Functional genomics identifies drivers of medulloblastoma dissemination

Abstract

Medulloblastomas are malignant brain tumors that arise in the cerebellum in children and disseminate via the cerebrospinal fluid to the leptomeningeal spaces of the brain and spinal cord. Challenged by the poor prognosis for patients with metastatic dissemination, pediatric oncologists have developed aggressive treatment protocols, combining surgery, craniospinal radiation, and high-dose chemotherapy, that often cause disabling neurotoxic effects in long-term survivors. Insights into the genetic control of medulloblastoma dissemination have come from transposon insertion mutagenesis studies. Mobilizing the Sleeping Beauty transposon in cerebellar neural progenitor cells caused widespread dissemination of typically nonmetastatic medulloblastomas in Patched(+/-) mice, in which Shh signaling is hyperactive. Candidate metastasis genes were identified by sequencing the insertion sites and then mapping these sequences back to the mouse genome. To determine whether genes located at transposon insertion sites directly caused medulloblastomas to disseminate, we overexpressed candidate genes in Nestin(+) neural progenitors in the cerebella of mice by retroviral transfer in combination with Shh. We show here that ectopic expression of Eras, Lhx1, Ccrk, and Akt shifted the in vivo growth characteristics of Shh-induced medulloblastomas from a localized pattern to a disseminated pattern in which tumor cells seeded the leptomeningeal spaces of the brain and spinal cord.

Figure 1

The Sleeping Beauty transposon system. SB consists of two components, the transposable element (transposon) and the transposase enzyme, which catalyzes transposon mobilization. A, Vector for expressing the transposase under control of a tissue-specific promoter/enhancer. B, The SB transposon contains a pair of inverted repeat/direct repeat elements (IRDR), flanking the mobile cargo sequence. For cancer gene identification, the cargo sequence is designed to mimic retroviral insertional mutagenesis. The transposon contains splice acceptor sites (SA) and polyadenylation (pA) sequences to disrupt the expression of genes into which the transposon integrates (mRNA1). The transposon also contains 5’ sequences from the murine stem cell virus (MSCV) long terminal repeat (LTR) to serve as promoter/enhancer elements, which increase expression of adjacent genes (mRNA2). The MSCV LTR is followed by a splice donor (SD). Thus, a transcript initiated in the LTR can splice into downstream exons of endogenous genes. The SB transposon schematized here is the T2/Onc vector [reviewed in (16)].

Figure 2

Histopathology of spinal leptomeningeal dissemination. A, classic cytoarchitecture of Shh-induced medulloblastoma, showing a uniform field of undifferentiated tumor cells (left) abutting the cerebellar cortex (right) (H&E). B, aggregates of Shh-induced medulloblastoma cells attached to the leptomeninges (arrows) of the spinal cord (below) and a spinal nerve (left) (H&E). C, metastasis of Shh+Lhx1–induced medulloblastoma cells to a spinal nerve (right) exiting the spinal cord (left) (H&E). D, immunoperoxidase staining of tissue section adjacent to that shown in C, showing GFP immunoreactivity in tumor cells, thus verifying expression of retrovirus-transferred Lhx1. E, metastasis of Shh+Ccrk–induced medulloblastoma to the surface of the spinal cord (H&E). F, tissue section adjacent to that shown in E, showing GFP immunoreactive staining in Shh+Ccrk–induced medulloblastoma cells attached to the spinal cord. Scale bar, 50 µm (A–D), 25 µm (E–F).

Figure 3

Histopathology of forebrain leptomeningeal dissemination. A, infiltration of Shh+Ccrk–induced medulloblastoma (arrow) into the brain stem and hippocampal fissure (H&E). B, Shh+Lhx1–induced medulloblastoma cells attached to the leptomeninges of the brain stem (H&E). C, Shh+Lhx1–induced medulloblastoma cells in the hippocampal fissure. Granule neurons of the dentate gyrus are visible below (H&E). D, GFP immunoreactive staining in metastasizing cells from Shh+Lhx1–induced medulloblastoma. Tissue section is adjacent to that shown in C. E, Shh+Ccrk–induced medulloblastoma cells in the subependymal space of the lateral ventricle (arrows) (H&E). F, Shh-induced medulloblastoma infiltrating the fourth ventricle (H&E). Scale bar, 500 µm (A, F), 100 µm (B–D), 50 µm (E).

Figure 4

Survival analysis of mice following retroviral transfer of Shh and metastasis-inducing oncogenes. Kaplan-Meier survival analysis of mice injected with RCAS-Shh, alone or in combination with RCAS-Ccrk and RCAS-Lhx1 (A) or RCAS-Eras and RCAS-Akt (B). The mice were injected on day zero and sacrificed at the indicated time points.

Figure 5

Expression of gCIS–associated genes in human medulloblastoma subgroups. Box plots showing relative expression of ERAS (A), CCRK (B), and LHX1 (C) in normal cerebella (CB; fetal n=9, adult n=5) and medulloblastoma samples (MB; n=103) profiled on Affymetrix exon arrays. The 103 medulloblastomas were divided into subgroups (WNT, SHH, Group 3, Group 4) and analyzed separately. Log2 expression is a measure of the luminosity of the gene probe signal, corrected for the background luminosity of each array and normalized using control probes across different arrays.