and MET, within primary medulloblastoma specimens. This screen identified several single nucleotide polymorphisms (SNPs) and two novel variations, one in each SPINT1 and SPINT2 genes. In my second objective, I generated a transgenic mouse model with cerebellar-specific aberrant MET signalling. These mice developed extensive cerebellar abnormalities but formed no tumours. These results indicate that mutations in the HGF/MET pathway components alone are not sufficient to initiate medulloblastoma formation and must coincide with additional genetic insults to promote tumour formation, maintenance, and progression.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor. Little is known about aggressive forms of this disease. In order to identify pathways mediating aggressiveness in MB, we performed microarray experiments. Primary human MBs were compared to their patient matched recurrent or metastatic counterparts. Murine tumors from two MB mouse models that present with differing clinical severities were also evaluated. We identified the Transforming Growth Factor-beta (TGF-β) as a potential contributor to MB pathogenesis in both species. Smad3, a major downstream component of the TGF-β pathway, was shown to correlate with MB metastasis and survival in human tissue. Similarly, Smad3 expression during development identified a subset of cerebellar neuronal precursors as putative cells of origin for the Smad3 positive MBs. To our knowledge, this is the first study that links TGF-β to MB pathogenesis. Our research suggests that canonical activation of this pathway leads to better prognosis for patients.

Medulloblastoma cells do not contain detectable amounts of the bZIP protein Zhangfei. However, previous work has shown that expression of this protein in cells of the ONS-76 line, derived from a human medulloblastoma, causes the cells to stop growing and develop processes that resemble neuritis (a characteristic of differentiated neurons). Zhangfei-expressing cells eventually die. My objective was to determine the molecular mechanisms by which Zhangfei influences ONS-76 cells. My strategy was to infect ONS-76 cells with adenovirus vectors expressing either Zhangfei or the control E. coli protein â-galactosidase (LacZ) and then to compare the following parameters in Zhangfei and LacZ-expressing cells: a) markers of apoptosis, autophagy and macropinocytosis (the three main pathways of cell death); b) transcripts for genes involved in neurogenesis and apoptosis; c) phosphorylation of peptide targets of selected cellular protein kinases; and d) active transcription factors. Zhangfei-expressing cells appeared to succumb to apoptosis as determined by the expression of phosphatidylserine on the cell surface and intensity of nuclear staining with the DNA dye Hoechst. Increased staining for autophagic vesicles and upregulated expression of autophagy response genes in these cells indicated that they were undergoing autophagy, possibly associated with apoptosis. My analysis of steady-state transcripts for genes involved in apoptosis and neurogenesis and functional protein kinases in Zhangfei-expressing cells indicated that the mitogen-activated protein kinase (MAPK) pathway was active in these cells. In addition, I found that the transcription factor Brn3a as well as factors implicated in differentiation were also active. These observations led me to hypothesize that Zhangfei enhances the expression of Brn3a, a known inducer of TrkA, the high-affinity receptor for nerve growth factor (NGF). TrkA then binds in an autocrine manner to NGF, triggering the MAPK pathway and leading to differentiation of ONS-76 cells into neuron and glia-like cells, eventually bringing about cell death by apoptosis and autophagy. I tested this hypothesis by showing that Zhangfei could enhance transcription from the isolated Brn3a promoter, that ONS-76 cells produce NGF as detected in a bioassay, and that antibodies against NGF and inhibitors of TrkA and selected components of the MAPK pathway could partially restore the growth of Zhangfei-expressing ONS-76 cells. My work supports previous work highlighting the importance of NGF-TrkA signaling in the outcome of medulloblastomas and shows how Zhangfei is able to trigger this pathway.

<p>Studying the early stages of cancer can provide important insight into the molecular basis of the disease. In many human cancers, such as prostate, pancreatic, and colon cancer, a pre-neoplastic, or intermediate, stage of the disease has been identified. The pre-neoplastic stage is presumed to be a transition during which normal cells undergo malignant transformation. However, the link between the pre-neoplastic cells and end-stage disease has never been formally established. To investigate the fate of such cells, the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma was used. Pre-neoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Genetic fate mapping and orthotopic transplantation provided definitive evidence that PNCs give rise to tumors and showed that the predominant fate of PNCs that do not form tumors is differentiation. Moreover, N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer.</p> / Dissertation

<p>Cytosolic cytochrome c promotes apoptosis by triggering caspase activation. In healthy cells cytochrome c localizes to mitochondria, where it participates in the electron transport chain. Apoptotic stimuli induce permeabilization of the outer mitochondrial membrane and release of cytochrome c. Once cytosolic, cytochrome c binds Apaf-1, inducing the formation of a protein complex that recruits and activates caspases, which serve to dismantle the dying cell. Although the steps of this signaling pathway have been described, many of the regulatory mechanisms influencing the cellular response to cytosolic cytochrome c remain unclear. Using apoptosis assays and microinjection techniques, we investigated the response of several cell-types to cytosolic cytochrome c.</p> <p>First, we demonstrate that cytosolic cytochrome c kills brain tumor cells but not normal brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in brain tumors compared with negligible Apaf-1 in brain tissue. These differences in Apaf-1 abundance correlate with differences in E2F1, a previously identified activator of Apaf-1 transcription. Chromatin immunoprecipitation assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to Apaf-1 expression in brain tumors. These results demonstrate an unexpected sensitivity of brain tumors to cytochrome c and raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancers.</p> <p>Secondly, we develop a method for monitoring caspase activity in Xenopus laevis oocytes and early embryos. The approach, utilizing microinjection of a near-infrared dye that emits fluorescence only after its cleavage by active caspases, has enabled the elucidation of subtleties in the apoptotic program. We demonstrate that brief caspase activation is sufficient to cause death. We illustrate the presence of a cytochrome c dose threshold, which is lowered by neutralization of inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that eventual death of these oocytes is caspase-independent. Imaging caspase activity in the embryo suggests that apoptosis in early development is not cell-autonomous. Finally, we believe this method presents a useful screening modality for identifying novel apoptotic regulators as well as pro-apoptotic small-molecules that could be useful in treating brain tumors.</p> / Dissertation

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

Medulloblastoma is the most common malignant pediatric brain tumour. Although survival rates have improved in recent years, long-term survivors exhibit a significantly diminished quality of life complicated by neurological, endocrine, intellectual, and social sequelae as a result of conventional therapies. In order to improve the current outlook for patients with medulloblastoma, rational, targeted therapies that are more efficient and less toxic are required. Despite insight gained from the study of hereditary tumour syndromes and candidate gene approaches, the molecular basis of medulloblastoma remains poorly defined, with more than half of all cases remaining unaccounted for at the genetic level. The intent of my PhD research program was to use high-resolution genomics in an attempt to gain an improved understanding of the medulloblastoma genome and potentially uncover novel genes and pathways driving its pathogenesis. By applying a combination of single nucleotide polymorphism (SNP) arrays, exon arrays, and microRNA arrays to a large cohort of primary medulloblastoma samples, we have identified novel oncogenes and tumour suppressors, implicated deregulation of the histone code as an important event in the pathogenesis of medulloblastoma, and refined the definition of medulloblastoma subgroups. This thesis demonstrates the extent of heterogeneity that exists in the medulloblastoma genome, showing that relatively few genomic aberrations are common when studying medulloblastoma as a single disease. In spite of this heterogeneity, we have identified novel candidate genes and processes that may serve as potential targets for future therapies. Importantly, we have established an improved method of classifying medulloblastomas into distinct molecular variants, showing that certain genomic changes are enriched and occasionally restricted to a specific subgroup. Finally, in addition to genomic differences, we have confirmed that medulloblastoma subgroups differ in their demographics and clinical behavior, and propose that medulloblastoma subgroup affiliation should become an integral component of patient stratification in the future.

Medulloblastoma is the most common malignant pediatric brain tumour. Although survival rates have improved in recent years, long-term survivors exhibit a significantly diminished quality of life complicated by neurological, endocrine, intellectual, and social sequelae as a result of conventional therapies. In order to improve the current outlook for patients with medulloblastoma, rational, targeted therapies that are more efficient and less toxic are required. Despite insight gained from the study of hereditary tumour syndromes and candidate gene approaches, the molecular basis of medulloblastoma remains poorly defined, with more than half of all cases remaining unaccounted for at the genetic level. The intent of my PhD research program was to use high-resolution genomics in an attempt to gain an improved understanding of the medulloblastoma genome and potentially uncover novel genes and pathways driving its pathogenesis. By applying a combination of single nucleotide polymorphism (SNP) arrays, exon arrays, and microRNA arrays to a large cohort of primary medulloblastoma samples, we have identified novel oncogenes and tumour suppressors, implicated deregulation of the histone code as an important event in the pathogenesis of medulloblastoma, and refined the definition of medulloblastoma subgroups. This thesis demonstrates the extent of heterogeneity that exists in the medulloblastoma genome, showing that relatively few genomic aberrations are common when studying medulloblastoma as a single disease. In spite of this heterogeneity, we have identified novel candidate genes and processes that may serve as potential targets for future therapies. Importantly, we have established an improved method of classifying medulloblastomas into distinct molecular variants, showing that certain genomic changes are enriched and occasionally restricted to a specific subgroup. Finally, in addition to genomic differences, we have confirmed that medulloblastoma subgroups differ in their demographics and clinical behavior, and propose that medulloblastoma subgroup affiliation should become an integral component of patient stratification in the future.

<p>Medulloblastoma is the most common malignant brain tumor in children. The understanding of the genetic alterations in this tumor is emergent, and many such genetic driver events have yet to be functionally-characterized. Our studies have sought to understand the causes and consequences of OTX2 dysregulation in established medulloblastomas and in its putative cellular origins. Using a tumor genetic approach, we have uncovered frequent OTX2 copy number gains driving expression of this oncogene in a subset of medulloblastomas. However, OTX2 is frequently expressed in medulloblastomas independent of genomic copy number gain, and we thus sought to understand the transcriptional regulation of this gene in these tumors. We have found that chromatin accessibility, promoter DNA methylation, and activity of a distal downstream enhancer is distinct between OTX2-expressing and -nonexpressing medulloblastomas. Notably, autoregulation serves to maintain OTX2 expression in some medulloblastomas, whereas DNA methylation actively suppresses OTX2 in tumors not expressing this gene. Finally, we describe the effect of expressing Otx2 (the mouse homolog of OTX2) aberrantly in the developing mouse hindbrain, revealing that Otx2 disrupts spatiotemporal restrictions of neuronal progenitor cell proliferation. The effect of Otx2 in vivo is transient, with ectopically-proliferating cells give way to differentiated neurons. We found that OTX2 expression was not able to give rise to high penetrance medulloblastoma when combined with P53 deletion or double heterozygosity for P53 and PTEN. Thus, although Otx2 alters migration and proliferation dynamics of hindbrain neuronal progenitor cells, further studies are needed to identify the genetic alterations that cooperate with this oncogene to give rise to medulloblastoma.</p> / Dissertation