Arl13b is a Target for the Treatment of Medulloblastoma Open Access

Bay, Sarah (2017)

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The signaling pathways responsible for proper development are often the same ones implicated in cancer formation. Sonic Hedgehog (Shh) signaling is a critical developmental pathway that directs cerebellar development and ventral neural tube patterning. Shh ligand binds the receptor Patched, removing Patched's inhibition of Smoothened and allowing for activation of Gli transcription factors. When ligand is absent, Gli proteins are processed to their repressor form. In vertebrates, this signaling cascade requires the primary cilium, a microtubule-based projection found on every vertebrate cell. The ciliary GTPase Arl13b regulates Shh signaling at multiple levels and affects the production of Gli activator but not Gli repressor. Overactivated Shh signaling can cause medulloblastoma (MB), a tumor of the cerebellum, and new molecular therapies are needed. Genetic ablation of cilia prevents tumors in mice, but cilia are a poor therapeutic target. Arl13b is a strong candidate since its loss reduces Shh activity while preserving Gli repressor, which protects against MB formation.

This dissertation covers my work on the requirement of Gli repressor for ventral neural tube patterning. By deleting Arl13b--and thus disrupting Gli activator function--we uncovered a patterning defect in the neural tube that corrected over time; I showed this recovery was dependent on Gli repressor. I also describe the generation of a mouse model of MB in which I could delete Arl13b in the cerebellum before tumor formation or in an existing tumor. However, after the generations of crosses required to make the model, the tumor phenotype disappeared, leading to the conclusion that a genetic modifier was interfering with tumor formation.

The core of this dissertation demonstrates that the loss of Arl13b in a mouse model of MB inhibits tumor formation. By disrupting Arl13b function in cell culture, I demonstrate that the loss of Arl13b results in reduced Shh signaling--and related growth and proliferation phenotypes. I show that Arl13b is not an essential gene in adulthood in mice. Taken together, my results provide the proof of principle that Arl13b is a promising target for the treatment of MB as its loss counteracts tumorigenic signaling while leaving cilia function largely intact.

Table of Contents

Chapter 1 Medulloblastoma, Cilia, and Shh Signaling ____________________ 1

1.1 Introduction_________________________________________________ 2

1.2 Brief overview of Shh signaling__________________________________ 4

1.3 Brief overview of cilia__________________________________________ 5

1.4 Medulloblastoma______________________________________________ 5

1.4.1 Overview, subtypes, and demographics__________________________________ 5

1.4.2 The SHH Subtype__________________________________________________ 7

1.4.3 PTCH1/Ptch1 in MB, BCC, and Gorlin syndrome___________________________ 8

1.4.4 Activating and antagonizing the oncogene SMO/Smo________________________ 8

1.4.5 Mouse models of MB______________________________________________ 10

1.5 Dual roles of cilia in oncogenic signaling___________________________ 10

1.6 Cerebellar development________________________________________ 12

1.7 Shh signaling and neural tube patterning_________________________ 13

1.7.1 Neural tube development and patterning________________________________ 13

1.7.2 Shh ligand and its receptors__________________________________________ 16

1.7.3 Smo, the obligate transducer of Shh signaling_____________________________ 17

1.7.4 Gli processing___________________________________________________ 20

1.7.5 SuFu, PKA, and Kif7 are critical regulators of GliR and GliA__________________ 21

1.8 Cilia_______________________________________________________ 23

1.8.1 Cilia are more than just axonemes______________________________________ 23

1.8.2 Human ciliopathies emphasize the importance of cilia in human development_____ 24

1.8.3 Vertebrate Shh signaling occurs at the cilium_____________________________ 24

1.8.4 IFT mutants connect cilia transport and Shh signaling_______________________ 25

1.8.5 Other proteins localized to cilia regulate Shh signaling______________________ 26

1.9 Arl13b and Shh signaling_______________________________________ 27

1.9.1 Discovery and characterization of Arl13b________________________________ 27

1.9.2 Arl13b is a regulator of neuronal identity in the developing neural tube__________ 28

1.9.3 Arl13b is an atypical GTPase_________________________________________ 29

1.9.4 Arl13b regulates ciliogenesis and trafficking in the primary cilium______________ 29

1.9.5 Cerebellar hypoplasia is a consequence of human ARL13B mutations____________ 31

1.10 Summary and objectives______________________________________ 31

Chapter 2 Materials & Methods _____________________________________ 43

2.1 Cell culture_________________________________________________ 44

2.1.1 General cell maintenance____________________________________________ 44

2.1.2 Cloning________________________________________________________ 44

2.1.3 Creation of stable lines_____________________________________________ 44

2.1.4 Generation of Shh-conditioned media (Shh CM)__________________________ 45

2.1.5 Cre treatment and Smo agonists/antagonists______________________________ 46

2.1.6 KD of hARL13B in MB cell lines_____________________________________ 46

2.1.7 Colony forming assay______________________________________________ 47

2.1.8 Propagation of adenovirus___________________________________________ 47

2.1.9 Lentivirus production and infection____________________________________ 49

2.2 qPCR_____________________________________________________ 50

2.3 Mouse work________________________________________________ 51

2.3.1 Mouse lines_____________________________________________________ 51

2.3.2 Genotyping by PCR_______________________________________________ 52

2.3.3 Tamoxifen treatment_______________________________________________ 52

2.3.4 Embryo dissection________________________________________________ 53

2.3.5 Clearing and cardiac perfusion________________________________________ 53

2.3.6 MB isolation and culturing___________________________________________ 53

2.3.7 Terminal blood collection via cardiac puncture for RNA-seq__________________ 54

2.4 Immunofluorescence and immunohistochemistry___________________ 54

2.4.1 Paraffin embedding and sectioning____________________________________ 54

2.4.2 Hemotoxylin and eosin staining_______________________________________ 55

2.4.3 Immunofluorescence_______________________________________________ 55

2.4.4 Proliferation markers_______________________________________________ 56

2.4.5 Antibodies used__________________________________________________ 56

2.5 Western blotting_____________________________________________ 56

2.5.1 Cell and tissue lysis________________________________________________ 56

2.5.2 Gels, transfer, blotting______________________________________________ 57

2.6 Image analysis______________________________________________ 57

2.7 Statistics___________________________________________________ 58

Chapter 3 Temporal deletion of Arl13b reveals that a mispatterned neural tube corrects cell fate over time _________________________________________67

3.1 Summary___________________________________________________ 68

3.2 Abstract____________________________________________________ 68

3.3 Introduction_________________________________________________ 69

3.4 Materials and Methods_________________________________________ 71

3.5 Results______________________________________________________ 74

3.5.1 Modulation of Shh activity level and regulation of Smo localization_____________ 74

3.5.2 Neural progenitors are sensitive to changes in Shh activity at E9.5, but not at E10.5__________________________________________________________________76

3.5.3 Rescue of neural tube patterning over time in Arl13bΔE9.5 embryos demonstrates an active role for low-level Shh activity ________________________________________77

3.5.4 Recovery of patterning is complete____________________________________ 79

3.5.5 The recovery of patterning in Arl13bΔE9.25 and Arl13bΔE9.5 neural tubes is Gli3 dependent____________________________________________________________80

3.6 Discussion_________________________________________________ 81

3.6.1 The initial role of GliA as an instructive signal____________________________ 82

3.6.2 Recovery of patterning_____________________________________________ 82

3.6.3 The role of Gli3 in neural patterning___________________________________ 84

Chapter 4 Identifying a cancer-suppressing modifier __________________ 98

4.1 Summary_________________________________________________ 99

4.2 Introduction_______________________________________________ 99

4.3 Results___________________________________________________ 100

4.3.1 Generation of SAMg and SAMc mice__________________________________ 100

4.3.2 Generation of SAMe mice__________________________________________ 101

4.3.3 SAMe mice no longer develop MB at the expected rate____________________ 101

4.3.4 Identifying the genetic backgrounds combined in SAMe mice________________ 102

4.3.5 Mapping the genetic modifier via low-density SNP mapping_________________ 103

4.3.6 Mapping the genetic modifier via whole genome sequencing and RNAseq_______ 104

4.4 Discussion__________________________________________________ 105

Chapter 5 Disruption of the ciliary GTPase Arl13b suppresses Sonic Hedgehog overactivation and inhibits medulloblastoma formation __________________113

5.1 Abstract___________________________________________________ 114

5.2 Introduction________________________________________________ 114

5.3.1 Generation of SmoWT-GFP and SmoA1-GFP stably expressing cell lines________ 118

5.3.2 The loss of Arl13b reduces Shh pathway output regardless of SmoA1_________ 118

5.3.3 Knockdown of ARL13B reduces Shh signaling in human MB cells lines________ 119

5.3.4 Knockdown of Arl13b reduces proliferation of mouse MB cells_______________ 120

5.3.5 Loss of Arl13b in the developing cerebellum inhibits MB formation___________ 121

5.3.6 Postnatal function of Arl13b________________________________________ 122

5.4 Discussion________________________________________________ 123

Chapter 6 Discussion and Perspectives _____________________________ 138

6.1 Summaries________________________________________________ 139

6.2 Arl13b and Shh signaling_____________________________________ 139

6.2.1 Arl13b, Inpp5e, and PDEs__________________________________________ 140

6.2.2. Arl13b and Smo_________________________________________________ 142

6.2.3 Other possibilities for Arl13b's mechanism of action______________________ 144

6.3 Arl13b and MB______________________________________________ 144

6.3.1 Efficacy of Arl13b deletion in extant tumors_____________________________ 144

6.3.2 Safety considerations of disrupting ARL13B in human patients______________ 146

6.3.3 Developing an anti-Arl13b therapy____________________________________ 147

6.4 Final conclusions____________________________________________ 148

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