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Cerebellar development is a highly regulated process involving numerous factors acting with high specificity, both temporally and by location. Part of this process involves extensive proliferation of cerebellar granule neuron precursors (CGNPs) induced by Sonic Hedgehog (SHH) signaling, but downstream effectors of mitogenic signaling are still being elucidated. Using primary CGNP cultures, a well-established model for SHH-driven proliferation, we show that SHH-treated CGNPs feature high levels of hypoxia-inducible factor 1α (HIF1α), which is known to promote glycolysis, stemness, and angiogenesis. In CGNPs cultured under normoxic conditions, HIF1α is posttranslationally stabilized in a manner dependent upon reactive oxygen species (ROS) and NADPH oxidase (NOX), both of which are also upregulated in these cells. Inhibition of NOX activity resulted in HIF1α destabilization and reduced levels of cyclin D2, a marker of CGNP proliferation. As CGNPs are the putative cells of origin for the SHH subtype of medulloblastoma and aberrant SHH signaling is implicated in other neoplasms, these studies may also have future relevance in the context of cancer. Taken together, our findings suggest that a better understanding of nonhypoxic HIF1α stabilization through NOX-induced ROS generation can provide insights into normal cell proliferation in cerebellar development and SHH-driven cell proliferation in cancers with aberrant SHH signaling.

Sonic hedgehog (Shh) signaling is closely coupled with the bioenergetics of medulloblastoma, the most common malignant pediatric solid tumor. We have reported earlier that Shh causes deregulation of mitochondrial biogenesis by suppressing mitofusins, leading to fragmented mitochondria in vitro as well as in SmoA1 mouse medulloblastomas (MB) in vivo. Ectopic expression of mitofusins restored mitochondrial fusion accompanied by a rescue in proliferation to the non-proliferative phenotype. In the present study, we report that a rescue in proliferation is also observed in organotypic slice cultures implanted with mitofusin overexpressing SmoA1 neurospheres. When mitofusin overexpressing tumor cells were injected into the cerebella of postnatal day 2 mice, tumor development was significantly delayed. We have significant evidence indicating that Shh induces high increases levels of Reactive Oxygen Species (ROS) in Cerebellar Granule Neuron Precursor cells (CGNPs). When total ROS in a cell was scavenged by treating with N-Acetyl Cytosine (NAC), the fragmented mitochondrial morphology was rescued to their fused morphology. NADPH Oxidase 4 (NOX4) is a known producer of ROS in cells. When we treated CGNPs with apocynin, an NADPH oxidase inhibitor, a significant reduction in proliferation was observed. NOX4 was also found to localise in the peri-vascular niche in SmoA1 tumors. This could have possible implications for a role of ROS in promoting the proliferation of tumor re-populating cells post irradiation. Our goal is to determine if manipulating ROS-mediated mitochondrial dynamics can restore the metabolic profile of tumor cells to that of non-transformed, non-proliferating cells. This would suggest a potential novel treatment paradigm for medulloblastoma that may reduce the requirement for high dose radiation.

Medulloblastoma is the most common solid malignant pediatric brain tumor. These tumors arise in the cerebellum and can be molecularly subdivided into 4 consensus subgroups, one of which is marked by amplification and activation of Sonic hedgehog (Shh) pathway components and downstream targets. This subclass is proposed to arise from the oncogenic transformation of cerebellar granule neuron precursors (CGNPs), whose expansion during post-natal brain development requires activation of the Shh pathway and downstream targets. These tumors often demonstrate similarities with normal cerebellar development at the molecular level, thus allowing us to use primary CGNP cultures as a model system for the Sonic hedgehog (Shh) driven subclass of medulloblastoma. In addition to mitogens driving proliferation in cancer, it has been shown in the past that low levels of intracellular reactive oxygen species (ROS) can contribute to proliferation through, amongst other methods, phosphatase inhibition and subsequent deregulation of key pathways including pathways that collaborate with Shh signaling. To this end we've studied a reported ROS-generating effector of insulin and insulin-like growth factor signaling, NADPH oxidase 4 (Nox4). It is thought that Nox4 is both upregulated by insulin signaling and synergizes with it downstream via ROS-induced sustained phosphorylation of Akt. Our work in CGNPs revealed a marked induction of Nox4 in response to Shh at the mRNA and protein levels as well as an increase in total reactive oxygen species content. Subsequent studies suggest that Nox4 activity is critical to sustaining proliferation of Shh driven CGNPs. Western blots of shRNA knockdowns of Nox4 showed reduction of proliferative marker CyclinD2. The knockdowns also precipitated a drop in phosphorylated Akt perhaps leaving the Shh pathway without the full effects of one of its major signaling partners, the PI3K/Akt pathway.

Medulloblastoma is the most common solid malignant pediatric brain tumor. These tumors arise in the cerebellum and can be molecularly subdivided into 4 consensus subgroups, one of which is marked by amplification and activation of Sonic hedgehog (Shh) pathway components and downstream targets. This subclass is proposed to arise from oncogenic transformation of cerebellar granule neuron precursors (CGNPs), whose expansion during post-natal brain development is driven by and requires activation of the Shh pathway. These tumors often demonstrate similarities with normal cerebellar development at the molecular level, thus allowing us to use primary CGNP cultures as a model system for the Sonic hedgehog (Shh) driven subclass of medulloblastoma. In addition to mitogens driving proliferation, it has been shown in the past that low levels of intracellular reactive oxygen species (ROS) are required for proliferation, through mechanisms as diverse as inhibition of receptor tyrosine phosphatases, stabilization of proliferation proteins, and modifications of metabolites, thus indicating additional roles for ROS in proliferation and perhaps tumor growth beyond their known capacity to cause DNA damage, thereby contributing to genomic instability and apoptosis. Although the Shh ligand does not bind to a receptor tyrosine kinase (RTK), it is known that Shh signaling cooperates with RTK- activated pathways such as the insulin-like growth factor pathway to drive proliferation. To determine whether intracellular ROS play a role in Shh-driven CGNP proliferation, we treated CGNPs with the ROS scavenger lipoic acid (LA) in the presence of Shh, and observed a significant decrease in proliferation. Conversely, addition of the ROS inducer tert-Butyl hydroperoxide to Shh treated CGNPs led to enhanced proliferation over Shh treatment alone. These results indicate that a certain level of ROS are required to support Shh-driven CGNP proliferation, and enhancing their levels can increase proliferation. To investigate whether Shh signaling may affect expression of ROS regulatory genes we carried out a qPCR analysis. We identified up-regulation of sod2, gstm1, and gsto1: genes known to respond to ROS and whose products neutralize ROS, over vehicle-treated CGNPs. Paradoxically, when we examined ROS regulatory enzyme expression in Shh-driven mouse medulloblastomas, we noted sharp drop in the expression of gstm1, gsto1, and sod2 compared to the adjacent cerebellum suggesting a reduced ability to inactivate ROS, which could be contributing to proliferation or DNA damage in this transgenic model. These findings suggest that normal Sonic hedgehog pathway activation contributes to the production and tight regulation of ROS via downstream effectors in addition to synergizing with ROS to drive proliferation, a highly regulated balance that may be lost in medulloblastoma. Citation Format: Chad R. Potts, Rachel D. Rotenberry, Anna M. Kenney. Reactive oxygen species in Sonic hedgehog-driven proliferation of cerebellar granule neuron precursors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2014-3978

A subclass of Medulloblastoma shows considerable dependence on the Sonic Hedgehog (Shh) pathway. Since cellular functions are a consequence of coordinated networks involving protein-protein interactions, it is imperative to explore different events cooperating with Shh entities in order to explore novel targets for medulloblastoma therapy. The Yes Associated Protein (YAP), a component of the Hippo pathway, has been previously established as a downstream target of the Shh pathway (Fernandez et al 2011). Several interesting gene targets featured in the networks developed by us using YAP overexpression in CGNPs. A concurrent upregulation of IGF2 and H19, both imprinted genes, was observed. Possible links between the Shh/Hippo pathways and IGF2 signaling also featured in the networks developed in the study. Important links to lipid metabolism, a process highly active in medulloblastomas and essential for the proliferation of CGNPs, were also predicted. The study includes results of signaling interactions between the Shh/Hippo pathways and entities from other relevant pathways that together play a role in medulloblastoma development and proliferation. Citation Format: Anshu Malhotra, Chad R. Potts, Africa Fernandez-Lopez, Rachel Rotenberry, Anna M. Kenney. Signaling network-based analyses of sonic hedgehog pathway components: predictions and possibilities. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2013-4105