Exploring Focal Adhesion Kinase (FAK)

278 Pages·2017·16.01 MB·English

Exploring Focal Adhesion
Kinase (FAK) as a therapeutic
target in triple negative breast
cancer
A thesis submitted in accordance with the

conditions governing candidates for the degree
of:
Doctor of Philosophy

Samuel Rhys Jones

September 2017

Breast Cancer Molecular Pharmacology Research group
Cardiff University School of Pharmacy and Pharmaceutical
Sciences
Cardiff University

Cardiff, Wales
I
DECLARATION
This work has not been submitted in substance for any other degree or award at this or any
other university or place of learning, nor is being submitted concurrently in candidature for
any degree or other award.
Signed ………… ……………………… (candidate) Date …………28/09/2017…….………

STATEMENT 1
This thesis is being submitted in partial fulfillment of the requirements for the degree of
………(insert MCh, MD, MPhil, PhD etc, as appropriate)
Signed ………… ………….…………… (candidate) Date …………28/09/2017…….………

STATEMENT 2
This thesis is the result of my own independent work/investigation, except where otherwise
stated, and the thesis has not been edited by a third party beyond what is permitted by
Cardiff University’s Policy on the Use of Third Party Editors by Research Degree Students.
Other sources are acknowledged by explicit references. The views expressed are my own.
Signed ………… ……….……….…… (candidate) Date …………28/09/2017…….………

STATEMENT 3
I hereby give consent for my thesis, if accepted, to be available online in the University’s
Open Access repository and for inter-library loan, and for the title and summary to be made
available to outside organisations.
Signed ……… ………………..…..….. (candidate) Date …………28/09/2017…….………
II
Abstract
Triple-negative breast cancer (TNBC) is an aggressive cancer subtype that displays poor
prognosis due to a lack of targeted therapies and an early pattern of spread. Recent evidence
also points to a correlation between cancer “stem-like” cells (CSCs) and the inherently
aggressive traits of TNBC. As such, targeting signalling pathways which support metastasis
and CSC populations may represent an important therapeutic strategy to treat these
tumours and improve current patient outcomes. The non-receptor tyrosine kinase FAK (focal
adhesion kinase) is known to influence cancer development and progression, with its
upregulation common in several cancer types. Indeed, FAK can regulate various cellular
processes associated with disease progression including, cell survival, migration and stem-
like behaviours. Therefore, we explored the influence of FAK in TNBC cells and the potential
benefit of its targeting in this subtype.
Whilst assessment of FAK expression and activity across a panel of breast cancer cell lines
representing the major clinical subtypes revealed that FAK was not significantly augmented
in MDA-MB-231 cells (model of TNBC) versus other models, MDA-MB-231 cells displayed a
FAK-dependent migratory and invasive behaviour involving FAK-mediated activation of Akt
and STAT3. These observations also extended to cell proliferation, with pharmacological or
genetic FAK inhibition leading to perturbed cell cycle progression. Whilst FAK did not
contribute to the maintenance of a CSC subpopulation, FAK was necessary for their anoikis
resistance and mammosphere self-renewal, the latter regulated by FAK-dependent
modulation of β-catenin through GSK3β and interaction between the FAK/Wnt signalling
pathways.
Using computational modelling, several novel FAK inhibitors that targeted FAK kinase-
independent scaffolding function were developed and screened to assess in vitro efficacy in
TNBC cells. Of all 45 compounds, ‘compound 9’ showed significantly improved ability to
reduce cell proliferation and migration versus the lead compound, chloropyramine. As
expected, this agent had little effect of FAK phosphorylation but appeared to reduce focal-
adhesion targeting and subcellular distribution of FAK and significantly inhibited cell
migration and growth. Our in vitro data support a case for FAK as a promising therapeutic
target in TNBC with an ability to suppress both tumorigenic events and those associated with
metastasis. Targeting FAK scaffolding function may represent a novel approach to developing
FAK inhibitors that can circumvent resistance traditionally associated with kinase inhibitors.

III
Acknowledgments
Foremost, I wish to extend my sincerest gratitude to Dr. Stephen Hiscox for allowing me to
undertake this PhD and for the constant support, guidance and encouragement he has
provided at all stages throughout the last four years. I also wish to thank my co-supervisors
Professor Andrew Westwell and Professor Andrea Brancale for their contributions to this
project.
I am also grateful for the insightful comments provided by Dr Julia Gee and Dr Kathryn Taylor
which have aided in the completion of this study. Importantly, I owe a great deal to the past
and present members of the Breast Cancer Molecular Pharmacology group, whose teaching
and assistance (particularly in technical matters) was so valuable in the completion of this
work. I would also like to thank them for being such a warm and friendly group, creating an
amazing and conductive atmosphere in which to undertake this project, as well as keeping
me topped up with coffee, cake and crosswords.
A special thanks must also be extended to Dr Richard Clarkson, Dr Rhiannon French and
members of the Clarkson Group for their assistance and support during my final year of
undergraduate study. I feel that without this time in the group, I may not have continued to
take on a PhD.
Many thanks must also go to all the friends I have made over the last four years, with whom
I have had some amazing times which served to keep us all balanced. Importantly, thanks
must go to my parents and family for their continuous encouragement and support, and the
keen interest they have shown in my work.
Lastly, I would like to acknowledge my girlfriend Sammie for being a rock and keeping me
sane through all the tough times, as well as tolerating me (and my constant mess)
throughout.
IV
Contents
List of Abbreviations…………………………………………………………………………………………………………. 11
Chapter 1 – General Introduction………………………………………………………………………………….. 15
1.1. Breast cancer…………………………………………………………………………………………………………….. 16
1.1.1. Incidence and Mortality………………………………………………………………………………………….. 16
1.1.2. Clinical Subtypes…………………………………………………………………………………………………….. 16
1.1.2.1. Molecular stratification of breast cancer subtypes………………………………………………. 17
1.1.3. Therapeutic strategies for breast cancer…………………………………………………………………. 19
1.1.3.1. Targeting ER in breast cancer therapy………………………………………………………………….. 19
1.1.3.1.1. Selective oestrogen receptor modulators (SERMs)……………………………………………. 20
1.1.3.1.2. Selective oestrogen receptor degraders (SERDs)……………………………………………….. 20
1.1.3.1.3. Aromatase inhibitors (AIs)………………………………………………………………………………… 21
1.1.3.2. Targeting HER2 in breast cancer therapy……………………………………………………………… 21
1.1.3. Triple-negative breast cancer………………………………………………………………………………….. 23

1.2. Focal Adhesion Kinase (FAK)………………………………………………………………………………………. 25
1.2.1. FAK discovery and structure……………………………………………………………………………………. 25
1.2.2. FAK activity and signalling……………………………………………………………………………………….. 27
1.2.2.1. FERM domain regulation of activity……………………………………………………………………… 29
1.2.2.2. Central kinase domain…………………………………………………………………………………………. 30
1.2.2.3. C-terminus FAT region…………………………………………………………………………………………. 30
1.2.3. FAK family members……………………………………………………………………………………………….. 31
1.2.4. Functional relevance of FAK signalling…………………………………………………………………….. 32

1.3. FAK and cancer………………………………………………………………………………………………………..… 34
1.3.1. FAK and tumour initiation – Proliferation and survival…………………………………………….. 35
1.3.2. FAK and metastatic processes – Migration and invasion………………………………………….. 37
V
1.3.3. FAK and the “cancer stem cell” hypothesis………………………………………………………………. 39
1.3.4. Chemotherapy and the influence of FAK…………………………………………………………………. 41

1.4. Aims and Objectives…………………………………………………………………………………………………… 42

Chapter 2 – Methodology……………………………………………………………………………………………… 43
2.1. Materials and reagents………………………………………………………………………………………………. 44
2.1.1. Key drugs and reagents used…………………………………………………………………………………… 44
2.1.2. Other materials………………………………………………………………………………………………………. 45

2.2. Cell Culture………………………………………………………………………………………………………………… 48
2.3. Optimisation of RNAi transfection……………………………………………………………………………… 49

2.4. Functional Cell Assays………………………………………………………………………………………………… 51
2.4.1. Cell proliferation assays………………………………………………………………………………………….. 51
2.4.1.1. MTT assay……………………………………………………………………………………………….. 51
2.4.1.2. Cell counting assay………………………………………………………………………………….. 51
2.4.2. In vitro migration/invasion assays…………………………………………………………………………… 52
2.4.2.1. Wound healing assay………………………………………………………………………………. 52
2.4.2.2. Boyden Chamber assay……………………………………………………………………………. 52
2.4.3. In vitro mammosphere assays…………………………………………………………………………………. 53

2.5. Antibody-based detection…………………………………………………………………………………………. 54
2.5.1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western
Blotting……………………………………………………………………………………………………………………………. 54
2.5.1.1. Cell lysis for protein extraction………………………………………………………………… 54
2.5.1.2. Protein Quantification and sample preparation………………………………………. 55
2.5.1.3 SDS-PAGE…………………………………………………………………………………………………. 55
VI
2.5.1.4. Densitometry………………………………………………………………………………………….. 59
2.5.1.5. Stripping and re-probing blots…………………………………………………………………. 59
2.5.2. Immunofluorescent staining…………………………………………………………………………………… 59
2.5.3. Flow cytometry analysis………………………………………………………………………………………….. 60
2.5.3.1. Evaluation of cell cycle distribution…………………………………………………………. 60
2.5.3.2. Assessment of stem-like subpopulations…………………………………………………. 60
2.5.3.2.1. CD24/44 analysis……………………………………………………………………… 60
2.5.3.2.2. ALDH analysis………………………………………………………………………….. 62
2.5.4. Immunocytochemistry (ICC) evaluation of Ki67………………………………………………………. 62

2.6. Statistical analysis of data………………………………………………………………………………………….. 63

Chapter 3 – Characterisation of FAK expression and activity in breast cancer subtypes…. 64
3.1. Introduction………………………………………………………………………………………………………………. 65
3.1.1. Aims and objectives……………………………………………………………………………………. 66
3.2. Analysis of FAK expression with breast cancer patient outcome………………………………… 67
3.3. Exploration of FAK and Py2 in breast cancer models…………………………………………………… 72
3.4. Analysis of subcellular distribution of FAK in breast cancer cell models……………………… 75
3.5. Analysis of proliferative and migratory capacity of breast cancer cell models…………….. 80
3.6. Breast cancer cell models exhibit differential susceptibility to the FAK inhibitor PF271
………………………………………………………………………………………………………………………………………… 83
3.7. The effects of FAK inhibition on cellular proliferation…………………………………………………. 86
3.8. The effects of FAK inhibition on TNBC migration and invasion……………………………………. 88
3.9. Discussion…………………………………………………………………………………………………………………. 91
3.9.1. Conclusions……………………………………………………………………………………………….. 97

VII
Chapter 4 – Mechanistic exploration of FAK in TNBC proliferation and migration………….. 98
4.1. Introduction………………………………………………………………………………………………………………. 99
4.1.1. Aims and objectives……………………………………………………………………………………. 99
4.2. Assessment of the contribution of FAK scaffold functions to proliferation………………… 101
4.2.1. Optimisation of FAK siRNA……………………………………………………………………….. 101
4.2.2. Effects of FAK depletion on MDA-MB-231 cell proliferation………………………. 103
4.3. Cell growth inhibition is accompanied by a decrease in proliferative marker Ki67…….. 105
4.4. PF271-inhibited proliferation results from perturbed cell cycle proliferation…………… 107
4.5. Inhibiting FAK kinase function significantly impairs downstream pathways associated
with proliferation and cell cycle………………………………………………………………………………………. 108
4.6. Evaluation of FAK scaffold function in regulating TNBC migration and invasion………… 111
4.7. Assessing the role of the FAK-family member Pyk2 in regulating migration………………. 115
4.8. Evaluation of changes in cell morphology and FAK distribution in migrating cells following
FAK inhibition…………………………………………………………………………………………………………………. 119
4.9. Evaluation of signalling changes following PF271-mediated FAK inhibition in migrating
cells……………………………………………………………………………………………………………………………..… 132
4.10. Validation of FAK-mediated regulation of cell proliferation and migration in a second
model of TNBC……………………………………………………………………………………………………………….. 135
4.11. Inhibition or suppression of FAK has limited effects on MDA-MB-468 proliferation… 139
4.12. Levels of Ki67 are unaltered following FAK inhibition in MDA-MB-468 cells…………… 140
4.13. FAK inhibition does not affect MDA-MB-468 cell migration……………………………………. 142
4.14. Exploration of FAK-regulated signalling pathways in MDA-MB-468 cells…………………. 144
4.15. siRNA-mediated PTEN suppression in MDA-MB-231 cells………………………………………. 147
4.15.1. PTEN-siRNA optimisation……………………………………………………………………….. 147
4.15.2. Functional assessment of PF271 in PTEN suppressed MDA-MB-231 cells… 149
4.16. Exploration of signalling changes in PTEN-suppressed cells following PF271 treatment
………………………………………………………………………………………………………………………………………. 151
4.17 Discussion………………………………………………………………………………………………………………. 157
4.17.1. Conclusions……………………………………………………………………………………………. 173
VIII
Chapter 5 – Mechanistic evaluation of FAK in TNBC cancer stem cells (CSCs)………………… 174
5.1. Introduction……………………………………………………………………………………………………………. 175
5.1.1. Aims and objectives…………………………………………………………………………………. 176
5.2. Evaluation of FAK contribution to maintenance of the CSC population……………………… 177
5.3. Functional assessment of the role of FAK in stem-like characteristics……………………… 182
5.4. Analysis of CSC signalling changes in response to FAK inhibition……………………………….. 187
5.5. Exploration of the FAK/Wnt relationship in CSC self-renewal……………………………………. 190
5.6. Discussion……………………………………………………………………………………………………………….. 192
5.6.1. Conclusions……………………………………………………………………………………………… 198

Chapter 6 – Development and screening of novel FAK inhibitors for TNBC……………………. 199
6.1. Introduction……………………………………………………………………………………………………………. 200
6.1.1. Chloropyramine as a potential starting point for novel FAK-compound
development…………………………………………………………………………………………………….. 200
6.1.2. Outline of design processes used for development of novel FAK inhibitors in
TNBC…………………………………………………………………………………………………………………. 202
6.1.3. Aims and objectives…………………………………………………………………………………. 203
6.2. Screening of novel anti-FAK agents against MDA-MB-231 cell proliferation……………… 204
6.3. Several Batch 1 compounds can significantly inhibit wound healing in triple-negative
cells……………………………………………………………………………………………………………………………….. 208
6.4. Compound 9 inhibits TNBC cell proliferation and migration…………………………………….. 210
6.5. Compound 9 does not alter the activity and expression of FAK and associated
kinases…………………………………………………………………………………………………………………………… 212
6.6. Treatment with compound 9 causes changes in cellular morphology and cellular
localisation of active FAK………………………………………………………………………………………………… 214
6.7. Discussion……………………………………………………………………………………………………………….. 221
6.7.1. Conclusions……………………………………………………………………………………………… 227

IX
Chapter 7 – General discussion……………………………………………………………………………………. 228
7.1. TNBC……………………………………………………………………………………………………………………..… 229

7.2. Relevance of FAK as a therapeutic target in cancer…………………………………………………… 229
7.2.1. Targeting FAK as a strategy to reduce tumour metastasis………………………… 231
7.2.1.1. FAK as an antimigratory target………………………………………………………………. 231
7.2.1.2. FAK as an anti-CSC target………………………………………………………………………. 233
7.2.2. Clinical challenges of FAK inhibition………………………………………………………………………. 238

7.3. Therapeutic strategies for anti-cancer targeting of FAK……………………………………………. 239
7.3.1. Current FAK-targeting therapeutics………………………………………………………………………. 239
7.3.2. Strategies for targeting FAK…………………………………………………………………………………… 241
7.3.2.1. Targeting scaffold function and kinase activity………………………………………. 241
7.3.3. Alternative strategies for targeting FAK…………………………………………………………………. 243
7.3.3.1. Impairing the FAK FERM-domain…………………………………………………………… 243
7.3.3.2. Targeting the FAT-region and subcellular localisation……………………………. 244
7.3.4. FAK inhibitors in combination treatment strategies………………………………………………. 246

7.4. General study limitations and considerations…………………………………………………………… 248

7.5. Final Conclusions……………………………………………………………………………………………………… 249

Chapter 8 – Appendix…………………………………………………………………………………………………. 250

Chapter 9 – References……………………………………………………………………………………………….. 252
X