Targeted expression of MYCN causes medulloblastoma in mice Free

LB-79

Background: Medulloblastoma, a tumor composed of both neuronal and glial elements, is the most common primary pediatric brain tumor. Aberrant signaling through the Sonic Hedgehog (SHH) pathway is associated with the desmoplastic subtype of medulloblastoma and drives transformation, likely involving cerebellar granule cell precursors (cGNP). MYCN is a major regulator of cGNP proliferation and differentiation during normal development, and is an indirect target of the SHH pathway. To characterize the importance of MYCN in the pathogenesis of medulloblastoma, we targeted MYCN to neuroectodermal cells of the cerebellum using the tetracycline (Tet) system to conditionally express both MYCN and luciferase transgenes.

Methods: We targeted expression of the tetracycline transactivator protein (tTA) to brain astrocytes using the murine Glt-1 (glutamate transporter-1) promoter. We then generated a bi-directional Tet-response element (TRE) driving expression of both human MYCN and firefly luciferase (Luc). The tTA molecule is active in the absence of tetracycline derivatives, allowing transcription of MYCN and Luc from the TRE. In the presence of tetracycline derivatives, TRE expression is suppressed. Animals doubly transgenic for Glt1-tTA and TRE-MYCN/Luc were serially screened (in the absence of doxycycline) for tumor formation using Luc bio-imaging.

Results: We characterized expression of the Glt1 transgene in the brain, demonstrating high levels of expression specifically in astrocytes, with maximal expression in the cerebellum. We then crossed Glt1-tTA mice with TRE-MYCN/Luc mice. Serial bio-imaging of Luc expression in doubly transgenic animals revealed increased hindbrain signal intensity. We sacrificed these animals (at 2-4 months of age) for pathologic studies. Grossly, structural abnormalities of the cerebellum were apparent, with histology characteristic of large-cell, anaplastic medulloblastoma. Strong Mycn protein expression was confirmed by immunohistochemistry, along with expression of the astrocytic marker GFAP, and the neuronal markers synaptophysin and neuron specific enolase. Levels of angiogenesis, apoptosis, and proliferation were characterized. Penetrance data and survival data will be presented.

Significance: This model validates the functional importance of MYCN in medulloblastoma, and supports a role for cerebellar astrocytes in the origin of the disease. The anaplastic pathology observed was surprising, suggesting that dysregulation of MYCN may be a common feature of medulloblastomas in the absence of SHH signaling abnormalities. This model is amenable to non-invasive imaging for biologic analyses, to monitor malignant progression, and to measure real-time therapeutic responses. Furthermore, the regulable nature of MYCN expression, when combined with luciferase imaging, lends this model to temporal studies of tumor initiation and oncogene dependence.