Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.

ABSTRACT

Toward the goal of generating a mouse medulloblastoma model with increased tumor incidence, we developed a homozygous version of our ND2:SmoA1 model. Medulloblastomas form in 94% of homozygous Smo/Smo mice by 2 months of age. Tumor formation is, thus, predictable by age, before the symptomatic appearance of larger lesions. This high incidence and early onset of tumors is ideal for preclinical studies because mice can be enrolled before symptom onset and with a greater latency period before late-stage disease. Smo/Smo tumors also display leptomeningeal dissemination of neoplastic cells to the brain and spine, which occurs in many human cases. Despite an extended proliferation of granule neuron precursors (GNP) in the postnatal external granular layer (EGL), the internal granular layer formed normally in Smo/Smo mice and tumor formation occurred only in localized foci on the superficial surface of the molecular layer. Thus, tumor formation is not simply the result of over proliferation of GNPs within the EGL. Moreover, Smo/Smo medulloblastomas were transplantable and serially passaged in vivo, demonstrating the aggressiveness of tumor cells and their transformation beyond a hyperplastic state. The Smo/Smo model is the first mouse medulloblastoma model to show leptomeningeal spread. The adherence to human pathology, high incidence, and early onset of tumors thus make Smo/Smo mice an efficient model for preclinical studies.

NeuroD2 is sufficient to induce cell cycle arrest and neurogenic differentiation in nonneuronal cells. To determine whether this bHLH transcription factor was necessary for normal brain development, we used homologous recombination to replace the neuroD2 coding region with a beta-galactosidase reporter gene. The neuroD2 gene expressed the reporter in a subset of neurons in the central nervous system, including in neurons of the neocortex and hippocampus and cerebellum. NeuroD2(-/-) mice showed normal development until about day P14, when they began exhibiting ataxia and failure to thrive. Brain areas that expressed neuroD2 were smaller than normal and showed higher rates of apoptosis. Cerebella of neuroD2-null mice expressed reduced levels of genes encoding proteins that support cerebellar granule cell survival, including brain-derived neurotrophic factor (BDNF). Decreased levels of BDNF and higher rates of apoptosis in cerebellar granule cells of neuroD2(-/-) mice indicate that neuroD2 is necessary for the survival of specific populations of central nervous system neurons in addition to its known effects on cell cycle regulation and neuronal differentiation.

The mechanisms of retinoid activity in tumors remain largely unknown. Here we establish that retinoids cause extensive apoptosis of medulloblastoma cells. In a xenograft model, retinoids largely abrogated tumor growth. Using receptor-specific retinoid agonists, we defined a subset of mRNAs that were induced by all active retinoids in retinoid-sensitive cell lines. We also identified bone morphogenetic protein-2 (BMP-2) as a candidate mediator of retinoid activity. BMP-2 protein induced medulloblastoma cell apoptosis, whereas the BMP-2 antagonist noggin blocked both retinoid and BMP-2-induced apoptosis. BMP-2 also induced p38 mitogen-activated protein kinase (MAPK), which is necessary for BMP-2- and retinoid-induced apoptosis. Retinoid-resistant medulloblastoma cells underwent apoptosis when treated with BMP-2 or when cultured with retinoid-sensitive medulloblastoma cells. Retinoid-induced expression of BMP-2 is thus necessary and sufficient for apoptosis of retinoid-responsive cells, and expression of BMP-2 by retinoid-sensitive cells is sufficient to induce apoptosis in surrounding retinoid-resistant cells.

Gene expression studies conducted with mouse models of Huntington's disease (HD) have revealed profound modifications in gene transcription. However, the complexity of in vivo tissue hampers definition of very early transcriptional modifications and does not allow discrimination between cell-autonomous changes and those resulting from intercellular activity processes. To identify early, cell-autonomous transcriptional changes, we compared gene expression profiles of clonal striata-derived cells expressing different N-terminal 548-amino-acid huntingtin fragments (with 26, 67, 105 or 118 glutamines) under the control of a doxycycline-regulated promoter. In these cells, mutant huntingtin did not form aggregates or cause cell death; therefore, the gene expression profiles report transcriptional changes reflecting early pathogenic events. We found that genes involved in cell signaling, transcription, lipid metabolism and vesicle trafficking were affected, in some cases, within 12 hours of mutant protein induction. Interestingly, this study revealed differential expression of a number of genes involved in cholesterol and fatty acid metabolism, suggesting that these metabolic pathways may play a role in HD pathogenesis.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It arises in the cerebellum and has been associated with a variety of genetic alterations, including genes in the Sonic hedgehog (Shh), Notch and Wnt signaling pathways. This study focuses on the Shh pathway, which is activated in a subset of MBs [1] [2] [3]. In this pathway, Shh binds to the receptor, Patched (PTCH), which liberates the Smoothened (SMO) protein, allowing GLI and MYCN transcription factors to turn on target genes, including, in a negative feedback loop, PTCH itself. Mutations in the PTCH gene are observed in 10-20% of sporadic MBs and are associated with a familial predisposition to MB, known as Gorlin's syndrome [4]. Additional Shh pathway activating mutations have been identified in SMO and Suppressor of Fused (SUFU) genes. Yet these mutations account for less than 25% of tumors that show Shh pathway activation [2]. The purpose of the current study was to begin exploring epigenetic mechanisms by which the Shh pathway could be activated.Methylation of tumor suppressor genes is increasingly recognized as a causative mechanism in tumorigenesis [5]. In MB, hypermethylation has been consistently identified in several genes, including, but not limited to RASSF1A, a multi-faceted tumor-suppressor [6], Caspase 8 (CASP8), whose disruption alters apoptosis and tissue homeostasis [7] and Hypermethylated in cancer 1 (HIC1) [8], a target of p53 (see [9] for detailed review). Notably, additional studies on CASP8 and HIC1 [10] have also revealed methylation in control samples (either adult or fetal cerebellum), illustrating the importance of using appropriate tissue matched samples for comparison in methylation studies. Moreover, none of these genes display a clear role in the molecular pathogenesis of MB. Hypermethylation has been identified in regulatory components of activating pathways such as Wnt, a pathway strongly associated with tumorigenesis in other cancers [11]. The present study is motivated by the hypothesis that methylation of the PTCH1 promoter is causative in some MB cases.We focused on PTCH1 for several reasons: 1) PTCH1 is a negative regulator of the Shh pathway. Thus, constrained transcription or translation of this gene would activate Shh activity. 2) Several MB cases display elevated GLI expression and concordant low PTCH1 expression, suggesting a loss of PTCH1 inhibition. 3) In a mouse model of MB in which one allele of PTCH1 was genetically disabled along with the p53 gene, the remaining allele was naturally silenced by methylation [12]. These findings and the overall scarcity of established *Corresponding Author:

No abstract