Abstract 1419: HIF1a downstream of Sonic hedgehog mitogenic and oncogenic signaling and brain development and medulloblastoma

Medulloblastoma, the most common solid malignant pediatric tumor, originates during post-natal development in the cerebellum. It is postulated that cerebellar granule neuron precursors (CGNPs) are cells-of-origin for medulloblastomas associated with increased activity of the Sonic hedgehog (Shh) pathway. In mice and humans, these cells undergo a rapid peri-natal expansion phase in the external granule layer (EGL) of the cerebellum, and this expansion requires Shh, which is secreted by the Purkinje neurons underlying the EGL. Shh promotes CGNP proliferation and maintains these cells in an undifferentiated state. To carry out these functions, the Shh pathway interacts with and/or regulates cooperating intracellular signalling pathways. We have previously shown that interactions between Shh and insulin-like growth factor (IGF) are required for the full mitogenic effect of Shh in CGNPs, and IGF activity is prominent in human SHH medulloblastomas. Both IGF and Shh drive mTOR activity, which is associated in certain contexts with increased translation of hypoxia inducible factor 1α (HIF1α). HIF1α promotes angiogenesis and is also associated with maintenance of a stem cell-like state, i.e inhibition of differentiation. We asked whether Shh signaling regulates HIF1α in CGNPs during cerebellar development and in mouse models of SHH medulloblastoma. Using western blot and immunfluorescence analysis, we observed increased HIF1a in CGNPs upon Shh treatment. In vivo, HIF1α was localized to the nuclei of cells in the EGL. There was no significant effect of Shh treatment on HIF1α mRNA expression, but exposure of CGNPs to the mTOR inhibitor rapamycin resulted in reduced HIF1a protein levels, indicating that the effects of Shh on Hif1α are post-transcriptional. We also observed high levels of HIF1α in SmoA1 medulloblastomas, where it co-localized with its known targets pyruvate kinase M2 (PKM2, a metabolic gene) and Oct4 (a hallmark of stem cells) both in the perivascular niche (PVN) and tumor bulk. IGF2, an established HIF1α target, was highly expressed in the PVN. mTOR activity promotes increased HIF1α translation, and that HIF1α induces expression of IGF2, forming a feed-forward loop leading to mTOR activation and increased HIF1α translation. When SmoA1 tumor-bearing mice were treated with the Wyeth mTOR inhibitor CCI-779, HIF1α levels were reduced. These results are consistent with HIF1α regulating VEGF, which we have previously shown to be elevated in SmoA1 medulloblastomas, in an mTOR-dependent manner. Moreover, the expression of HIF1α in both the PVN and tumor bulk is consistent with mTOR activity being widely distributed throughout SmoA1 medulloblastomas, as determined by phosphorylation of the downstream mTOR effector ribosomal protein S6. Taken together, these results suggest a role for HIF1α in Shh mitogenic/oncogenic signaling in the developing cerebellum and medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1419. doi:1538-7445.AM2012-1419