The Role of Growth Differentiation Factor 15 in Breast Cancer Open Access

Peake, Bridgette Franshar (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/cn69m500p?locale=en
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Abstract

Abstract

The Role of Growth Differentiation Factor 15 in Breast Cancer




This dissertation seeks to uncover the mechanism by which the inflammatory cytokine
growth differentiation factor 15 (GDF15) drives tumorigenesis in breast cancer. GDF15
has been implicated as a biomarker of poor prognosis and decreased survival in several
malignancies. However, few studies have examined GDF15 expression, signaling, or
function in breast cancer. GDF15 is significantly elevated in many tumor types in
association with epithelial mesenchymal transition (EMT), drug resistance, and
progressive disease. Our studies demonstrate that GDF15 overexpression is associated
with upregulation of mitogenic signaling pathways (TGF-ß, MAPK, and FOXM1).
GDF15 expression is further associated with high tumor grade, ER-negativity, and HER2
overexpression in patients with breast cancer. Stable overexpression of GDF15 resulted
in upregulation of mesenchymal markers and transcription factors, including FOXM1,
and increased cellular invasion. GDF15 stable clones and breast cancer cells stimulated
with recombinant human GDF15 demonstrated activation of insulin-like growth factor-1
receptor (IGF-1R). Pharmacologic inhibition of IGF-1R blocked GDF15-mediated
FoxM1 up-regulation, EMT, and invasion. Knockdown of FOXM1 dramatically reduced
GDF15-mediated invasion and suppressed expression of matrix metalloproteinases
MMP2 and MMP9. Finally, knockdown of GDF15 significantly inhibited invasion of
HER2-overexpressing and triple-negative breast cancers. Preliminary optimization
studies using DZGDF15NPs for targeted GF15 mRNA neutralization show therapeutic
promise in the future. The collective findings presented herein provide further insight
into the mechanism by which GDF15 drives cancer progression, and suggest potential
therapeutic strategies that could benefit patients with breast cancer.


Table of Contents

Page
TABLE OF CONTENTS
Chapter 1. Introduction and Background ........................................................................... 1
1. Breast cancer .................................................................................................................... 2
2. Subtypes of breast cancer ................................................................................................. 4
i. Luminal type breast cancer ................................................................................................ 4
ii. Basal-like and triple-negative breast cancer (TNBC) ....................................................... 5
iii. HER2 and breast cancer .................................................................................................... 6
3. HER2-targeted therapies ................................................................................................ 10
i. Trastuzumab .................................................................................................................... 10
ii. Lapatanib ......................................................................................................................... 11
iii. Other HER2 targeting agents .......................................................................................... 14
iv. Resistance to HER2-targeted therapy .............................................................................. 15
4. FOXM1 signaling and breast Cancer ............................................................................. 18
i. FOXM1 a downstreat target of HER2 signaling…………………………………..……...18
ii. Targeting FOXM1 in resistant breast cancer………..…………………….……………..25
5. Scope of Dissertation…………………………………………………………………….....26
Chapter 2. Materials and Methods............................................................................................ 29
1. General Methods ............................................................................................................ 30
i. Reagents .......................................................................................................................... 30
ii. Bacterial transformation…………………………………………………………………31
iii. Cell Culture ..................................................................................................................... 32
iv. Creation of trastuzumab-resistant clones………………………………………………...32
v. Creation of stable GDF15-over-expressing clones……………………………………....32
vi. Creation of stable GDF15-knockdown clones…………………………………………..33
vii. Creation of stable IGF-1R-knockdown clones………………………………………….33
viii.DNA/siRNA transfection…………………………………………………………….…34
ix. Cell cycle analysis………………………………………………………………………..34
x. Immunofluorescence ....................................................................................................... 35
xi. Immunohistochemistry .................................................................................................... 36
xii. Spheroid migration assays………………………………………………………………37
xiii. Invasion chamber assays…………………………………………………………….....37
xiv. Stimulation experiments………………………………………………………………..38
xv. Quantitative RT-PCR analyses ........................................................................................ 38
xvi. Western Blotting .............................................................................................................. 39
xvii. ELISA ......................................................................................................................... 40
xviii. Statistics………………………………………………………………………………40
2. GDF15 DNAzyme assays .............................................................................................. 41
i. Synthesis of gold nanoparticles ....................................................................................... 41
ii. Preparationof deoxyribozyme-functionalized gold nanoparticles ................................... 42
iii. Confirmation of DNAzyme (DZNP) catalytic activity ................................................... 43
iv. Cell culture and DZNP-mediated gene knockdown ........................................................ 43
Chapter 3. Characterization of GDF-15 in Breast Cancer ..................................................... 45
1. Introduction .................................................................................................................... 46
2. Results ............................................................................................................................ 58
i. Receptors and signaling pathways associated with overexpression of GDF15 in breast 6cancer ................................................................................................................................... 58
ii. Sustained MEK/ERK signaling is associated with GDF15 overexpression ................... 64
iii. GDF15 and TGF-β1 co-localize with TGF-βRII ............................................................ 67
iv. TGF-βR kinase inhibition blocks IGF-1R signaling in stable GDF15 overexpressing cells...67
3. Discussion ...................................................................................................................... 70
Chapter 4. Correlative Analyses of Growth Differentiation Factor 15 in Breast Cancer ... 73
1. Introduction .................................................................................................................... 74 Results ............................................................................................................................ 78
i. GDF15 exprssion ocrrelates with ER-negative and HER2-positive status ..................... 78
ii. GDF15 expression corretlates with reduced overall survival ......................................... 85
iii. GDF15 transcript and secretion are increased in an in vitro model of HER2-positive/ERnegative breast cancer ........................................................................................ 85
iv. GDF15 and TGFβ1 colocalize with TGFβRII in HCC 1954 breast cancer cells .......... 88
v. GDF15 pharmacologic inhibition has no effect on downstream signaling in HCC1954 breast cancer cells ................................................................................................................ 89
3. Discussion ...................................................................................................................... 95
Chapter 5. GDF15 Mediates EMT and invasion of breast cancer through IGF-1R/FoxM1 signaling…………………………………………………………………………………………98
1. Introduction .................................................................................................................... 99
2. Results .......................................................................................................................... 100
i. GD15 overexpression alters cell cycle profiles and induces EMT in breast cancer ... 100
ii. GD15 activates IGF-1R signaling ................................................................................ 110
iii. Matrix metalloproteinases MMP2 and MMP9 are upregulated in GDF15- overexpressing breast cancer cells ............................................................................... 110
iv. Knockdown of FoxM1 or GDF15 inhibits invasion of GDF15-overexpressing cells 111
3. Discussion .................................................................................................................... 111
Chapter 6. Targeting GDF15 in Breast Cancer Using Catalytic Deoxyribozyme Down-Regulation……………………………………………………………………………………...121
1. Introduction .................................................................................................................. 122
2. Results .......................................................................................................................... 123
i. GDF15 gene silencing in breast cancer ........................................................................ 126
ii. DNAzyme catalytic activity ......................................................................................... 130
iii. GDF15 DNAzyme-HER2 aptamer cellular uptake ...................................................... 132
iv. DZNP regulation of GDF15 gene expression .............................................................. 132
3. Discussion .................................................................................................................... 136
Chapter 7. Conclusions ............................................................................................................. 138
i. Summary & Conclusions ............................................................................................... 139
ii. Clinical implications ...................................................................................................... 145
iii. Future Directions ........................................................................................................... 149
Chapter 8. References...……………………………………………………………………….152

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