Reactive Oxygen Species Signaling and Hypoxia-Independent Regulation of HIF1α in Sonic Hedgehog-Driven Medulloblastoma and Cerebellar Progenitor Proliferation Open Access

Eyrich, Nicholas (2017)

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Medulloblastoma (MB) is the most common solid pediatric malignancy of the central nervous system. 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 Shh pathway activation. CGNP cultures offer an excellent model system for studying Shh-driven MB, given its similarities with normal cerebellar development. Ex vivo tumor slice cultures also allow us to study MB cellular processes and pharmacological interventions with enhanced physiological relevance since tumor architecture and cell-cell interactions are preserved.

In addition to mitogens driving proliferation, it has been shown that low levels of intracellular reactive oxygen species (ROS) are required for proliferation through a myriad of mechanisms. In other cancers, increased ROS levels have been shown to affect other signaling events including the stabilization of Hypoxia-Inducible Factor-1-Alpha (HIF1α) by interfering with prolyl hydroxylase's ability to tag HIF1α for VHL-mediated degradation. HIF1α, in addition to its oxygen-sensing role, has been implicated in the Warburg effect, which causes an increase in glycolytic activity and relative decrease in oxidative phosphorylation in oftentimes normoxic cancer cells. The current literature and preliminary studies led us to investigate potential sources of ROS within the NADPH Oxidase (Nox) family of proteins and possible consequences involving HIF1α downstream of Shh. We've shown that HIF1α is stabilized following Shh pathway induction, and under normoxic conditions, Nox activity is required to maintain a minimum level of ROS. Taken together, our findings suggest an axis mediated by Nox-generated ROS that helps understand hypoxia-independent regulation of HIF1α in Shh medulloblastoma that could be contributing to tumor recurrence.

Table of Contents

Table of Contents

Introduction & Background - 1

Medulloblastoma Clinical Characteristics - 2

Cerebellar Development and Shh Medulloblastoma - 5

Hypoxia Inducible Factor-1-Alpha in Medulloblastoma and Its Hypoxia-Independent Regulation - 8

Reactive Oxygen Species (ROS) in Cancer and HIF1α Regulation - 15

NADPH Oxidases: Key Sources of ROS in Cancer - 17

Scope of the Thesis - 21

Materials and Methods - 22

Animal Studies - 23

Cerebellar Granule Neuron Precursor Culture - 23

Protein Collecting and Immunoblotting - 24

Ex Vivo Organotypic SmoA1 Tumor Slice Culture - 24

Immunofluorescence - 25

Reactive Oxygen Species Assay - 25

Results - 27

HIF1α Colocalizes with Stem Cell Markers in the MB Perivascular Niche - 28

HIF1α is Up-regulated in SmoA1 MB Tumors - 30

HIF1α Protein is Up-regulated in Shh-treated CGNPs - 32

Shh Induces ROS Production in CGNPs - 34

Antioxidant-Mediated Sequestering of ROS Reduces HIF1α Protein Stabilization in Shh-treated CGNPs and in SmoA1 Medulloblastomas Ex Vivo - 36

NADPH Oxidase Inhibition Reduces HIF1α Protein Stabilization in Shh-treated CGNPs and in SmoA1 Medulloblastomas Ex Vivo - 39

NADPH Oxidase 4 is Upregulated Downstream of Shh - 39

Discussion - 42 References - 48 Table of Figures

Figure 1. Medulloblastoma and its Molecular Subgroups - 3

Figure 2. Frequency of Each Molecular Subgroup of Medulloblastoma - 4

Figure 3. Shh Signaling in Post-Natal Development of The Cerebellum - 6

Figure 4. HIF-1 Transcriptional Activity and Its Correlation with Cancer Prognoses - 10

Figure 5. Elevated HIF1α at the Protein Level in Medulloblastoma compared to Normal Cerebellum - 11

Figure 6. HIF1α Expression in the Medulloblastoma Perivascular Niche - 13

Figure 7. HIF1α Protein Levels Increase in Shh-Treated CGNPs in an mTOR-dependent Manner - 14

Figure 8. Factors that affect Cellular ROS Homeostasis and Pathway Signaling Relative to ROS Levels - 16

Figure 9. Inducing ROS Increases Shh-Treated CGNP Proliferation and ROS Scavenging Attenuates Proliferation - 16

Figure 10. Mitochondrial ROS Can Lead to HIF1α Stabilization by Interfering with PHDs - 18

Figure 11. Nox4 Structure and Screen of Potential ROS-Promoting Genes in Shh-Treated CGNPs - 19

Figure 12. Elevated Levels of Nox4 Protein in Shh-Treated CGNPs and Mouse MB - 20

Figure 13. HIF1α Protein is up-regulated in SmoA1 MB - 29

Figure 14. HIF1α Colocalizes with Stem Cell Markers in the Medulloblastoma Perivascular Niche - 31

Figure 15. HIF1α Protein is Up-regulated in Shh-treated CGNPs - 33 Figure 16. Shh Induces ROS Production in CGNPs - 35

Figure 17. Immunoblot Analysis Following ROS Scavenging using NAC in Shh-Treated CGNPs and SmoA1 Medulloblastomas Ex Vivo - 37

Figure 18. Immunoblot Analysis Following ROS Scavenging with GSH in Shh-Treated CGNPs and SmoA1 Medulloblastomas Ex Vivo - 38

Figure 19. Immunoblot Analysis Following NADPH Oxidase Inhibition in Shh-Treated CGNPs and SmoA1 Medulloblastomas Ex Vivo - 40

Figure 20. Nox4 Protein is Up-regulated in MB and Shh-treated CGNPs - 41

Figure 21. Proposed Nox4--ROS--HIF1α Model - 46

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