c-Myc enhances sonic hedgehog-induced medulloblastoma formation from nestin-expressing neural progenitors in mice

Abstract

Medulloblastomas are malignant brain tumors that arise in the cerebella of children. The presumed cells-of-origin are undifferentiated precursors of granule neurons that occupy the external granule layer (EGL) of the developing cerebellum. The overexpression of proteins that normally stimulate proliferation of neural progenitor cells may initiate medulloblastoma formation. Two known mitogens for neural progenitors are the c-Myc oncoprotein and Sonic hedgehog (Shh), a crucial determinant of embryonic pattern formation in the central nervous system. We modeled the ability of c-Myc and Shh to induce medulloblastoma in mice using the RCAS/tv-a system, which allows postnatal gene transfer and expression in a cell type-specific manner. We targeted the expression of Shh and c-Myc to nestin-expressing neural progenitor cells by injecting replication-competent ALV splice acceptor (RCAS) vectors into the cerebella of newborn mice. Following injection with RCAS-Shh alone, 3/32 (9%) mice developed medulloblastomas and 5/32 showed multifocal hyperproliferation of the EGL, possibly a precursor stage of medulloblastoma. Following injection with RCAS-Shh plus RCAS-Myc, 9/39 (23%) mice developed medulloblastomas. We conclude that nestin-expressing neural progenitors, present in the cerebellum at birth, can act as the cells-of-origin for medulloblastoma, and that c-Myc cooperates with Shh to enhance tumorigenicity.

Figure 1

Histopathology of cerebellar lesions induced by Shh. (A) Western blot analysis of Ntv-a mouse brain cultures infected with RCAS-Shh. Two major bands (60 and 37 kDa) corresponding to full-length Shh and carboxy-terminal peptide derived from autoproteolysis were detected with anti-HA antibody in infected cells (+) but not uninfected cells (-). (B) Focal hyperproliferation of EGL induced by Shh (Mo—molecular layer; Pu—Purkinje cell layer; IGL—internal granule layer). (C) Shh-induced medulloblastoma infiltrating cerebellar cortex. (D) Cytoplasmic expression of endogenous c-Myc in tumor cells in Shh-induced medulloblastoma. (E) Human medulloblastoma specimen showing expression of c-Myc in nuclei (arrow) and cytoplasm (arrowhead) of tumor cells. Scale bar = 110 µm (B,C), 55 µm (D), 27 µm (E).

Figure 2

Histopathology of medulloblastomas induced by c-Myc plus Shh. (A,B) Medulloblastomas infiltrating the lateral pons and cerebellum (Cb) and filling the fourth ventricle (4V). (C) Cytoarchitecture of medulloblastoma induced by c-Myc plus Shh. Arrows indicate mitotic figures. (D,E) Immunoperoxidase staining of virally transduced Shh and c-Myc in tumor cells with antibodies against HA (D) and Myc (E) epitopes. (F) Double immunofluorescence staining of Shh (green) and c-Myc (red) shows colocalization (yellow) of both proteins in the cytoplasm of a subpopulation of medulloblastoma cells. Nuclei are counterstained with propidium iodide (blue). Scale bar = 474 µm (A,B), 27 µm (C–E), 27 µm (F).

Figure 3

Expression of neural antigens in medulloblastomas induced by c-Myc plus Shh. Expression of synaptophysin (A), βIII tubulin (B), and NeuN (C) in tumor cells and GFAP (D) in processes of entrapped astrocytes. Scale bar = 27 µm.