Abstract IA27: Functional genomics identifies drivers of medulloblastoma dissemination

Abstract

Abstract The pediatric brain tumor medulloblastoma shows a characteristic pattern of metastasis, called leptomeningeal dissemination (LMD), in which tumor cells disseminate via the cerebrospinal fluid to the leptomeningeal surfaces of the brain and spinal cord. This pattern distinguishes medulloblastomas from other solid tumors, which metastasize through the bloodstream or lymphatic channels. The invasion-metastasis cascade, a widely accepted paradigm derived from studies of hematogenous spread of carcinomas, is envisioned as a multistep process, governed by three set of genes: initiation, progression, virulence genes. By contrast, our knowledge of the molecular mediators of medulloblastoma LMD is rudimentary. Initial insight into the genetic drivers of LMD came from a transposon insertion mutagenesis study (Wu et al, Nature 482: 529-33, 2012). Mobilizing the Sleeping Beauty transposon in cerebellar neural progenitor cells caused widespread dissemination of typically localized, nonmetastatic medulloblastomas in Patched+/- mice, in which Sonic Hedgehog (Shh) signaling is hyperactive. Candidate LMD 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 and examined histological sections of brain and spinal cord during a 4-month observation period. Ectopic expression of the following Sleeping Beauty common insertion site genes shifted the in vivo growth characteristics of Shh-induced medulloblastomas from a localized pattern to a disseminated pattern: Eras (embryonic stem cell-expressed Ras), Lhx1 (LIM homeobox protein), Ccrk (cell cycle-related kinase), Akt (PI3K signaling protein), Arnt (hypoxia-inducible factor-1β), Gdi2 (Rab-GDP dissociation inhibitor), and Smad5 (TGF-β signal transducer). To elucidate the physiological traits conferred by these genes, we overexpressed them in a human medulloblastoma cell line (DAOY) and an immortalized, nontransformed cell line derived from mouse granule neuron precursors (SHH-NPD) and then quantified migration, invasiveness, and anchorage independence, growth characteristics that are closely associated with metastatic competence in carcinomas. Although the effects were quantitatively different in DAOY compared with SHH-NPD, all genes that induced LMD in vivo conferred these traits to cells in culture. We have used the set of genes discovered through Sleeping Beauty mutagenesis to begin constructing an LMD cascade, analogous to the invasion-metastasis cascade. Some LMD driver genes (Eras and Akt) enhanced the growth of the primary tumor, as indicated by shortened animal survival, while others did not. Therefore, we propose a two-category scheme for the LMD cascade, in which genes that increase primary tumor growth (Eras and Akt) are analogous to initiation/progression genes, whereas genes that only promote the growth and survival of disseminating cells (Lhx1, Ccrk, Arnt, Gdi2, and Smad5) are analogous to virulence genes. This simplified scheme will serve as a framework, upon which future research can build more sophisticated versions that better reflect the complexity of LMD. Citation Format: Daniel W. Fults. Functional genomics identifies drivers of medulloblastoma dissemination. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA27.