Novel Downstream Effectors of Mitogenic Sonic Hedgehog Signaling in Cerebellar Development and Sonic Hedgehog Medulloblastoma Open Access

Robinson, Mary Hope (Fall 2018)

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The most common central nervous system malignancy in children, medulloblastoma arises from neural progenitor cells in the cerebellum and is diagnosed in over 300 children each year in the United States. While the current standard of care has brought the survival rate to ~70%, the treatments often leave patients with long-term negative side effects. The need for therapeutics that are specific, effective, and less toxic requires a greater understanding of the molecular underpinnings of medulloblastoma. Sonic Hedgehog (SHH) medulloblastoma, driven by aberrant activation of the SHH pathway, constitutes ~30% of cases and is the focus of this work. The major components of the SHH pathway have been identified, but there is still much to learn about the downstream effectors.

Pediatric cancers have few genetic mutations, providing a relatively uncluttered background to dissect molecular signaling pathways. At the same time, this paucity of mutations presents few targets and has led researchers to look beyond mutations. Epigenetics, changes in gene expression without changes in DNA sequence, has become an important avenue for research. We have identified Helicase, Lymphoid Specific (HELLS) as an epigenetic factor that is upregulated downstream of the SHH pathway in SHH-N stimulated CGNPs, in the developing murine cerebellum, and in both human and murine SHH MB when compared to non-tumor cerebellar tissue. HELLS upregulation was modulated by inhibition of the interaction between TEAD, a transcription factor, and the oncogenic co-activator YAP1, an established downstream effector of SHH. To ascertain direct regulation of HELLS by YAP/TEAD, a ChIP-qPCR assay was utilized, revealing YAP1 binding to Hells upstream DNA. These results indicate YAP1 mediates the SHH induced upregulation of HELLS in our systems. Because epigenetic modifications can be reversed, identification of an epigenetic factor upregulated by the SHH signaling pathway in medulloblastoma could have important implications for future therapeutics, not only in SHH MB but also in the myriad of other cancers with aberrant SHH signaling. 

In a separate project, we have identified high levels of HIF1α protein downstream of SHH signaling in CGNPs cultured in normoxic conditions. Rather than increased transcription, our results indicate stabilization of HIF1α, likely through ROS signaling. In addition, we identified increased levels of NOX4 protein, a potent producer of ROS. Taken together, our results demonstrate increased HIF1α protein levels as a result of ROS signaling induced stabilization. Similar to epigenetic regulation, ROS levels can be modulated, presenting an intriguing possibility for therapeutics development.

These studies lay the foundation for further research and may help fill in some of the gaps between SHH signaling and tumor formation. Importantly, both of these avenues of research could result in targeted therapies to treat SHH MB.

Table of Contents

Abstract. iv

Acknowledgments. vii

Table of Contents. x

Table of Figures. xiii

List of Abbreviations. xv

Chapter 1    Introduction. 1

1.1        Medulloblastoma. 1

1.1.1         Medulloblastoma Overview.. 1

1.1.2         Medulloblastoma Subgrouping. 4

1.2        Sonic Hedgehog. 11

1.2.1         The Hedgehog Pathway. 11

1.2.2         Hedgehog in Cerebellar Development 16

1.2.3         Hedgehog in Cancer. 19

1.2.4         Therapeutic Targeting of Hedgehog. 20

1.2.5         Hedgehog Model Systems. 21

1.3        Epigenetics. 23

1.3.1         What is epigenetics?. 23

1.3.2         DNA Methylation. 26

1.3.3         Histone post-translational modification. 27

1.3.4         Chromatin remodeling. 29

1.3.5         Other epigenetic mechanisms. 29

1.4        HELLS. 32

1.4.1         Helicase, Lymphoid Specific. 32

1.4.2         HELLS in Development and Disease. 37

1.5        In search of novel downstream effectors of SHH.. 39

Chapter 2         Upregulation of the Chromatin Remodeler HELLS is mediated by YAP1 in Sonic Hedgehog Medulloblastoma. 41

2.1        Author’s Contribution and Acknowledgement of Reproduction. 41

2.2        Abstract. 42

2.3        Introduction. 43

2.4        Results. 47

2.4.1         HELLS expression is upregulated in SHH-N stimulated CGNPs and in the developing murine cerebellum   47

2.4.2         Elevated expression of HELLS in Human SHH MB. 50

2.4.3         HELLS levels are increased in SHH murine MB. 52

2.4.4         HELLS expression is dependent on YAP1 activity. 54

2.4.5         YAP1/TEAD binding to DNA upstream of HELLS confirms direct regulation of HELLS. 58

2.5        Discussion. 61

2.6        Methods. 66

2.7        Acknowledgments. 71

Chapter 3         Reactive Oxygen Species Signaling Promotes HIF1α Stabilization in Sonic Hedgehog-Driven Cerebellar Progenitor Cell Proliferation. 73

3.1        Author’s Contribution and Acknowledgement of Reproduction. 73

3.2        Abstract. 74

3.3        Introduction. 75

3.4        Results. 78

3.4.1         HIF1α is Upregulated at the protein level in SHH-treated CGNPs. 78

3.4.2         SHH Increases ROS Production in CGNPs. 81

3.4.3         Scavenging of ROS reduces HIF1α stabilization in SHH-treated CGNPs. 83

3.4.4         NADPH Oxidase 4 is Upregulated in SHH-Treated CGNPs. 83

3.4.5         NOX Regulates HIF1α at the Protein Level and Appears Necessary for Proliferation. 86

3.5        Discussion. 88

3.6        Materials and Methods. 91

3.7        Acknowledgments. 93

3.8        Additional Preliminary Findings. 94

Chapter 4    Summary and Future Directions. 97

4.1        Upregulation of the epigenetic factor HELLS downstream of SHH signaling in cerebellar development and in SHH MB  97

4.1.1         Summary of findings. 97

4.1.2         Discussion and Future Directions. 99

4.2        Stabilization of HIF1α downstream of SHH signaling. 103

4.2.1         Summary of findings. 103

4.2.2         Discussion and Future Directions. 105

4.3        Next Challenges. 105

Chapter 5    References. 108

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