ii A furin/MMP-14 proteolytic cascade generates Vasculostatin-40 from extracellular BAI1: Implications for angiogenesis and tumorigenesis Open Access

Cork, Sarah M (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/rb68xc11m?locale=en
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Abstract


Vascular integrity or function is compromised in many neurological diseases,
underscoring the importance of elucidating fundamental mechanisms of brain
angioregulation. Notably, the exuberant vascular proliferation of glioblastoma multiforme
(GBM), the most common brain malignancy in adults, significantly contributes to its rapid,
aggressive growth and dismal prognosis. Improved understanding of brain-specific
angiogenesis may thus prove a source of therapeutic potential for GBM, and the seven-
transmembrane receptor Brain angiogenesis inhibitor 1 (BAI1) presents a promising target
for such investigation. A potent anti-tumor agent in in vivo models, we show that expression of
BAI1 is reduced in human gliomas in proportion to tumor severity, and higher levels of BAI1
are significantly associated with prolonged survival in astrocytoma but not GBM patients.
While the forces driving BAI1 loss in gliomas are incompletely understood, BAI1 mRNA
expression is inversely correlated with levels of pathological GBM vasculature, implicating an
anti-angiogenic mechanism. Previous work has shown that the anti-tumor effect of BAI1
derives from the proteolysis and secretion of its large extracellular domain, named
Vasculostatin-120 (Vstat120), a potent angiogenesis inhibitor. Here we show that
extracellular BAI1 is proteolytically cleaved to generate an abundant 40-kilodalton product
named Vasculostatin-40 (Vstat40), and identify the site of processing. We demonstrate that
cleavage of Vstat40 from BAI1 depends on a two-step process of activation. In the final step
of the cascade, Vstat40 is directly processed from extracellular BAI1 by matrix
metalloproteinase 14 (MMP-14), and this proteolytic event is greatly facilitated by the
upstream activity of proprotein convertases, particularly furin. Vstat40 is observed to inhibit
endothelial cell migration and cord formation in a CD36-dependent manner, and interferes
with vascularization in vivo. While expression of Vstat40 in an LN229-derived inducible
orthotopic xenograft mouse model does not significantly increase subject survival, tumors
expressing Vstat40 show significantly inhibited vascularization as well as altered tumor cell
morphology and reduced levels of apoptosis markers. Taken together, these findings
represent an important advance in our understanding of BAI1 as both a therapeutic molecule
and a receptor with diverse roles in neurobiology.

Table of Contents

CONTENTS

Chapter 1. Introduction. 1

1.1. An overview of glioma molecular biology 2

1.2. Clinical development of novel glioma therapies 5

1.3. Overview of angiogenesis and pathology of tumor-associated vasculature 7

1.4. Anti-angiogenic therapy in the clinic: promise and pitfalls 11

1.5. Endogenous angiogenesis inhibitors: generation and significance 14

1.6. An overview of the BAI family proteins 16

1.7. BAI family gene regulation and expression in development 17

1.8. Proteolysis of BAI proteins and functional implications 19

1.9. Intracellular signaling and C-terminal binding partners 23

1.10. A role for BAIs in neurovascular function and disease 26

1.11. Conclusions. 28

Chapter 2. Mechanisms of Vstat40 proteolYSiS. 35

Introduction. 36

Materials and Methods. 38

Results. 43

N-terminal BAI1 is processed to yield a major 40-kilodalton secreted fragment. 43

Identification of the site of Vstat40 processing. 44

Inhibition of furin activity reduces Vstat40 processing. 45

A furin-activated metalloproteinase processes extracellular BAI1 into Vstat40. 47

MMP-14 directly processes extracellular BAI1 into Vstat40. 48

Discussion. 50

Chapter 3. Vstat40 is a potent regulator of angiogenesis. 63

Introduction. 64

Materials and Methods. 68

Results. 74

Vstat40 inhibits endothelial cell migration in culture in a CD36-dependent manner. 74

The Vstats interfere with endothelial cord formation. 76

Vstat40 inhibits angiogenesis in vivo. 76

Vstat40 interferes with VEGF-induced vascular permeability. 79

Discussion. 79

Chapter 4. Vstat40 and BAI1 IN gliomagenesis. 91

Introduction. 92

Materials/Methods. 94

Results. 97

BAI1 is lost in gliomas in proportion to tumor severity. 97

BAI1 expression is associated with a survival benefit to astrocytoma but not GBM patients. 97

Elevated BAI1 is associated with reduced angiogenesis and necrosis. 98

Patterns of angioregulator expression in brain tumors. 99

Association of BAI1 expression with that of other cell adhesion molecules. 100

Validation of the in vivo tumor model. 100

Vstat40 does not promote survival in an in vivo tumorigenesis experiment. 101

Vstat40-expressing tumors have reduced angiogenesis and differential levels of cleaved caspase 3. 103

Discussion. 105

Chapter 5. TOWARDS the Further CHARACterization of extracellular bai1. 126

Introduction. 127

Materials and Methods. 130

Results. 134

Expression of GST-Vstat40 in inclusion bodies. 134

Expression of GST-Vstat120 in inclusion bodies. 135

Initial results of purifying dialyzed GST-Vstat40 with reduced glutathione and thrombin. 135

Factors complicating GST-Vstat120 purification. 136

Identification of a desired antigenic region in the hormone-binding domain of human BAI1. 137

Characterization of antiserum against the hormone-binding domain. 138

Detection of BAI1 fragments in CM. 138

Discussion. 140

Chapter 6. Discussion and future directions. 152

6.1. Summary of results. 154

6.2. Conclusions. 159

6.3. Future directions. 160

References. 164

Appendices. List of protocols used and REAGENTS generated for experiments. 199

Protocols. 200

Plasmid constructs. 248

Cell lines. 287

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