Investigating the Role for Y-Box Binding Protein 1 in Medulloblastoma Radiation Resistance and Post-transcriptional Regulation of Migration and Metastasis Open Access
Mcswain, Leon (Summer 2023)
Abstract
Brain tumors represent a vast landscape of ailments ranging from the genomic and transcriptomically simple to the very complex and therapeutically adaptable. While relatively genetically straightforward, Medulloblastoma (MB) still represents a formidable tumor in pediatric patients for which treatments consist of total resection, harsh chemotherapy, and radiation resulting in numerous sequelae lasting into adulthood. From the mid to late 2010s the advent of transcriptomic technology, such as microarray and RNA sequencing afforded a closer look into the cell biology and, thus, potentially exploitable protein dependencies within these tumors. However, even considering this, modern therapies continue to consist of these antiquated techniques with a lack of modernization. This dissertation is an attempt to understand not only the sensitization of Medulloblastoma to the standard of care therapies mentioned above, but also to explore post-transcriptional regulation of proteins where differential RNA sequencing analysis may not detect oncoprotein dependencies due to a lack of transcriptional differences amongst patients.
The introductory chapters of this dissertation are an exploration of proteins medulloblastomas depend upon for mediating resistance to radiation and chemotherapy, some of which are druggable and have clinical trials pending. Y-Box Binding Protein 1 (YBX1) binding and signaling through some of these proteins, including Poly-ADP-Ribose Polymerase (PARP) and TP53, is thought to be responsible for mediating the radiation resistance phenotypes seen across many cancer types. Key DNA damage repair signaling proteins, such as RAD50 and MRE-11, are also published as binding partners for YB1. The 4th chapter of this dissertation explores the findings of those studies, and in some cases challenges them, in an investigation of sensitizing MB to radiation through YB1 silencing.
The final chapters of this dissertation challenge the frequently utilized transcriptomic technologies mentioned previously by exploring the RNA binding protein (RBP) functions of YB1 and introduce novel MB targets for reducing migration, metastasis, and proliferation. RNAs targeted by RBPs may show a lack
of differential RNA levels between samples while the corresponding proteins coded for by these RNAs show drastically different levels. Here we have elucidated a novel signaling axis whereby YB1 regulates PlexinD1 protein levels by post-transcriptional mRNA binding and translational regulation. We also explore a binding partner, Sema3E, through which PLXND1 signals to mediate migration and proliferation.
Altogether, these studies are a step forward in sensitizing MB to standard of care, and a contribution to understanding tumor biology and mRNA translational regulation as a whole, potentially applicable outside of medulloblastoma.
Table of Contents
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Abbreviations
Chapter 1. Medulloblastoma and Sonic Hedgehog Signaling
1.1 Current Clinical Approaches to Medulloblastoma
1.2 Medulloblastoma: Developmental Biology Gone Awry 1.3 Metastasis in Medulloblastoma
1.4 Conclusions
Chapter 2. A Review: Targeting Proteins Involved in the DNA Damage Response to Radiation and Chemotherapy in Medulloblastoma
2.1 Abstract
2.2 DNA Damage Signaling
2.3 Targeting Proteins Involved in the DNA Damage Response to Radiation and Chemotherapy
2.3.1 ATR-CHK1 2.3.2 PARP
2.3.3 APE1
2.3.4 CK2 and MGMT 2.3.5 AKT and PI3K
2.4 p53
2.4.1 p53 Status as a Predictor of Chk1 and PARP Inhibition
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2.4.2 p53 and Medulloblastoma Development
2.4.3 Targeting the p53 Response 2.5 Conclusions
Chapter 3. Y-Box Binding Protein 1: a Pleiotropic Oncogene
3.1 Abstract
3.2 Structure and Regulation of YB1
3.3 YB1 in Embryonic and Tumor Development
3.4 YB1 Drives Proliferation and EMT
3.5 YB1 in the DNA Damage Response and Genomic Stability 3.6 YB1 in the Clinic: Resistance and Targeting
3.7 Conclusions
Chapter 4. YB1 Modulates the DNA Damage Response in Medulloblastoma
4.1 Abstract
4.2 Introduction 4.3 Results
4.3.1 YB1 is expressed across all MB subgroups and overexpression is associated with decreased survival in an SHH primary mouse model
4.3.2 YB1 depletion results in differential cell cycling and nuclear morphology after IR damage
4.3.3 Irradiation of YB1 depleted cells results in differential γH2AX resolution and CHK 2 phosphorylation in SHH and Group3 MB cells
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4.3.4 YB1 depletion results in accelerated DSB and SSB repair, γH2AX resolution, and a lack of RPA32 phosphorylation at Serines 4/8
4.3.5 YB1 depleted cells accumulate less RAD51 and more TP53BP1 nuclear bodies during and after S-Phase repair
4.3.6 YB1 depletion results in greater canonical NHEJ and lower HR
4.3.7 YB1 knockdown results in decreased proliferation and increased senescence 4.4 Discussion
4.5 Materials and methods
Chapter 5. An Introduction to Plexins and Semaphorins in Development and Oncogenesis
5.1 Abstract
5.2 Discovery and structure of PlexinD1
5.3 PlexinD1-Sema3E during development: attraction and repulsion 5.4 PlexinD1-Sema3E in cancer
5.5 Discussion
Chapter 6: YB1 Regulates PlexinD1 Translation in Sonic Hedgehog Medulloblastoma
6.1 Abstract
6.2 Introduction 6.3 Results
6.3.1 PlexinD1 is enriched in YB1 RIPseq of primary murine SHH MB cells 6.3.2 PlexinD1 is expressed in SHH MB and Membrane PlexinD1 is expressed an colocalizes with NRP1 and Sema3E in SHH MB cells
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6.3.3 YB1 Positively Regulates PlexinD1 Translation in Human and Mouse SHH Models
6.3.4 PlexinD1 and Sema3E Regulate SHH MB Cell Migration
6.3.5 Peptide inhibition of PlexinD1 reduces proliferation and migration
6.4 Discussion
6.5 Materials and methods
Chapter 7: Conclusions
About this Dissertation
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