UNIVERSITY OF CALGARY
The Role of PIK3CA in Cisplatin Resistance of Cervical Cancer
by
Cole Merry
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
GRADUATE PROGRAM IN MEDICAL SCIENCE
CALGARY, ALBERTA
JUNE, 2016
© Cole Merry 2016
Abstract
Cervical cancer is a significant public health issue. A recent study (McIntyre et al; 2013)
suggested that phosphatidyl inositol 3 kinase (PI3K) catalytic subunit α (PIK3CA) is an
important marker for the prognosis of cervical cancer patients treated with chemoradiotherapy
(CRT), with PIK3CA-mutant tumour bearing patients having poorer survival than PIK3CA-wt
patients. Activating mutations in PIK3CA promote increased PI3K signalling, and tumorigenesis
in vivo. I investigated the role that E545K, identified by McIntyre et al (2013) as the most
common mutation in PIK3CA in cervical cancer, may play in radiation and cisplatin resistance of
cervical cancer cell lines. This study indicated a potential role of PIK3CA in cisplatin resistance,
although cisplatin resistance was not a universal characteristic of cells expressing PIK3CAE545K, and was not reversed with the use of the PI3K inhibitor GDC-0941. The cisplatin
resistant cell line showed sensitivity to GDC-0941, suggesting PI3K inhibitors as an alternative
to cisplatin.
ii
Acknowledgements
I would like to acknowledge my Supervisor Dr. Susan Lees-Miller for providing me the
opportunity to work in her lab, and for being an excellent mentor and advocate throughout my
studies. A special thank you to my supervisory committee members Dr. Corinne Doll and Dr.
Savraj Grewal for taking the time to provide me guidance and feedback for my research, and to
Dr. Randal Johnston and Dr. Guido van Marle for participating in my evaluation. I would also
like to acknowledge Dr. Arjumand Wani for being a great support and excellent teammate
throughout my studies, I enjoyed our time working together. Thank you to Dr. Aru Narendran
and Manika Perinpan for providing the inhibitors, reagents, and assistance for my drug screens.
Thank you Dr. Karen Kopciuk for assisting me with my statistical analyses. Thank you to all of
my other past and present fellow researchers, associates, and technicians in the Lees-Miller lab
for your support: Dr. Pauline Douglas, Ruiqiong Ye, Shujuan Fang, YapingYu, Shilpa Salgia,
Carin Pihl, Dr. Chen Wang, Dr. Edward Bartlett, Dr. Lucy Swift, Dr. Sarvan Kumar, Nick Jette,
Maryam Ataeian, Cortt Piett, Elias Saba, Daniel Moussienko, and Michelle Love, thank you all
for making the Lees-Miller lab a great place to do research. Lastly, I would like to thank my
Family: David, Melanie, Ross and Brittany Merry, and Corinna Liu for supporting me
throughout my education, for always believing in me, and for pushing me to do my greatest.
iii
Dedication
I would like to dedicate this work to a brilliant young mind who was taken too soon.
“Good friends are hard to find, harder to leave, & impossible to forget.”
Rest in Peace
Max Milaney
24/02/1993 - 11/02/2016
iv
Table of Contents
Abstract ............................................................................................................................... ii
Acknowledgements ............................................................................................................ iii
Dedication .......................................................................................................................... iv
Table of Contents .................................................................................................................v
List of Tables ................................................................................................................... viii
List of Figures and Illustrations ......................................................................................... ix
List of Symbols, Abbreviations and Nomenclature ........................................................... xi
CHAPTER ONE: INTRODUCTION ..................................................................................1
1.1 Cervical cancer ..........................................................................................................1
1.1.1 Cervical cancer is a significant global health problem. .....................................1
1.1.2 Human papilloma virus (HPV) plays a large role in the development of cervical
cancer. ................................................................................................................1
1.2 Radiation and cisplatin are currently the standard treatments for locally advanced
cervical cancer, given concurrently as chemoradiotherapy (CRT). .........................5
1.2.1 DNA damage from Ionizing Radiation (IR) ......................................................5
1.2.2 Cisplatin damage to DNA .................................................................................5
1.2.3 Cisplatin combined with radiation shows increased efficacy in killing cancer
cells. ...................................................................................................................6
1.3 Several repair pathways are involved in the repair of cisplatin and IR damage to cells.
..................................................................................................................................9
1.3.1 DNA damage response to IR involves PI3K related kinases. ...........................9
1.3.2 NHEJ .................................................................................................................9
1.3.3 HR......................................................................................................................9
1.3.4 NER (global NER and TCR) ...........................................................................10
1.3.5 Fanconi Anemia pathway ................................................................................11
1.3.6 MMR ...............................................................................................................13
1.3.7 BER .................................................................................................................15
1.4 Phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks) ......................................16
1.4.1 PI3Ks are a large group of receptor associated lipid kinases that regulate
important growth, metabolism and survival pathways. ...................................16
1.4.2 Active AKT has many nuclear and cytosolic targets that control cell growth, cell
death, and metabolism. ....................................................................................19
1.4.3 Mutations leading to up-regulation of PI3K/AKT/mTOR signalling have been
implicated in tumour progression and resistance to drug therapy. ..................21
1.4.4 Recent clinical studies have identified PI3K as a novel prognostic marker for
clinical outcome in cervical cancer patients. ...................................................22
1.4.5 PI3K inhibitors have been used in vitro and in vivo to increase sensitivity to
radiation and cisplatin. .....................................................................................24
1.5 Cisplatin resistance ..................................................................................................24
1.5.1 Several mechanisms of cisplatin resistance have been identified. ..................24
1.5.2 Potential mechanistic links between PI3K and cisplatin or radiation resistance25
1.6 Hypothesis ...............................................................................................................26
1.7 Objectives ................................................................................................................27
v
CHAPTER TWO: MATERIALS AND METHODS ........................................................29
2.1 Cell Culture ..............................................................................................................29
2.2 Cell Proliferation Assay ...........................................................................................30
2.3 Cell Lysis .................................................................................................................30
2.4 SDS-PAGE and Western Blots of WCEs ................................................................31
2.5 Ionizing Radiation (IR) and Drug Treatment of Cells .............................................33
2.6 Clonogenic Survival Assays (CSAs) .......................................................................36
2.7 Alamar Blue® (AB) Cell Survival Assays ..............................................................37
CHAPTER THREE: RESULTS ........................................................................................38
3.1 Cervical cancer cell lines vary in growth rate. PIK3CA-E545K status does not
necessarily correlate with faster growth rate in a cell line panel. ..........................38
3.2 Expression of major PI3K markers showed higher levels of p85 and lower levels of
PTEN in PIK3CA-E545K cell lines. AKT expression was lowest in HeLa cells.39
3.3 Cervical cancer cell lines with PIK3CA-E545K do not show significant resistance to
IR compared with PIK3CA-wt cervical cell lines. .................................................40
3.4 Cisplatin treatment showed no effect on cervical cancer cell lines initially using both
clonogenic survival and Alamar Blue® cell survival assays. ................................40
3.5 Clinical cisplatin and 154 mM NaCl 1X PBS stock cisplatin have similar effects on
cell survival while DMSO stock cisplatin has a relatively little effect on cell survival.
154 mM NaCl 1X PBS stock is an effective vehicle for maintaining cytotoxicity of
cisplatin using both Alamar Blue® assays and Clonogenic Survival Assays. ......45
3.6 CaSki cells are relatively more resistant to cisplatin than other cervical cancer cell lines
using clonogenic survival assays. ..........................................................................45
3.7 Serum starvation results in decreased RSK pT359 in HeLa cells and increased RSK
pT359 in CaSki cells. .............................................................................................49
3.8 GDC-0941 treatment results in decreased AKT pS473 in CaSki cells but not HeLa
cells. .......................................................................................................................49
3.9 GDC-0941 combined treatment does not increase CaSki cell sensitivity to cisplatin.
CaSki, but not HeLa cells, are sensitive to GDC-0941 as a single agent. .............53
CHAPTER FOUR: DISCUSSION ....................................................................................55
4.1 Conclusions ..............................................................................................................55
4.1.1 Cervical cancer cell lines display diverse growth characteristics and expression
profiles. ............................................................................................................55
4.1.2 PIK3CA-E545K cells are not radiation resistant.............................................56
4.1.3 CaSki cells are resistant to cisplatin. ...............................................................57
4.1.4 Serum starvation had distinct effects on CaSki and HeLa cells, and did not
reliably reduce levels of phospho-AKT or phospho-RSK. ..............................58
4.1.5 GDC-0941 treatment decreased phospho-AKT levels in CaSki cells. ............59
4.1.6 GDC-0941 is an effective single agent against CaSki cells, but does not reverse
cisplatin resistance in CaSki cells. ...................................................................60
4.1.7 Other findings and future experiments ............................................................61
4.2 Working hypothesis .................................................................................................61
4.3 Considerations .........................................................................................................62
4.3.1 Survival Assays ...............................................................................................62
4.3.2 Drug formulation .............................................................................................63
vi
4.3.3 Western Blots ..................................................................................................63
4.3.4 Antibodies........................................................................................................64
4.4 PI3K inhibitors as therapeutics ................................................................................65
REFERENCES ..................................................................................................................67
APPENDIX A: SUPPLEMENTARY FIGURES ..............................................................72
APPENDIX B: FIGURE FROM MANUSCRIPT BY DR. ARJUMAND WANI
(PUBLICATION PENDING) ...................................................................................75
APPENDIX C: COPYRIGHT AND PERMISSIONS ......................................................77
vii
List of Tables
Table 1. Conditions and parameters of antibodies used for Western blot analysis in this study. . 33
Table 2. PI3K inhibitors screened for this study........................................................................... 35
Table 3. Information on HeLa, SiHa, CaSki, and Me-180 cell lines obtained from ATCC ......... 38
viii
List of Figures and Illustrations
Figure 1. Suppression of p53 and pRB by HPV E6 and E7 proteins, respectively. ...................... 4
Figure 2. Repair of DSBs by NHEJ or HR. ................................................................................... 7
Figure 3. Cisplatin structure and mechanism of adduct and crosslink damage to DNA. ............... 8
Figure 4. NER pathway through TCR or GGR............................................................................. 11
Figure 5. FA pathway core components and DDR proteins that interact with FA pathway to
repair Interstrand crosslinks. ................................................................................................. 12
Figure 6. Mechanism of MMR. .................................................................................................... 14
Figure 7. BER pathway. ................................................................................................................ 16
Figure 8. Domains of p110, p85, AKT and p90 RSK and kinase activity of p110 on PIP2 ....... 18
Figure 9. Downstream signalling of PI3K. AKT is a major regulator of PI3K signalling. ......... 20
Figure 10. Prognosis for 5-year survival of cervical cancer patients based on PIK3CA status
(wt or mutant). ...................................................................................................................... 23
Figure 11. Growth rates of cervical cell lines. .............................................................................. 39
Figure 12A. Expression levels of PI3K markers in Hela (H), CaSki (C), SiHa (S), and Me180 (M) cell lines. ................................................................................................................. 41
Figure 12B. Expression levels of PI3K markers in Hela (H), CaSki (C), SiHa (S), and Me180 (M) cell lines. ................................................................................................................. 42
Figure 13. Clonogenic survival of HeLa (wt PIK3CA), SiHa (wt PIK3CA), CaSki (wt/E545K
PIK3CA), and Me-180 (wt/E545K PIK3CA) cervical cancer cell lines following
treatment with ionizing radiation. ......................................................................................... 43
Figure 14. Effect of cisplatin formulated in DMSO on HeLa (wt PIK3CA), SiHa (wt
PIK3CA) and CaSki (wt/E545K PIK3CA) viability using A) Clonogenic survival assay
and B) Alamar Blue® assay. ................................................................................................. 44
Figure 15. Effect of clinical, 154mM saline, and DMSO formulated cisplatin on HeLa cell
viability using Alamar Blue® viability assays. .................................................................... 46
Figure 16. Viability of SiHa (wt PIK3CA), CaSki (wt/E545K PIK3CA), and Me-180
(wt/E545K PIK3CA) treated with cisplatin formulated in 1X PBS (154 mM NaCl) using
Alamar Blue® viability assay. .............................................................................................. 47
ix
Figure 17. Clonogenic survival of HeLa (wt PIK3CA), SiHa (wt PIK3CA), CaSki (wt/E545K
PIK3CA), and Me-180 (wt/E545K PIK3CA) cervical cancer cell lines following
cisplatin treatment (154 mM NaCl 1xPBS) (24 hrs). ........................................................... 48
Figure 18. Effect of serum starvation on AKT and RSK activity of HeLa (H) and CaSki (C)
cells. ...................................................................................................................................... 50
Figure 19. Treatment of CaSki cells with GDC-0941. ................................................................. 51
Figure 20. Treatment of HeLa cells with GDC-0941. .................................................................. 52
Figure 21. Clonogenic survival of HeLa (wt PIK3CA), and CaSki (wt/E545K PIK3CA),
cervical cancer cell lines following combined treatment with cisplatin and GDC-0941...... 54
x
List of Symbols, Abbreviations and Nomenclature
Symbol:
E6-AP
AB
ABD
AKT
APE
APLF
ATM
ATR
BAD
BER
BRCA1/2
CDK
CHK2
CRT
CSA
DDR
DMSO
DNA-PKcs
DSB
dsDNA
EBD
EGFR
eIF4F
ERCC1
ERK
FA
FAAP24
FANC
FBS
FEN1
FOXO
GPCR
GSH
HD
HPV
HR
IGFR
IP
IR
MAPK
MDM2
MT
MMR
Definition:
E6 activating protein
Alamar blue®
Adapter binding domain
Protein kinase B
Apurinic/apyrimidinic endonuclease
Aprataxin and PNKP-like factor
Ataxia telangiectasia mutated
Ataxia telangiectasia and Rad3-related
Bcl-2-associated death promoter
Base excision repair
Breast cancer type associated protein 1&2
Cyclin-dependant kinase
Checkpoint kinase 2
Chemoradiotherapy
Clonogenic survival assay
DNA damage response
Dimethyl sulfoxide
DNA-dependant protein kinase catalytic subunit
Double strand break
Double stranded DNA
ERK binding domain
Epidermal growth factor receptor
Eukaryotic initiation factor 4F (complex)
Excision repair cross-complementation group 1
Extracellular regulated kinase
Fanconi anemia
FA core complex associated protein 24
Fanconi anemia, complementation group proteins
Fetal Bovine Serum
Flap endonuclease 1
Forkhead box O proteins
G-protein coupled receptor
Glutathione
Hydrophobic domain
Human papilloma virus
Homologous recombination
Insulin-like growth factor receptor
Immunoprecipitation
Ionizing radiation
Mitogen-activated protein kinase
Murine double minute 2 homolog
Metallothionein
Mismatch repair
xi
mTOR
mTORC1
mTORC2
NER
NHEJ
p53
PBS
PCNA
PE
PDK1
PH
PI3K
PIKK
PIP2
PIP3
PNKP
pRb
PTEN
RAS
RBD
RFC
RPA
RpS6
RTK
SDS
SH2/3
SSB
ssDNA
SCC
TCR
TFIIH
TSC1/2
UV
WCE
WRN
XLF
XP
XRCC4
Mammalian target of rapamycin
Mammalian target of rapamycin complex 1
Mammalian target of rapamycin complex 2
Nucleotide excision repair
Non-homologous end joining
Tumour protein p53
Phosphate buffered saline
Proliferating cell nuclear antigen
Plating efficiency
Phosphoinositide-dependent protein kinase-1
Pleckstrin homology (domain)
Phosphatidylinositol-4,5-bisphosphate 3-kinase
PI3K-related protein kinase (PIKKs plural)
Phosphatidylinositol-4,5-bisphosphate
Phosphatidylinositol-3,4,5-triphosphate
Polynucleotide kinase 3’-phosphatase
Retinoblastoma protein
Phosphatase and tensin homolog
Rat sarcoma protein
Ras binding domain
Replication factor C (complex)
Replication protein A
Ribosomal protein S6
Receptor tyrosine kinase
Sodium dodecyl sulphate
Src homology 2/3 domains
Single strand break
Single stranded DNA
Squamous cell carcinoma
Transcription-coupled repair
Transcription factor II human
Tuberous sclerosis complex 1/2
Ultraviolet radiation
Whole cell extract
Werner syndrome ATP-dependent helicase
XRCC4-like factor
Xeroderma pigmentosum proteins
X-ray repair cross-complementing protein 4
xii
Chapter One: Introduction
1.1 Cervical cancer
1.1.1 Cervical cancer is a significant global health problem.
Cervical cancer is the third most common newly diagnosed cancer in women worldwide 1. Each
year, over 500,000 new cases of cervical cancer are diagnosed, and 270,000 patients die from the
disease 1. Progress has been made in the way of preventative action through early screening and
human papillomavirus (HPV) vaccinations. However, developing countries and poorer
communities may lack adequate early cytology screening and/or sexual health education and
often don’t have good access to HPV vaccinations. Approximately 80% of cervical cancers are
squamous cell carcinoma (SCC), while the remaining 20% are mostly adenocarcinoma with a
small number of other rare subtypes 2. Surgery and chemoradiotherapy (CRT) are the current
standard treatments, with typical CRT consisting of concurrent cisplatin and radiation treatments.
Challenges with patients whose tumours do not respond well to CRT and a lack of options in the
way of new treatments stress the importance of finding novel regimens to improve outcomes for
women diagnosed with cervical cancer. Thus, cervical cancer continues to be a significant health
problem that impacts women and their families worldwide, and will continue to be so in the
foreseeable future.
1.1.2 Human papilloma virus (HPV) plays a large role in the development of cervical cancer.
HPV has been detected in over 90% of cervical cancers 3–5. High-risk HPV sub-types for
cervical cancer include 16, 18, 31, and 45. The HPV genome encodes for 8 genes in total, two of
which, E6 and E7, are crucial for cellular transformation leading to cancer. E5 also has
1
oncogenic properties but hasn’t been as well studied in human HPV carcinogenesis6. Expressed
by high-risk virus types, E6 and E7 negatively regulate regulatory proteins of the DNA damage
response including cell cycle control protein p53 and retinoblastoma protein (pRb), respectively
(Figure 1). p53 acts as a major tumour suppressor gene and response element to cellular stress
such as DNA damage, and can regulate genes that control the cell cycle, apoptosis, and
senescence. Cellular p53 levels are kept low by continuous targeting to the proteasome by E3
ubiquitin ligase murine double minute 2 homolog (MDM2) (Figure 1)7. Upon exposure to stress
or damage, p53 is phosphorylated by several different protein kinases that increase its half-life,
making p53 more stable. Stabilized p53 acts as a transcription factor and promotes decreased
transcription of proliferative genes, while promoting expression of genes such as p21 that control
cell cycle progression. While p53 does function diversely, its major function appears to be the
repression of genes that promote cell growth, allowing a healthy cell that has undergone stress or
damage to halt growth and repair DNA damage before committing to replication8. Therefore,
p53 plays a major role in preserving the cell’s genomic integrity and is one of the most common
genes mutated in cancer. In HPV-associated cervical cancer, E6 functions by forming a complex
with p53 and E6 activating protein (E6-AP), an E3 ubiquitin ligase responsible for p53
ubiquitination, both expressed by the host cell (Figure 1A). This complex promotes silencing of
p53, and blocks p53-induced cell cycle arrest, and initiation of apoptosis. On the other hand, Rb
is a repressor of E2F-1 transcription factors that regulate genes such as cyclin E that are involved
in the G1/S transition in the cell cycle9. In HPV-associated cervical cancer, E7 is able to bind to
the pocket region of Rb and prevent association with E2F-1 (Figure 1B). Normally, cyclindependant kinases (CDKs) inactivate Rb by phosphorylation to promote cell cycle progression.
2
Both p53 and Rb are tumour suppressors that prevent inappropriate growth of damaged cells. By
down regulating p53 and Rb, the HPV virus stably replicates, and prevents the cell from
initiating apoptosis or arresting the cell cycle to stop viral replication10. HPV is a type I double
stranded DNA (dsDNA) virus capable of integrating, and in some cases, replicating itself inside
the host cell genome. A recent study mapped the integration sites of HPV in cervical cancer
patient genomes and confirmed earlier findings that HPV integrates in a targeted fashion into
fragile sites of the genome11, leading to a population of stably infected HPV-positive cells
remaining in the patient tissue that develop transformative properties that promote cancer
growth. Although up to 80% of women have at least one HPV infection of the genital mucosa
during their life time, the majority of infected people are able to clear HPV infections within two
years while a small number of infected women (10-20%) fail to clear the virus12. While a strong
humoral immune response to HPV is linked to lower incidence of persistent HPV infection and
squamous lesions, the precise mechanism of immune recognition and clearance of HPV in
unimmunized women is not fully understood13. Cervical cell lines used in our study all contain
integrated genome(s) of HPV (Table 3).
3
Figure 1. Suppression of p53 and pRB by HPV E6 and E7 proteins, respectively.
Under normal conditions, p53 is targeted for ubiquination by MDM2 (A), and pRb is controlled
by cell cycle kinases (B). E6 expression leads to abrogation of p53 cell cycle control, and E7
expression leads to activation of E2F genes that promote cell cycle progression. Curved black
arrows indicate ubiquitination.
4
1.2 Radiation and cisplatin are currently the standard treatments for locally advanced
cervical cancer, given concurrently as chemoradiotherapy (CRT).
1.2.1 DNA damage from Ionizing Radiation (IR)
Ionizing radiation (IR) causes damage to cells by directly interacting with the sugar phosphate
DNA backbone and/or by damaging or altering bases. Indirect DNA damage by IR also occurs
by the ionization of water in the cell to create reactive oxygen species and cause oxidative DNA
damage. The most toxic forms of IR-induced DNA damage are double strand breaks (DSBs)
(see Figure 2). DSBs typically arise from two single strand breaks (SSBs) on opposite strands
within a short distance from one another. DSBs initiate large signalling responses and structural
changes in the DNA and surrounding chromatin, and become repaired by non-homologous endjoining (NHEJ) throughout the cell cycle and HR during G2 and S phases14 (see sections 1.3.2
&1.3.3).
1.2.2 Cisplatin damage to DNA
Cisplatin is a potent DNA damaging agent commonly used in cancer treatment along with
radiation for cancers such as cervical cancer. Cisplatin, carboplatin, and oxaliplatin are all
platinum compounds consisting of a platinum ion with four covalent ligands. Cisplatin contains
two amine (NH2) groups and two cis-chloride (Cl) groups that make cisplatin cytotoxic (Figure
3). Cisplatin can enter the cell through passive diffusion, facilitated diffusion, or active
transport. When cisplatin enters the cell, lower intracellular levels of chloride ions lead to
displacement of Cl- ligands by water, and this activates cisplatin. Water and cisplatin form a
hydrated complex that is highly reactive towards purine bases of DNA to form platinum-DNA
mono-adducts (Figure 3). These mono-adducts form further intra- and inter-strand crosslinks,
and platinum protein adducts that impede access of DNA enzymes and interfere with replication
5
and transcription. Major types of repair for cisplatin-induced damage include nucleotide
excision repair (NER), homologous recombination (HR), and the Fanconi Anemia (FA)
pathways15. The mismatch repair (MMR) pathway is also activated and has been shown to
mediate cisplatin cytotoxicity along with base excision repair (BER) 15,16 (See sections 1.3.31.3.7).
1.2.3 Cisplatin combined with radiation shows increased efficacy in killing cancer cells.
Cisplatin and IR are repaired by distinct pathways mentioned above and outlined below, but
share the HR repair pathway. Using cisplatin in conjunction with radiation increases the
cytotoxicity of radiation, possibly due to impairing the NHEJ pathway or creating more complex
damage that overwhelms the cell’s capacity to repair17–19.
6
Figure 2. Repair of DSBs by NHEJ or HR.
In NHEJ, Ku70/80 and DNA-PKcs play key roles. The steps are: detection of the break,
modification of the broken ends, and ligation of the break together. NHEJ is error prone repair,
and can occur throughout the cell cycle. BRCA1/2, MRN complex, and RAD 51 are crucial in
mediating HR. The steps of HR are resection of the ends around the break, homology search,
repair using template DNA, and resolution of Holliday junctions. HR is error free, and limited to
S and G2 phase. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews
Molecular Cell Biology, Chowdhury et al20 ©2013. http://www.nature.com/nrm/index.html
7
Figure 3. Cisplatin structure and mechanism of adduct and crosslink damage to DNA.
Aquation of cisplatin is a key step in activation of the drug. Aquated cisplatin exerts damage by
binding DNA. The majority of damage arises from intrastrand cross-links between two purine
bases, which are more common than interstrand crosslinks. Reprinted by permission from
Macmillan Publishers Ltd: Nature Reviews Cancer, 21 ©2003.
http://www.nature.com/nrc/index.html
8
1.3 Several repair pathways are involved in the repair of cisplatin and IR damage to cells.
1.3.1 DNA damage response to IR involves PI3K related kinases.
Two key players that initiate downstream pathways and amplify DNA damage response are
ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit
(DNA-PKcs)14. Both proteins belong to the family of phosphatidyl inositol 3 kinase (PI3K)related protein kinases (PIKKs) and share sequence similarity to the lipid kinase, phosphatidyl
inositol 3 kinase (PI3K). Both DNA-PKcs and ATM are important in mediating DSB repair22.
1.3.2 NHEJ
NHEJ is an especially important three-step pathway because it is active throughout the cell cycle,
and repairs the majority of IR-induced DSBs (Figure 2). First, Ku and DNA-PKcs tether and
detect the DSB. Then, Artemis, polynucleotide kinase 3’-phosphatase (PNKP), aprataxin and
PNKP like factor (APLF), and DNA polymerases γ and μ process the end of the breaks to create
ligatable 5’ phosphate and 3’ hydroxyl ends. Lastly, ligation involving X-ray repair crosscomplementing protein 4 (XRCC4), DNA ligase IV, and XRCC4-like factor (XLF) occurs23.
1.3.3 HR
HR is another DDR pathway for repairing cisplatin and IR-induced DSBs (Figure 2). The HR
pathway utilizes homologous DNA templates to rewrite damaged sequences of DNA and is only
possible in the presence of a sister chromatid in S and G2 phases. This highly error-free
mechanism, compared to NHEJ, involves detection of DSBs by the MRN complex containing
MRE11, RAD50, and NBS1, which is important in initiating HR-mediated DSB repair and in
generating single stranded DNA (ssDNA). ssDNA is then detected and bound by replication
protein A (RPA), required for HR24,25. RPA is displaced by BRCA2, allowing Rad51 to bind
9
ssDNA. The latter steps of HR are poorly characterized, however they involve a search for a
homologous template, strand invasion, and synthesis of new DNA from the template. Finally,
Holliday junctions must be resolved to complete the recombination. RAD51, BRCA1, and
BRCA2 are crucial factors in mediating HR.
1.3.4 NER (global NER and TCR)
NER is an important repair pathway for cisplatin adducts that removes bulky lesions from the
DNA such as thymine dimers caused by ultraviolet radiation (UV). Xeroderma pigmentosum
(XP) proteins play a central role in NER and are named after the medical condition that is caused
by mutations occurring in the group of genes encoding for these proteins. XP patients are highly
sensitive to UV and frequently develop skin cancers associated with UV. NER is initiated
through two mechanisms: damage can be sensed by a global response in transcriptionallyinactive genes through XPC and XPE (Global-NER), or by transcription coupled repair (TCR)
through a stalled RNA polymerase and subsequent recognition of the lesion by Cockayne
syndrome (CS) proteins A and B. After the damage recognition step, TCR and global NER
proceed through the same steps. Following damage recognition, XPA and RPA associate at the
lesion with the Transcription factor II Human (TFIIH) complex comprised of many subunits
including seven core subunits, XPB, XPD, p62, p52, p44, p34, and TTDA, and opens the DNA
for access by excision enzymes. XPF-Excision repair cross-complementation group 1 (ERCC1)
complex excises the DNA 5’ towards the lesion, while XPG cuts 3’ towards the lesion, and a
single stranded piece of DNA between 24 and 32 base pairs (bp) long is removed. RPA
stabilizes the ssDNA while PCNA-RFC complex is recruited. Polymerase δ/ε/κ fill in the gap,
and DNA ligase III-XRCC1 seals the DNA backbone (Figure 4).
10
Figure 4. NER pathway through TCR or GGR.
NER can be initiated globally by XPC and XPE, or in actively transcribed genes by CS proteins
A and B (A). Excised section is then filled and ligated through common steps (B). Reprinted by
permission from Macmillan Publishers Ltd: Nature Cell Research, 26 ©2013.
http://www.nature.com/cr/index.html
1.3.5 Fanconi Anemia pathway
The Fanconi Anemia (FA) pathway is important in the repair of interstrand cross-links. The
main activating kinase is ataxia telangiectasia and Rad3-related (ATR). Interstrand cross-links
lead to a stalled replication fork. Fanconi anemia core complex associated protein 24 (FAAP24)
and Fanconi anemia, complementation group M (FANCM) recruit a core complex of proteins
including FANCL (Figure 5). FANCL acts as an E3 ubiquitin ligase that mono-ubiquitinates
FANCD2 and FANCI. The pathways downstream of FANCD2-FANCI are not well
11
characterized, however they involve endonuclease activity which generates a DSB at the site of
the cross-link. HR factors are subsequently required to resolve the DSB27.
Figure 5. FA pathway core components and DDR proteins that interact with FA pathway
to repair Interstrand crosslinks.
The FA core complex recognizes a stalled replication fork and recruits FANCD2 and FANCI.
HR proteins participate in completing the repair of interstrand cross-links during replication.
Reprinted by permission from American Society for Clinical Investigation: The Journal of
Clinical Investigation, Kee and D’Andrea28 ©2012. http://www.jci.org/
12
1.3.6 MMR
MMR is another important pathway in the recognition of cisplatin lesions that repairs two
different types of mismatched lesions (Figure 6). MutS complex (MSH2/MSH6) recognizes
small mismatches 1-2 nucleotides in size, while MutS complex (MSH2/MSH3) recognizes
larger loops in DNA that typically arise during replication. MutL complex (MLH1/PMS2) is
then recruited to the MutS complex. After the hydrolysis of ATP and conformational change,
the MutL-MutS complex can migrate away from the break, making way for exonuclease I (a 5’
to 3’ exonuclease) to remove the lesion. Other proteins required include RFC, PCNA, and RPA.
MutL has been identified as having cryptic endonuclease activity that nicks the newly
synthesized strand either 5’ or 3’ from the lesion29 . The gap is filled by DNA polymerase δ,
following which DNA ligase 1 seals the ends. MMR recognizes cisplatin lesions and, rather than
repair, mediates apoptotic signalling in response to cisplatin damage.
13
Figure 6. Mechanism of MMR.
MutS recognizes the lesion and recruits MutL. MutL has cryptic endonuclease activity and cuts
5’ or 3’ from the lesion. Reprinted by permission from Macmillan Publishers Ltd: Nature
Reviews Molecular Cell Biology, Jiricny30 ©2006. http://www.nature.com/nrm/index.html
14
1.3.7 BER
BER can also recognize cisplatin damage to DNA. Initially, a DNA glycosylase removes the
damaged base (Figure 7). Next, apurinic/apyrimidinic endonuclease (APE) cuts the DNA
backbone and DNA polymerase β adds the correct nucleotide. Finally, DNA ligase I in long
batch BER or DNA ligase III in short batch BER fills in the nick. Long batch BER requires
additional enzymes including Flap endonuclease 1 (FEN1), RPA, PCNA, RFC, and Werner
syndrome ATP-dependent helicase (WRN), and occurs when DNA polymerase β is blocked by a
5’-deoxyribose phosphate (dRP) group. Current literature suggests that like MMR, BER is
important in mediating cisplatin cytotoxicity, and that reduction in BER can contribute to
cisplatin resistance. BER and MMR are thought to compete with productive repair pathways,
leading to non-productive repair of cisplatin lesions, and activation of cell death16.
15
Figure 7. BER pathway.
Reprinted by permission from Taylor and Francis Group: Critical Reviews in Biochemistry and
Molecular Biology, Baute and Depicker31 ©2008.
http://www.tandfonline.com/loi/ibmg20?open=43&repitition=0#vol_43
1.4 Phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks)
1.4.1 PI3Ks are a large group of receptor associated lipid kinases that regulate important
growth, metabolism and survival pathways.
There are three classes of PI3Ks: I, II, and III. In my study, I focused on the class IA PI3Ks,
which is one of two subtypes in class I. The genes PIK3CA, PIK3CB, PIK3CD, and PIK3R1, all
belong to class IA and encode for p110α, p110β, p110δ, and p85α respectively. p110 and p85
subunits form a heterodimer at the cytosolic side of the cell membrane and respond to growth
16
signals from G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and
membrane localized rat sarcoma protein (RAS). For example, epidermal growth factor receptor
(EGFR) and insulin-like growth factor receptor (IGFR) are RTKs that activate PI3K. The src
homology 2 (SH2) domains of the p85 subunit associate with phosphorylated tyrosine (p-Tyr) in
the RTK or RTK substrate and p110. The kinase activity of p110 is lower when its C2 domain is
associated with the iSH2 domain of p85 (Figure 8A). Thus, p85 has a dual role in localizing and
in controlling the kinase activity of p110. Active p110 catalyzes the phosphorylation of the
signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5triphosphate (PIP3) (Figure 8B). Phosphatase and tensin homolog (PTEN) is a major antagonist
of PI3K activity that catalyzes the reverse reaction (Figure 8B) and is frequently mutated in
cancer. PIP3 serves as a docking site, recruiting protein kinase B (AKT) and phosphoinositidedependent protein kinase-1 (PDK1) to the plasma membrane through interactions with their
Pleckstrin homology (PH) domains that have high affinity for PIP3. PDK-1 becomes colocalized with AKT and phosphorylates AKT in its kinase domain at threonine 308 (pT308).
PDK2 then phosphorylates AKT on its C terminal regulatory domain at serine 473 (pS473). The
PDK2 activity of the second phosphorylation event is catalyzed by mTOR in complex 2
(mTORC2) (Figure 9). Recent studies have also demonstrated that DNA-PKcs can perform the
function of PDK2 and that several other kinases could potentially function as PDK232,33. Nonphosphorylated AKT remains inactive and these two phosphorylation events leave AKT in its
active form.
17
Figure 8. Domains of p110, p85, AKT and p90 RSK and kinase activity of p110 on PIP2
A) Important proteins in PI3K signalling with domains, mutation hotspots, and phosphorylation
residues shown. p110 contains: Adapter binding domain (ABD), Ras binding domain (RBD), C2
domain, helical domain, and kinase domain. p85 contains: Src homology 3 (SH3) domain, GAP
domain, and n, inter, and c terminal Src homology 2 (SH2) domains. AKT contains: PH domain,
kinase domain, and an n-terminal hydrophobic domain (HD). RSK contains: n- and c-terminal
kinase domains, and ERK binding domain (EBD) B) PI3K phosphorylates the OH on the 3rd
position of PIP2, PTEN removes this phosphate (OH groups not shown).
18
1.4.2 Active AKT has many nuclear and cytosolic targets that control cell growth, cell death,
and metabolism.
When activated, AKT induces growth and translation, and down regulates apoptotic and cell
cycle inhibitory pathways 34. AKT targets, both nuclear and cytoplasmic, include: forkhead box
O proteins (FOXO) proteins, Bcl-2-associated death promoter (BAD), MDM2, and tuberous
sclerosis complex 2 (TSC2). TSC2 is a negative regulator of mTORC1, which promotes the
phosphorylation of subunits of the eukaryotic initiation factor 4F (eIF4F) complex as well as
ribosomal protein S6 (RpS6), which are essential proteins in initiating cap-dependant translation.
Furthermore, the mitogen-activated protein kinase (MAPKs) cascade is another major growth
signalling pathway commonly upregulated/activated in cancer. This pathway includes p90
RSKs, which act downstream of extracellular regulated kinase (ERK) in the MAPK cascade and
help phosphorylate eIF4F and RpS6, through inhibition of TSC2, and activation of MTORC1.
This serves as a functional point of cross-talk between MAPK and PI3K signalling in regulating
translation (Figure 9).
19
Figure 9. Downstream signalling of PI3K. AKT is a major regulator of PI3K signalling.
PIP3 recruits PDK1 and AKT to the cell membrane, where PDK1 phosphorylates AKT on kinase
domain residue Threonine 308. mTORC2 phosphorylates AKT on C terminal HD domain
residue Serine 473. Both phosphorylation events work to activate AKT which has many
downstream targets. PI3K/AKT converges with MAPK/ERK on TSC1/2 complex to promote
mTORC1 activity, a key mediator of translational effects downstream of both MAPK and PI3K.
AKT promotes growth and survival and suppresses apoptosis. AKT has many other targets
including: FOXO transcription factors, p27, p21, caspase 9, and Bad. Reprinted from
Multidisciplinary Digital Publishing Institute (open source): Cancers, Martelli35 ©2010.
http://www.mdpi.com/journal/cancers
20
1.4.3 Mutations leading to up-regulation of PI3K/AKT/mTOR signalling have been implicated
in tumour progression and resistance to drug therapy.
Class I p110 α/β subunits of PI3K are major upstream regulators of AKT activity. Increased
AKT signalling and associated phenotypic characteristics of increased AKT are present in cancer
cell lines and tumours, along with p110α and p110β gain of function and PTEN loss of function
mutations36–38. Class I p110γ and p110δ subunits facilitate cell-cell interactions/motility and the
maintenance of immune cells39,40. Alterations involving increased PI3K signalling such as
duplicated PIK3CA or PIK3CB and PTEN downregulation/deletion can be found in many types
of cancer. Stronach et al. (2011) demonstrated that, upon cisplatin treatment in cisplatin resistant
ovarian cancer cells, DNA-PKcs and AKT co-localize in the nucleus with a marked increase in
both total and nuclear AKT pS473, reversible by using siRNA to silence DNA-PKcs. This was
not observed in cells sensitive to cisplatin. Additionally, this study showed that the DNA-PKcs
inhibitor NU7026 could be used to reduce AKT pS473 and restore sensitivity to resistant cells by
increasing apoptosis. Kim et al. (2006) found that increased phosphorylated AKT levels
correlated with radiation resistance in a small sample of cervical cancer patients41. In addition,
the downstream targeting of p53 by AKT through mdm2 is well known, and additional studies
have suggested that p53 can participate in the negative regulation of AKT 42,43. It has also been
reported that the action of high-dose cisplatin may partially involve transient downregulation of
E6 oncoprotein and stabilization of p53 44. Upregulated AKT activity could abrogate this
temporary p53 stabilization and reduce the effects of cisplatin, potentially contributing to
resistance.
21
1.4.4 Recent clinical studies have identified PI3K as a novel prognostic marker for clinical
outcome in cervical cancer patients.
A recent study by Dr. Corinne Doll and associates at the Tom Baker Cancer Centre indicated that
mutant PIK3CA is a marker for poor survival outcomes in cervical cancer patients following
CRT45. In the study, 23% of patient samples in a retrospective cervical cancer study contained a
heterozygous mutation in the PIK3CA gene that encodes for the p110α catalytic subunit of PI3K.
Moreover, in 15 out of 19 patient samples, PIK3CA contained a single point mutation (E545K)
within exon 9 of PIK3CA45. Two recent patient studies also reported the prevalence of PIK3CA
mutations in cervical cancer to be 15% and 31.3% and confirmed the presence of PIK3CA
mutations in both adenocarcinoma and SCC11,46. Found in the helical domain of the encoded
protein p110α (Figure 9), E545K is a gain of function mutation that leads to increased PI3K
signalling, and has been demonstrated to be oncogenic in vitro and in vivo37,47,48. PIK3CAE545K is suggested to interrupt the inhibitory interaction of p85 SH2 domains on the kinase
domain of p11049. The kinase activity of p110 was shown to be higher in the E545K mutant than
the wild-type (wt)50. Moreover, multiple studies have shown that the difference in outcomes
between wt and mutant PIK3CA was apparent in early stage (FIGO IB-II) cervical cancer
patients45,46, suggesting that PIK3CA is a factor in poor response to treatment, and a more
aggressive cervical cancer phenotype after CRT. Stage II cervical cancer is locally advanced but
has not yet invaded the pelvic wall or the lower third of the vagina, according to the Canadian
Cancer Society.
22
Figure 10. Prognosis for 5-year survival of cervical cancer patients based on PIK3CA status
(wt or mutant).
A) Mutant PIK3CA patients diagnosed at early stages (stage 1B/II) have much poorer survival
probability for 5 years following CRT compared to wt PIK3CA. B) For patients diagnosed at
later stages, PIK3CA mutation does not have an effect on 5 year survival following CRT.
Reprinted from Gynecologic Oncology: 128 (3). McIntyre et al, PIK3CA mutational status and
overall survival in patients with cervical cancer treated with radical chemoradiotherapy45, Page
418, with permission from Elsevier. http://www.sciencedirect.com/science/journal/00908258
23
1.4.5 PI3K inhibitors have been used in vitro and in vivo to increase sensitivity to radiation
and cisplatin.
Wortmannin, an irreversible PI3K, DNA-PKcs and ATM inhibitor51, has been shown to increase
the efficacy of cisplatin treatment of ovarian cancer xenografts in nude mice 52. The PI3K and
DNA-PKcs inhibitor53 LY294002 can also cause cervical cancer cell lines to be more sensitive to
radiation. In both CaSki and Hela cells, pre-treatment with LY294002 had a synergistic effect
with radiation to reduce clonogenic survival and cell growth54. Herzog et al. (2013)
demonstrated, using mouse xenograft and syngeneic models, that the PI3K/mTOR inhibitor PF04691502 has anti-tumourigenic effects in vivo and that the inhibitor can be effective when
combined with radiation treatment55. These effects exhibited by PF-04691502 correlated with
increased p53 expression six hours after treatment in both wt low p53 expression UM-SCC1
cells and mutant p53 UM-SCC46 cells. Increased p53 expression was also seen in xenograft
immunohistochemistry after treatment with PF-04691502. In addition, many other PI3K
inhibitors are currently being developed or being tested in clinical trials (Table 2)56. AKT
inhibitors such as SC-66 have been used in cervical cell lines to promote cell death and inhibit
mTOR activity and glucose uptake57.
1.5 Cisplatin resistance
1.5.1 Several mechanisms of cisplatin resistance have been identified.
Cisplatin resistance typically falls under 2 broad mechanisms: exclusion of cisplatin from the
cellular environment and enhanced removal of cisplatin lesions from DNA. Cisplatin is excluded
from the cell in the following ways: reduced drug uptake, increased drug efflux, or chemical
inactivation of the drug. Multidrug resistance-associated protein 2, MRP2, encoded by gene
ABCC2, is associated with cisplatin resistance and is an example of active transport involved in
24
cisplatin resistance58. ATP7A and ATP7B are two other P-type ATPase copper transporter genes
that were overexpressed in cisplatin resistant cells. Another mechanism for cisplatin exclusion is
through increased levels of glutathione (GSH) and metallothionein (MT)59,60. GSH and MT are
nucleophilic and have high affinity for cisplatin.
Another broad mechanism of cisplatin resistance is the removal of cisplatin lesions by enhanced
repair. For example, NER is a major repair pathway for cisplatin damage, and upregulation of
NER proteins such as ERCC1, is associated with cisplatin resistance15. Since MMR and BER
mediate cisplatin cytotoxicity, downregulation of MMR genes is associated with cisplatin
resistance16. p53 also plays a crucial role in promoting apoptosis upon cisplatin treatment;
disruption in p53 has also been associated with cisplatin resistance. AKT has been identified as
a factor in suppressing p53-mediated apoptosis in ovarian cancer cells61.
1.5.2 Potential mechanistic links between PI3K and cisplatin or radiation resistance
There are several potential mechanisms by which PI3K enhances resistance to radiation and/or
cisplatin. As outlined above, active AKT has many targets, including p21, Mdm2, Bad, and
caspase-9. AKT is able to phosphorylate p21 and sequester it from the nucleus to prevent cell
cycle inhibition at G1/S. Cisplatin has been shown to decrease levels of E6 and increase levels
of p53 in SiHa62. Sequestration of p21 by active AKT could potentially abrogate the effect of
increased p53. PI3K-AKT can also activate Mdm2, which targets p53 for ubiquitin degradation.
Because cervical cancers express E6 and have suppressed p53 levels, upregulated PI3K-AKT
could further supress p53 and prevent re-induction upon cisplatin treatment. Meanwhile, direct
phosphorylation of pro-caspase 9 and Bad by AKT leads to a reduction in apoptosis. In addition
to the post-translational effects mentioned above, active PI3K/AKT phosphorylates FOXO1,
25
FOXO3, and FOXO4 to prevent transcription of genes that promote apoptosis and cell cycle
arrest63. Recent studies have shown the role of another gain of function mutation PIK3CAH1047R in promoting stem-like multipotency and heterogeneity in tumour development in
mouse breast cancer models64,65. Cancer stem cells (CSCs) are a controversial topic, but studies
have shown a link between multipotency and therapy resistance66,67. Another study of NSCLC
showed that PIK3CA mutations were highly represented in secondary cancers that arose after
prior cancer treatment suggesting a link between PIK3CA and recurrence after therapy68.
Finally, a study of HNSCC tumours indicated that tumours with mutations in PI3K carried twice
as many mutations in known cancer genes including many DDR genes. This group also showed
that multiple mutations within the PI3K pathway correlated strongly with increased tumour
stage69.
1.6 Hypothesis
The general hypothesis guiding this study is that mutant PIK3CA confers poor prognosis for
treatment outcome and long term survival in cervical cancer patients through several different
mechanisms. Because PIK3CA-E545K promotes the active conformation of the p110 kinase
subunit and leads to increased phosphorylation of PIP2 to PIP3, it increases the activation of
AKT, which has many downstream targets. These mechanisms are generally mediated by posttranslational or translational effects caused by the upregulation of PI3K and subsequent AKT
activity, such as altered checkpoint responses leading to inappropriate growth, inhibition of
apoptosis, and potentially increased multi-lineage potential and genomic instability. One or
26
more of these mechanisms serves to promote cisplatin resistance in cervical cancer, and
therefore, targeting PI3K signalling may be useful in overcoming IR and/or drug resistance.
1.7 Objectives
Previous studies have indicated that PI3K, mTOR, and AKT play a role in many different types
of cancer. McIntyre et al (2013) and Wright et al (2014) have demonstrated that a common
hotspot mutation PIK3CA-E545K is found in a significant number of cervical cancer patients and
correlates with poorer overall survival and response to treatment. Many PI3K, mTOR, AKT, and
dual/pan inhibitors are being and undergoing clinical trials (Table 2). Therefore, I planned to
investigate the use of these new classes of drugs in cervical cancer treatment.
The main objective of this study was to investigate the effectiveness of both clinically approved
agents currently in use, cisplatin and IR, as well as new PI3K inhibitors undergoing clinical trials
against a panel of cervical cancer cell lines. My goal was to determine whether or not PI3K
inhibitors are appropriate for further study as a therapeutic agent in treating cervical cancer and
to assess if PI3K inhibitors are more effective as a single agent or in combined therapy with
cisplatin. By using PI3K pathway inhibitors as a supplement or replacement for cervical cancer
treatment in patients with poor survival prognosis, research may be able to improve the outcome
for the subset of patients that harbour PIK3CA mutations and other PI3K pathway alterations or
mutations. I hope the findings in this study will justify further in vitro and in vivo studies to
explore the potential for PI3K inhibition as a viable option in the treatment of cervical cancer and
re-evaluate the suitability of cisplatin as a chemotherapy agent for cervical cancer patients
27
harbouring PIK3CA mutations and other potential biomarkers correlated with poor CRT
response.
Specific aims:
i)
To assess the relative sensitivity of cervical cancer cell lines expressing either
PIK3CA-wt or PIK3CA-E545K to cisplatin and IR, and assess the relative growth rate
and expression levels of PI3K markers in PIK3CA-wt and PIK3CA-E545K cervical
cancer cell lines.
ii)
To assess the activation of downstream AKT signalling pathway in CaSki cells
(PIK3CA-E545K) and HeLa cells (wt PIK3CA-wt) in the absence of mitogenic
stimuli.
iii)
To determine if the PI3K inhibitor GDC-0941 can modulate cisplatin resistance and
suppress the activity of AKT in cervical cancer cell lines.
28
Chapter Two: Materials and Methods
2.1 Cell Culture
Cervical cancer cell lines used in this project were maintained in a humidified incubator at 37°C
and 5% CO2. HeLa cells were cultured in Gibco® Dulbecco’s Modified Eagle Medium
(DMEM) (Thermo Fisher #11995) containing 5% (v/v) Hyclone® Fetal Bovine Serum (FBS)
(GE Healthcare #SH30109.03) and 1% (50U/mL) Penicillin-Streptomycin (PS) (Thermo Fisher
#15070063). SiHa cells were cultured in Minimum Essential Medium (MEM) (Thermo Fisher
#11095) containing 10% (v/v) FBS, 1% MEM Non-Essential Amino Acids (Thermo Fisher
#11140050), 1% (w/v) Sodium Pyruvate (Thermo Fisher #11360070), and 1% PS as above.
CaSki cells were cultured in RPMI Medium 1640 (Thermo Fisher #11875) containing 10% v/v
FBS and 1% PS. Me-180 cells were cultured in McCoy’s 5A (modified) Media (Thermo Fisher
#16600) containing 10% FBS and 1% PS. HeLa cells were confirmed as homozygous PIK3CAwt, and CaSki cells were confirmed as heterozygous PIK3CA-E545K by sequencing. To split
and seed cells, 10x Trypsin-EDTA (Thermo Fisher #15400054) was diluted to 1X in 1X
phosphate buffered saline (PBS) (AMRESCO #0780) (137mM NaCl, 2.7mM KCl, 9.8mM
phosphate buffer). To dissociate cells, 2 mL 1X trypsin was added per 10 cm plate, which were
then incubated at 37°C for 2 minutes for Hela, SiHa and Me-180 cells, and 5 minutes for CaSki
cells. Eight mL fresh media was added to neutralize trypsin, after which cells were aspirated off
the plate and resuspended in fresh media (with serum). Resuspended cells were used for
experiments or reseeded at a lower density to maintain cells in the incubator at 37°C under 5%
CO2 for a maximum of 3 months. After 3 months, fresh cells were seeded from lab stocks stored
in 10% dimethyl sulfoxide solution (DMSO) at -80°C, or in liquid nitrogen for storage past 6
29
months. Quick thawed cells were rinsed with 1X PBS, centrifuged at 2500 RPM (Fisher
Scientific Centrific centrifuge, model 225) for 5 minutes, resuspended, and plated in fresh media.
Thawed cells were given 3 passages (or 7 days) at least to recover before use in experiments.
2.2 Cell Proliferation Assay
HeLa, SiHa, CaSki, and Me-180 cells were seeded at 200,000 cells per 10 cm plate in triplicate.
Cells were grown for 1, 2, 3, 4, and 5 day time points, during which cells were trypsinized and
counted using trypan blue after each time point. Results were collected three times in independent
triplicate experiments. Statistical analysis was carried out by one-way ANOVA analysis on
median surviving fraction values from three separate experiments, using unpaired t-test as posthoc analysis between cell lines. P < 0.05 was taken as statistically significant.
2.3 Cell Lysis
Cells were rinsed with 1X PBS and incubated with 2 mL of 1X trypsin at 37°C. Cells were then
resuspended in media (2X or more volume of media compared to the original volume of trypsin
used) and pelleted by centrifuging at 2500 RPM (Allegra® X-15 Centrifuge by Beckman
Coulter) for 5 minutes at 4°C. Cell pellets were resuspended in ice cold 1X PBS and pelleted at
2500 RPM (Eppendorf 5417R centrifuge) for 5 minutes at 4°C. This step was repeated at least
once. Cell pellets were then resuspended in NETN lysis buffer (150 mM NaCl, 1 mM EDTA, 50
mM Tris-HCl pH 7.5 and 1% (v/v) NP40) with the following protease and phosphatase
inhibitors: 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 200 µM
phenylmethanesulfonyl fluoride (PMSF), and 1µM microcystin, and put on ice for 15 minutes.
30
Cells were sonicated on ice (Fisher Sonic Model 100 Ultrasonic Dismembrator) three times for
five seconds each with ten seconds of rest on ice between each five second pulse, and
centrifuged at 10,000 RPM (Eppendorf 5417R centrifuge) for 10 minutes at 4°C. The soluble
fractions (supernatant) of these whole cell extracts (WCEs) were collected and flash frozen,
along with the pellet in liquid nitrogen for storage at -80°C. Lysate protein concentrations were
determined using the Bio-Rad DC™ Protein Assay with bovine serum albumin (BSA) standard.
2.4 SDS-PAGE and Western Blots of WCEs
Aliquots of 50 µg protein from NETN lysate were prepared in sodium dodecyl sulphate (SDS)
sample buffer (80 mM TRIS-HCl pH 6.8, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 10 %
(v/v) glycerol, and 0.1% (w/v) bromophenol blue), boiled for 5 minutes, and run on 8% or 10%
acrylamide 0.75 mm polyacrylamide gels at 80 V for 25 minutes and 100 V for 1 hour and 40
minutes (8% gels for p110α blots) in SDS-Running Buffer (50 mM Tris, 384 mM glycine, 0.1%
(w/v) SDS, pH 8.3). Gels were transferred to nitrocellulose membrane for 1 hour at 100 V in
electroblot transfer buffer (25 mM Tris, 192 mM glycine, 20 % (v/v) methanol) at room
temperature with an ice pack in the buffer reservoir. Blots were blocked with 10% (w/v) skim
milk in TBS-T (1X TBS = [500 mM NaCl, 20 mM TRIS-HCl, pH 7.5] plus 0.1% (v/v) Tween)
for at least one hour at room temperature. They were then probed with primary antibodies made
up as indicated in Table.1 in 1X TBS-T with 0.1% gelatin and 0.05% sodium azide overnight at
4°C, and with secondary antibodies for 30 minutes or one hour (see Table 1). Blots were
developed with equal volumes of Western Lightning® Plus-ECL oxidizing reagent (Perkin
Elmer #ORT 2755) and Western Lightning® Plus-ECL Enhanced Luminol reagent (Perkin
31
Elmer #ORT2655). Next, they were exposed to Super RX-N Fuji Medical X-ray film (Fujifilm
#47410 19291) and visualized by manually developing and fixing exposed X-ray film.
Quantification of blots was done using ImageJ Version 1.50b where arbitrary expression values
were obtained by taking each band as a fraction of its respective Ku80 loading control or total
protein control in the case of phospho-markers. Relative expression was plotted using these
arbitrary values as follows. In blots from untreated cell lines (Figure 12), the highest expressing
lane was used as the internal control and set at a value of 1 and every other lane in the replicate
was plotted as fold-difference from the internal control. For serum starvation blots (Figure 18),
the arbitrary expression values of the samples from starved cells (0.2% serum for 48 hours) were
plotted as fold difference from the samples from pre-starved cells (2% serum for 48 hours). For
GDC-0941 treatment blots (Figures 19 & 20), the arbitrary expression values of each drug
treatment were plotted as fold-difference compared to vehicle control.
32
Table 1. Conditions and parameters of antibodies used for Western blot analysis in this
study.
Antigen (target) Host species Company
primary
Ku80
pS473 AKT
pT308 AKT
AKT
PTEN
p85
p110α
Mouse
Rabbit
Rabbit
Mouse
Rabbit
Rabbit
Mouse
Catalogue #
(dilution of
primary)
Overnight
Ab3107
1:3000
sc-7985-R
1:1000
sc-16646-R
1:500
sc-5298
1:1000
9188
1:1000
05-212
1:2000
Non commercial 1:1000
(Graupera lab)
Abcam
Santa Cruz
Santa Cruz
Santa Cruz
Cell signalling
Millipore
Mariona
Graupera
Garcia*
Cell signalling
Cell signalling
Cell signalling
Cell signalling
Bio-Rad
(dilution of
secondary)
time
1:3000 30 min
1:3000 1 hr
1:3000 1 hr
1:2000 1 hr
1:2000 1 hr
1:2000 1 hr
1:2000 1 hr
Rabbit
3011
1:1000
1:2000 1 hr
p110β
8753
1:500
1:3000 1 hr
pT359 p90RSK Rabbit
9341
1:500
1:3000 1 hr
pS380 p90RSK Rabbit
Rabbit
9333
1:1000
1:3000 1 hr
p90RSK
Goat
170-6515
Secondary to
rabbit
Goat
Bio-Rad
170-6516
Secondary to
mouse
*Prof. Bart Vanhaesebroeck and Dr. Mariona Graupera (Centre for Cell Signalling, University of
London, UK).
2.5 Ionizing Radiation (IR) and Drug Treatment of Cells
Cells were irradiated using a Gamma cell 1000 Tissue Irradiator (MDS Nordion) irradiator with
a Caesium-137 source at a dosage rate of approximately 3 Gy/minute (1 Gy = 21 seconds). In
preliminary experiments, cisplatin (Sigma-Aldrich #479306) was formulated in 20 mM DMSO
and stored as a stock solution at -20oC. Clinically formulated cisplatin (1 mg/mL [3.3 mM]
cisplatin (Hospira Healthcare Corporation) was obtained from the Tom Baker Cancer Centre
Pharmacy. A stock solution of 1 mM cisplatin in 1X PBS containing 154 mM NaCl was
prepared and used for all cisplatin experiments after it was determined to be the most effective
33
vehicle (Figures 15 & 16). A panel of PI3K inhibitors, kindly provided to us by the lab of Dr.
Aru Narendran, was screened for effects on cell viability and GDC-0941 (Selleckchem® S1065)
was selected for use in the study (Table 2 & Supplementary Figures 1-8). GDC-0941 was kept
as 10 mM stock in DMSO at -80 oC. Drugs were diluted from stock to the final concentrations
using the appropriate cell media.
34
Table 2. PI3K inhibitors screened for this study.
PI3K inhibitor Specificity
IC50 (in vitro)
Clinical trials
TGX-221
p110β, p110δ, p110α
5 nM, 0.1 μM , 5 μM n/a
PIK 75 HCl
p110α, γ, δ, β
n/a
GDC-0941
Pictilisib
p110α/δ
p110β,p110γ
5.8 nM, 76 nM, 0.51
μM , 1.3 μM
3 nM
11 nM, 75 nM
CAL-101
Idelalisib
p110δ,γ,β,α
2.5, 89, 565, 820 nM
Phase II MCL and CLL
LY294002
p110α/δ/β
0.50, 0.57, 0.97 nM
n/a
TG100-115
p110γ/δ
83,235 nM
Phase I myocardial
infarction
Xl-147
p110α/δ/γ/β
39,36,33,383 nM
Phase I (general)
99,166,116,262 nM
I and II multiple including
HNSCC and Squamous
cervical cancer
0.4 nM, 5.4 nM and
1.6 nM
Phase I and II Healthy,
Breast, NSCLC Ovarian,
Colorectal and Endometrial
Cancer
NVP-BKM120 p110α/β/δ/γ
(135)
PF-05212384
(PKI-587)
PI3Kα, PI3Kγ and
mTOR
PF-04691502
PI3K(α/β/δ/γ)/mTOR
1.8 nM/2.1 nM/1.6
nM/1.9 nM and 16
nM
*Information obtained from Selleckchem.com
35
Phase I and II
For NSCLC and Breast
cancer
Phase I (cancer)
2.6 Clonogenic Survival Assays (CSAs)
Cells were trypsinized, counted using trypan blue, seeded, allowed to adhere overnight, and treated
under their respective experimental conditions the next day (IR, drug or control). After IR
treatment, cells were immediately incubated under standard conditions and left undisturbed for the
duration of the experiment. After drug treatments, cells were incubated with fresh media under
standard conditions and left undisturbed for the duration of the experiment. Fourteen days
following the day when the first treatment was given, media was removed and plates were fixed
using a solution containing 3% (v/v) acetic acid, 8% (v/v) methanol, and 89% (v/v) water for 2
minutes. A stain solution consisting of 0.2% crystal violet and 10% (v/v) formalin in PBS was
added and removed after 5 minutes. Plates were rinsed 2X with distilled water, allowed to dry,
and the number of surviving colonies (defined as 50 cells or more) was counted using a ColcountTM
(Oxford Optronix) counting machine and software (version 4.3.5.1). Drugs were diluted into the
media for each cell line and incubated with cells under standard conditions. Cisplatin treatment
was always for 24 hours. In combined GDC-0941 experiments cells were treated first with GDC0941, drug and control plates were removed following 48 hours of treatment and immediately
replaced with fresh media containing cisplatin or control. For all CSA experiments using drugs:
drug and control plates were removed following treatment, rinsed with 1X PBS, and replaced with
fresh media for the remainder of the experiment. The surviving fraction of cells was determined
by dividing the surviving colonies by the number of cells initially plated, and multiplying by the
plating efficiency (PE). PE, which is the expected percentage of untreated cells surviving the
seeding process and forming visible colonies, was determined by dividing the average number of
surviving colonies on control plates by the number of cells seeded on the control plates. Dr. Karen
36
Kopciuk (University of Calgary, Faculty of Medicine) helped us determined the appropriate
statistical methods to analyze the findings in our CSAs and carried out the statistical analysis of
the results in Figure 11. Results from all other CSAs were analyzed using one-way ANOVA with
t-test as post-hoc analysis. P-values were obtained comparing the effect of drug or IR treatment on
individual cell lines at varying doses. For the cisplatin assay in Figure 11, Dunnett’s test was used
as post-hoc analysis in the same manner as the t-test to obtain p-values. P < 0.05 was taken as
statistically significant.
2.7 Alamar Blue® (AB) Cell Survival Assays
Cells were seeded at 20,000 cells per well in 96-well plates in triplicate sets of eight tenfold
dilutions of drugs ranging from 1 pM to 10 μM with matching triplicate vehicle controls. Drugs
were diluted from stock in 1X PBS to make 200 μM stock solutions that were then diluted into
the media to give the appropriate concentration. Cells were incubated at 37°C under 5% CO2
with drugs for 4 days. Five uL AB (Thermo Fisher #DAL1025) was then added to each well
containing cells and incubated for 2 hours. The absorbance/emission values at 560/590 nm of
each well were determined using a SpectraMax® M2e plate reader (VWR). The
absorbance/emission of each drug dilution was divided by the absorbance/emission of the
matching control value set as 100% survival and plotted as percent survival on a linear nonlogarithmic graph.
37
Chapter Three: Results
3.1 Cervical cancer cell lines vary in growth rate. PIK3CA-E545K status does not
necessarily correlate with faster growth rate in a cell line panel.
To study the effect of PIK3CA-E545K, a panel of four cervical cell lines was obtained from
ATCC. Two of the cell lines, CaSki and Me-180, in this panel were heterozygous for PIK3CAE545K, while the other two cell lines, HeLa and SiHa, were PIK3CA-wt (Table 3). Growth rate
analysis (Figure 11) indicated that, at 5 days after seeding, HeLa and Me-180 had proliferated
fastest under normal incubation conditions, significantly faster than both CaSki and SiHa (p <
0.05 and p < 0.01, respectively). At 5 days, CaSki proliferated significantly faster than SiHa (p <
0.05). SiHa showed slowest growth of the four cell lines in the panel 5 days after seeding.
Table 3. Information on HeLa, SiHa, CaSki, and Me-180 cell lines obtained from ATCC
Cell line
HeLa
HPV
type 18
PIK3CA
wt
Origin
Cervix
adenocarcinoma
SiHa
type 16
wt
Cervix
squamous
CaSki
type 16 (highly
amplified) and
possibly 18
E545K
heterozygous
Cervix
squamous
metastasized
(small intestine)
Me-180
type 39
E545K
heterozygous
Cervix
squamous
metastasized
(omentum)
38
Figure 11. Growth rates of cervical cell lines.
Logarithmically growing HeLa, SiHa, CaSki and Me-180 cells were seeded in triplicate at
200,000 cells per 10 cm plate and grown asynchronously under normal incubation conditions.
At 1, 2, 3, 4 and 5 days, cells were trypsinized and counted in triplicate using a hemocytometer
with trypan blue staining. The average of 3 separate experiments with standard deviation is
shown. *p < 0.05, **p < 0.01
3.2 Expression of major PI3K markers showed higher levels of p85 and lower levels of
PTEN in PIK3CA-E545K cell lines. AKT expression was lowest in HeLa cells.
Western blot analysis of the major PI3K proteins showed that p110α and p110β were expressed
in all cell lines, and that relative levels of p110α and p110β varied between experimental
replicates (Figure 12). Me-180 and SiHa expressed higher levels of p110β than HeLa and CaSki.
p85 showed higher expression in CaSki and Me-180, while PTEN was lower in these cell lines
compared to PIK3CA-wt cell lines. AKT levels were determined by quantifying the band which
was closest to the predicted size of 56 kDa (see arrows right of panels Figures 12, 18, 19, &20).
Importantly, pT308 AKT probe showed a band slightly higher than probes for AKT pS473 and
total AKT. AKT was expressed at much lower levels in HeLa compared to SiHa, CaSki, and
39
Me-180. Levels of AKT pS473 were consistently highest in Me-180, and also tended to be
higher in CaSki but varied considerably between replicates.
3.3 Cervical cancer cell lines with PIK3CA-E545K do not show significant resistance to IR
compared with PIK3CA-wt cervical cell lines.
To assess the sensitivity of cervical cell lines in the panel to IR, CSAs were used with 1 Gy, 2
Gy, 4 Gy, and 6 Gy doses of radiation (Figure 13). At a dose of 4 Gy, CaSki was significantly
more resistant than both HeLa and Me-180 (p < 0.05). At 6 Gy, Me-180 showed significantly
higher survival than SiHa (p < 0.01). However, there was no significant difference in dosedependent response between HeLa, Me-180, and CaSki survival following IR treatment.
PIK3CA-E545K cell lines (Me-180 and CaSki) produced larger sized colonies using CSAs.
HeLa cells formed small, dense, uniform sized colonies, while SiHa cells formed small faint
colonies.
3.4 Cisplatin treatment showed no effect on cervical cancer cell lines initially using both
clonogenic survival and Alamar Blue® cell survival assays.
Our initial results suggested that cisplatin, using a maximum dose of 10 µM, was ineffective as a
cytotoxic agent against the cervical cell lines using both AB assays and CSAs (Figure 14). A
paper published during our study showed that using DMSO as a stock solvent for cisplatin
interferes with the toxicity of cisplatin towards several cell lines due to an interaction between
DMSO and cisplatin. Therefore, I tested three cisplatin formulations on the cell lines in my
panel to determine which formulations are appropriate for maintaining cytotoxic activity of
cisplatin.
40
Figure 12A. Expression levels of PI3K markers in Hela (H), CaSki (C), SiHa (S), and Me180 (M) cell lines.
50 µg of protein from asynchronously growing cells was harvested via NETN extraction, run on
SDS PAGE and immunoblotted for PI3K markers shown on the left. Apparent molecular
weights are shown on the right (in kDa). Results from 3 separate experiments are shown. The
predicted position of AKT is indicated by the black arrows. * Indicates position of band actually
quantified.
41
Figure 12B. Expression levels of PI3K markers in Hela (H), CaSki (C), SiHa (S), and Me180 (M) cell lines.
Bands in Figure 12A were normalized to their respective Ku80 loading controls and represented
as a fraction of the cell line expressing the highest level of given marker in each individual
experimental replicate. Phospho-RSK and phospho-AKT markers were quantified by
normalization to total RSK, and total AKT markers respectively. Average and standard
deviation of 3 replicates is shown.
42
Figure 13. Clonogenic survival of HeLa (wt PIK3CA), SiHa (wt PIK3CA), CaSki (wt/E545K
PIK3CA), and Me-180 (wt/E545K PIK3CA) cervical cancer cell lines following treatment
with ionizing radiation.
Cells were seeded overnight at 100, 200, 400, 800, and 1600 cells per 6cm plate (in triplicate) for
the mock, 1 Gy, 2 Gy, 4 Gy, and 6 Gy treatments respectively. Cells were either unirradiated (0)
or irradiated as indicated and cells were incubated at 37°C under 5% CO2. 14 days following IR,
plates were fixed and stained, and surviving colonies were counted. A) Average surviving
fraction from 3 separate experiments, each done in triplicate, average and standard deviation is
shown. B) Representative plates at indicated doses of radiation. *p < 0.05 CaSki to HeLa, CaSki
to Me-180. **p < 0.01 Me-180 to SiHa.
43
Figure 14. Effect of cisplatin formulated in DMSO on HeLa (wt PIK3CA), SiHa (wt
PIK3CA) and CaSki (wt/E545K PIK3CA) viability using A) Clonogenic survival assay and
B) Alamar Blue® assay.
A) Cells were seeded overnight at 100, 200, 400, 800, and 1600 cells per 6cm plate (triplicate)
for DMSO, 0.01 μM, 0.1 μM, 0.5 μM, and 1 μM cisplatin treatments respectively (100, 200, 200,
400, 600 cells per plate, respectively, for SiHa). Cells were incubated for 24 hours with cisplatin
(or DMSO control). After 24 hours, media containing the drug or control was removed and fresh
media was added. 14 days later, cells were fixed and stained using crystal violet, and surviving
colonies were counted. Results were averaged and standard deviation is shown. 1 of 3
experimental replicates is shown. B) Cells were incubated with cisplatin (formulated in DMSO)
at the indicated doses for 4 days. Viability was determined using the Alamar Blue® assay. This
experiment was carried out once in triplicate. Results were averaged and standard deviation is
shown.
44
3.5 Clinical cisplatin and 154 mM NaCl 1X PBS stock cisplatin have similar effects on cell
survival while DMSO stock cisplatin has a relatively little effect on cell survival. 154
mM NaCl 1X PBS stock is an effective vehicle for maintaining cytotoxicity of cisplatin
using both Alamar Blue® assays and Clonogenic Survival Assays.
Using AB assays, the efficacy of cisplatin was tested using three different formulations: clinical,
DMSO stock, and 154 mM NaCl 1X PBS stock. While DMSO formulated cisplatin was
ineffective against HeLa up to a maximum dose of 10 µM, clinical and 154 mM NaCl 1X PBS
formulated cisplatin were both equally effective against HeLa at doses lower than 10 µM (Figure
15). 154 mM NaCl 1X PBS cisplatin was also effective against the remaining cell lines in the
panel (Figure 16). The effect of cisplatin on cell survival using AB assays produced similar
results between cell lines in the panel, with SiHa survival being slightly higher than other cell
lines with a maximum dose of 10 µM. 154 mM NaCl 1X PBS stock cisplatin was used for all
remaining experiments using cisplatin.
3.6 CaSki cells are relatively more resistant to cisplatin than other cervical cancer cell lines
using clonogenic survival assays.
The effect of 154 mM NaCl 1X PBS stock cisplatin was tested on the cervical cancer cell line
panel using CSAs. CaSki displayed relative resistance to cisplatin in comparison with other cell
lines in the panel (Figure 17). Significant values were obtained at 0.5 µM (p <0.05), 1 µM (p <
0.001), and 2 µM (p < 2.0 x 10-16). To further elucidate the role of PIK3CA in cisplatin
resistance, CaSki and HeLa were selected for further study given their difference in sensitivity to
cisplatin and favourable properties for the assays used in this study.
45
46
Figure 15. Effect of clinical, 154mM saline, and DMSO formulated cisplatin on HeLa cell viability using Alamar Blue®
viability assays.
Cells were incubated with cisplatin made up from either DMSO stock, clinical stock, or 154mM NaCl 1XPBS stock and incubated at
37°C under 5% CO2 for 4 days. Viability was then determined using Alamar Blue®. 3 separate experiments were each done in
triplicate. Results were averaged and standard deviation is shown.
47
Figure 16. Viability of SiHa (wt PIK3CA), CaSki (wt/E545K PIK3CA), and Me-180 (wt/E545K PIK3CA) treated with cisplatin
formulated in 1X PBS (154 mM NaCl) using Alamar Blue® viability assay.
Cells were incubated with cisplatin (formulated in Saline PBS) as indicated and incubated at 37°C under 5% CO2 for 4 days. Viability
was then determined using Alamar Blue®. 3 separate experiments were each done in triplicate. Results were averaged and standard
deviation is shown.
Figure 17. Clonogenic survival of HeLa (wt PIK3CA), SiHa (wt PIK3CA), CaSki (wt/E545K
PIK3CA), and Me-180 (wt/E545K PIK3CA) cervical cancer cell lines following cisplatin
treatment (154 mM NaCl 1xPBS) (24 hrs).
Cells were seeded overnight at 100, 200, 400, 800, and 1600 cells per 6 cm plate (in triplicate)
for the mock, 0.1 µM, 0.5 µM, 1.0 µM, and 2.0 µM. Cells were treated as indicated and
incubated at 37°C under 5% CO2 for 24 hours, then washed in 1XPBS and incubated in fresh
media. 14 days following treatment, plates were fixed and stained, and surviving colonies were
counted. A) Average surviving fraction from 3 separate experiments, each done in triplicate with
standard deviation. B) Representative plates at the indicated doses of cisplatin. *p = 0.007
(CaSki to Me-180), p= 0.004 (CaSki to SiHa), p < 0.001 (CaSki to HeLa). **p < 0.001 (CaSki to
all). ***p < 2.0 x 10-16 (CaSki to all).
48
3.7 Serum starvation results in decreased RSK pT359 in HeLa cells and increased RSK
pT359 in CaSki cells.
To assess whether cisplatin resistant CaSki cells have upregulation of PI3K signalling, I assessed
AKT phosphorylation under pre-starved and serum starved conditions. I also assessed the
phosphorylation of p90 RSK, a downstream target of ERK that should be highly regulated by
extracellular growth factors present in serum. The levels of RSK pT359 generally decreased
upon starvation in HeLa cells. However, CaSki cells showed an increase in RSK pT359 levels.
Phospho-AKT levels in both cell lines were inconsistent and didn’t follow a trend with starvation
(Figure 18). In both pre- and post-starved conditions, both phospho-RSK markers were much
higher in HeLa than in CaSki (quantification data not shown).
3.8 GDC-0941 treatment results in decreased AKT pS473 in CaSki cells but not HeLa cells.
To elucidate the effect of GDC-0941 on AKT signalling in PIK3CA-E545K and PIK3CA-wt
cervical cancer cell lines, cells were treated with 1 µM and 2 µM GDC-0941 for 24 hours and 48
hours. Western blot analysis of these samples showed that AKT pT308 varied considerably
upon GDC-0941 treatment in both CaSki (Figure 19) and HeLa (Figure 20) while AKT pS473
tended to be lower after 48 hours of GDC-0941 treatment only in CaSki cells. The degree to
which phospho-AKT changed varied significantly between experiments. RSK pS380 was lower
at 24 hours and 1 µM GDC-0941 in CaSki while RSK pT359 was lower at 48 hours and 2 µM
GDC-0941 in HeLa cells (Figures 19 & 20, quantification not shown).
49
Figure 18. Effect of serum starvation on AKT and RSK activity of HeLa (H) and CaSki (C)
cells.
Asynchronously growing cells were incubated in medium containing 2% serum for 48 hrs (+)
followed by 0.2% serum for 48 hrs (-) with WCEs taken at the end of each 48 hr time point.
WCEs were run on 10% acrylamide SDS-PAGE gels, transferred to nitrocellulose membranes
and immunoblotted for indicated RSK and AKT markers. A) Western blots of AKT and RSK
markers B) quantification of fold-difference in AKT and RSK phosphorylation between prestarved and post-starved HeLa and CaSki cells represented on a logarithmic scale. PhosphoRSK and phospho-AKT markers were quantified by normalization to total RSK, and total AKT
markers respectively. Total AKT and RSK markers were quantified by normalization to Ku80.
* Indicates position of band actually quantified.
50
Figure 19. Treatment of CaSki cells with GDC-0941.
Asynchronously growing CaSki cells were treated with 1 µM GDC-0941 or 2 µM GDC-0941 for
24 or 48 hours. Cell lysates were taken at end of given treatments by NETN cell lysis protocol.
WCEs were run on 10% acrylamide SDS-PAGE gels, transferred to nitrocellulose membranes
and immunoblotted for the indicated RSK and AKT markers. A) Western blots of AKT and RSK
markers at indicated treatments. B) Quantification of fold-difference in AKT pS473 and pT308
phosphorylation between DMSO control and GDC-0941 treatments in CaSki cells represented
on a logarithmic scale. Phospho-AKT markers were quantified by normalization to total AKT.
Total AKT was quantified by normalization to Ku80. * Indicates position of band actually
quantified.
51
Figure 20. Treatment of HeLa cells with GDC-0941.
Asynchronously growing HeLa cells were treated with 1 µM GDC-0941 or 2 µM GDC-0941 for
24 or 48 hours. Cell lysates were taken at end of given treatments by NETN cell lysis protocol.
WCEs were run on 10% acrylamide SDS-PAGE gels, transferred to nitrocellulose membranes
and immunoblotted for the indicated RSK and AKT markers. A) Western blots of AKT and RSK
markers at the indicated treatments. B) Quantification of fold-difference in RSK pT359
phosphorylation between DMSO control and GDC-0941 treatments in HeLa cells represented on
a logarithmic scale. Phospho-AKT markers were quantified by normalization to total AKT.
Total AKT was quantified by normalization to Ku80. * Indicates position of band actually
quantified.
52
3.9 GDC-0941 combined treatment does not increase CaSki cell sensitivity to cisplatin.
CaSki, but not HeLa cells, are sensitive to GDC-0941 as a single agent.
To determine if combining cisplatin with GDC-0941 can overcome cisplatin resistance, I
assessed clonogenic survival of HeLa and CaSki cell lines following a combined treatment of
GDC and cisplatin, comparing to each drug separately. Cisplatin by itself remained effective
towards HeLa, but not towards CaSki. GDC-0941 by itself showed efficacy towards CaSki, but
not HeLa. Combining GDC-0941 with cisplatin showed no additive effects in CaSki, compared
to GDC-0941 only, or in HeLa cells, compared to cisplatin only (Figure 21).
53
Figure 21. Clonogenic survival of HeLa (wt PIK3CA), and CaSki (wt/E545K PIK3CA),
cervical cancer cell lines following combined treatment with cisplatin and GDC-0941.
Cells were seeded at 100, 200, 200, and 400 cells (HeLa) or 200, 400, 400, 800 cells (CaSki) per
6 cm plate (in triplicate) for control (both vehicles), GDC-0941 (plus cisplatin vehicle), cisplatin
(plus GDC-0941vehicle), and combined GDC-0941 and cisplatin, respectively. 24 hours after
seeding, cells were treated with 2µM GDC or control for 48 hrs (incubated at 37°C under 5%
CO2) and immediately afterwards cells were treated with 1 µM cisplatin or control (154 mM
NaCl 1XPBS) for 24 hrs. After cisplatin treatment cells were washed in 1XPBS and incubated in
fresh media. 14 days following initial treatment, plates were fixed and stained, and surviving
colonies were counted. A) Average surviving fraction from 3 separate experiments, each done in
triplicate, with standard deviation shown. B) Representative plates for the indicated treatments in
panel A. *(p < 0.01)
54
Chapter Four: Discussion
4.1 Conclusions
A previous study indicated a significant clinical relationship between PIK3CA mutations in
cervical cancer patients and poor response to standard CRT45. In particular, cervical cancer
patients with heterozygous PIK3CA mutations in exons 9 and 20 diagnosed at stage I-II (locally
advanced but not extended to the pelvic wall) were shown to have a poorer prognosis compared
with early stage cervical cancer patients presenting wild type phenotype. While PIK3CA has
been well-established as an oncogene capable of driving malignant changes in cells and as a
marker for poor response to CRT, the specific role that PIK3CA plays in cisplatin resistance has
not been well established in a cervical cancer model.
4.1.1 Cervical cancer cell lines display diverse growth characteristics and expression profiles.
Before determining the effect of PIK3CA-E545K status on cervical cancer resistance to radiation
and cisplatin treatments, I first assessed basic growth characteristics of the cell lines and
similarities between PIK3CA-E545K cell lines, such as upregulation/downregulation of key
markers in the PI3K/AKT pathway and increased cell proliferation. These findings suggested
that while p110α and p110β levels varied, upregulation of p85 and a downregulation of PTEN
occurred in PIK3CA-E545K expressing cell lines, consistent between replicates. PIK3CAE545K cell lines may favour the upregulation of PI3K pathway proteins because p85 is an
important mediator of p110α activity and PTEN is an important negative regulator of p110α
kinase activity. Me-180 cells expressed high levels of p85 protein, which could affect the
activity of p110α since p85 is known to exhibit an inhibitory effect that is alleviated by PIK3CAE545K on p110α. Since PIK3CA-E545K cell lines express wt-PIK3CA, elevated p85 expression
55
could inhibit the activity of the wt enzyme. Moreover, differences in allelic expression of
PIK3CA in CaSki and Me-180 could augment the effects from PIK3CA-E545K; these two cell
lines have different genetic backgrounds and come from different metastatic sites. Activation of
a growth cascade such as the PI3K pathway would expectedly increase cell growth rate, and a
study in isogenic breast cancer cell lines confirmed that PIK3CA-E545K expression leads to
increased rate of growth70. In my study, the heterozygous PIK3CA mutant cell line, CaSki,
presented slow growth while another PIK3CA mutant cell line, Me-180, was fast growing.
Therefore, my results did not indicate a clear relationship between mutant PIK3CA and growth
rate in the cervical cancer cell line panel.
4.1.2 PIK3CA-E545K cells are not radiation resistant.
My findings did not indicate a clear trend of radiation resistance in cell lines expressing the
PIK3CA-E545K mutation, although CaSki cells did display higher survival at 4 Gy, and Me-180
cells at 6 Gy. Due to the lack of a consistent and dose dependant response, it is not clear whether
the PIK3CA-E545K cell lines are more resistant to radiation than wild type cells. While there
isn’t a direct link between PIK3CA-E545K and radiation resistance specifically, studies have
shown that PI3K inhibitors show a combined effect in cells with radiation, and cervical cancer
patients with elevated phospho-AKT respond poorly to radiation41,54,71. Clinically, patients are
given larger cumulative doses of radiation than cells used in this study, but receive doses over
longer periods of time and over multiple treatment sessions. High dose brachytherapy is one
type of intracavity radiation therapy often using an iridium-192 (Ir192) source, and can be
administered to patients at a rate of up to 14 Gy/hr. Brachytherapy is often administered along
with linear beam radiation, which uses an external focused beam of high-energy electrons rather
56
than a radioactive source, directed at the patients’ tumour. In comparison, cervical cancer cells
in this study were treated with a higher dose rate over a much shorter period of time. The
radiation used in this study came from a single source, caesium-137 (Cs137), and was
administered only once at a dose rate of approximately 2.9 Gy/min. Importantly, the radiation
given off by Cs137 has almost twice as much energy than that of Ir192. Thus, CSA experiments
may only show acute survival effects, different from those of radiation given at a lower dose rate
for a longer time. Development of more effective methods of delivering high dose radiation to
target cancer cells is ongoing in the field of radiobiology. In addition to the potential challenge
posed by a discrepancy in dose rates in this study’s methods compared with the clinic, patient
tumours present complex and dynamic microenvironment conditions that cannot be entirely
replicated in cell culture. While some of these issues are difficult to avoid with a cell culture
model, ideal conditions should mimic those of clinical radiation therapy as closely as possible in
order to generate results that bring more relevance to clinical outcomes for patients. An in vivo
xenograft model would help strengthen findings and possibly overcome some of the limitations
of the approaches used in this study.
4.1.3 CaSki cells are resistant to cisplatin.
CaSki cells were found to be highly resistant to cisplatin, indicating that PIK3CA mutation could
play a role in the development of cisplatin resistance. However, only one of two PIK3CA mutant
cell lines tested was cisplatin resistant. Dr. Arjumand Wani of our research group has developed
stable cell lines expressing PIK3CA-E545K in an isogenic background in HeLa cells, and has
shown that the exogenous expression of PIK3CA-E545K in a cell line expressing wt-PIK3CA or
no PIK3CA leads to increased cisplatin resistance (Wani et al. 2016 submitted manuscript). This
57
suggests that the difference in cisplatin sensitivity between CaSki and Me-180 may be due to
other background factors within individual cell lines. PIK3CA-E545K dependant cisplatin
resistance may depend on other factors not present in Me-180 cells. Given that only CaSki cells
displayed resistance to cisplatin in my initial aim, I further assessed CaSki cells in my second
and third aims alongside HeLa cells as the wt cell line chosen for comparison with CaSki. SiHa
cells were also wild type for PIK3CA but displayed high levels of AKT expression, second
highest relative resistance to cisplatin using CSAs, and higher relative cisplatin resistance than
both CaSki and Me-180 using AB. Of the cell lines expressing PIK3CA-wt, SiHa displayed
higher relative resistance to cisplatin.
4.1.4 Serum starvation had distinct effects on CaSki and HeLa cells, and did not reliably
reduce levels of phospho-AKT or phospho-RSK.
In addition, I studied the effect of PIK3CA-E545K mutation on the phosphorylation of AKT, a
surrogate for activation, in CaSki compared to wt HeLa cells. AKT pS473, under normal
untreated conditions was consistently higher in Me-180, but varied significantly between
replicates using CaSki cells. Therefore, I assessed AKT pS473 under conditions of serum
starvation. I expected to find constitutive activation of AKT pS473 in CaSki cells but not HeLa
cells in serum starved conditions because PIK3CA-E545K should drive continuous activity of
PI3K/AKT signalling even when treated with low serum levels lacking growth factors that
activate receptors upstream of PI3K. I also included phospho-RSK markers in this experiment,
with the rationale that RSK is a downstream target of ERK that is highly regulated by
extracellular growth receptors. I experienced difficulty in obtaining consistent results for my
serum starvation experiments. Between replicates, the fold-difference varied by orders of
58
magnitude in comparison to the control conditions between experiments. Since this variation
occurred for both RSK and AKT markers, and given the difference in expression between RSK
and AKT in HeLa and CaSki cells, it was unlikely to establish a starvation protocol that would
work for both cell lines and be consistent enough to replicate. Cancer cell lines often have
mutations that hijack normal growth signalling and may not respond predictably to starvation
conditions. Collectively, it is unclear whether PIK3CA-E545K in CaSki cells leads to
constitutive activation of AKT. Recent studies have shown difficulty in establishing starvation
conditions that effectively and consistently knock down active kinase markers in transformed
cell lines, and that signalling pathway responses under serum starvation can vary significantly
between cell lines72,73. One interesting finding from these starvation experiments was the higher
level of p-RSK in HeLa cells compared to CaSki cells. Because RSK and AKT can both activate
mTORC1, this finding helps to explain GDC-0941 lacking an effect on HeLa cell survival as a
single agent, and brings to light the issue of PI3K inhibitor resistance through upregulation of the
MAK/RSK pathway. Elevated expressions of specific RSK isoforms have been shown to
correlate with resistance to PI3K inhibitors, which can be overcome with the combined treatment
of PI3K inhibitor with a MEK or p90 RSK inhibitor74. Although my results regarding
constitutive activation of AKT were inconclusive, I continued to study the effects of treatment of
CaSki and HeLa cells using GDC-0941.
4.1.5 GDC-0941 treatment decreased phospho-AKT levels in CaSki cells.
When I treated CaSki and HeLa cells with GDC-0941, levels of phospho-RSK and phosphoAKT varied significantly. The treatment of CaSki cells with GDC-0941 reduced levels of AKT
pS473 at 48 hours, while pT308 AKT levels were lower at 24 hours compared to the control.
59
Levels of p110α varied significantly in my initial Western blots of the untreated cell line panel,
which explains at least one reason for the variable effects between replicates. GDC-0941 is a
pan-PI3K inhibitor, which targets all catalytic class IA PI3Ks and has highest affinity for both
p110α and p110δ75. Changes in cellular stoichiometry of GDC-0941 targets between
experimental replicates could give rise to variation in signalling response to inhibition.
Additionally, while GDC-0941 does have an IC50 under 100 nM for all class IA PI3Ks in vitro,
its effects in cells are most apparent at concentrations of at least 0.5 µM, preferably 1-5 µM.
GDC-0941 also targets other PIKKs at higher doses, which could alter the effect on p-AKT
levels.
4.1.6 GDC-0941 is an effective single agent against CaSki cells, but does not reverse cisplatin
resistance in CaSki cells.
Next, I tested the effect of GDC-0941 as a single agent, and in combination with cisplatin, on the
cell survival of CaSki and HeLa. In this case, I assessed if GDC-0941 could be used to modulate
the resistance that I saw in CaSki cells towards cisplatin. I determined that GDC-0941 is
effective as a single agent against CaSki but not against HeLa cells, and that GDC-0941 does not
modulate cisplatin resistance in CaSki. Importantly, the treatment of CaSki with GDC-0941 for
48 hours at 2 µM showed an effect comparable to that of cisplatin treatment in HeLa cells for 24
hours at 1 µM. This suggests that PI3K inhibitors have potential against PIK3CA mutant
tumours; however in this case, I showed that combined treatment of a PI3K inhibitor and
cisplatin had no added effect on cell survival. Initial findings in CSAs of isogenic derived
PIK3CA-E545K cell lines by Arjumand Wani have suggested that combining GDC-0941 and
cisplatin do show combined effects, but only at concentrations of GDC-0941 less than 1 µM, for
60
shorter time periods, and with both drugs administered at the same time. Accordingly, the
concentration of GDC-0941, or length of treatment used in the experiments of this study may
have been cytotoxic enough to negate any combination effects with cisplatin. A combination
index analysis of cisplatin and GDC-0941, in cervical cancer cell lines would help to clarify
whether cisplatin and GDC-0941 are synergistic or not. This approach could also be used to
optimize timing of the drug(s) treatment, or to identify other drugs that combine with PI3K
inhibitors.
4.1.7 Other findings and future experiments
In addition to the findings in the combination CSAs mentioned above, Dr. Arjumand Wani has
shown that isogenic cervical cancer cells lines stably expressing PIK3CA-E545K are more
resistant to cisplatin, and cisplatin combined with IR using CSAs. CaSki cells, and HeLa cells
expressing PIK3CA-E545K also display an increased migratory phenotype compared to PIK3CA
-wt cell lines using both scratch (wound healing) assays, and transwell migration assays. In
these experiments, GDC-0941 treatment of the cells resulted in reduced cell migration. Future
experiments will include orthotopic xenograft transplantation of luciferase marked human
cervical cell lines (HeLa and SiHa) stably expressing PIK3CA-E545K into mice. Xenograft
tumour formation will be assessed, and mice will be treated with cisplatin and/or GDC-0941 to
assess the effects of these drugs on xenograft tumour growth, and survival of mice.
4.2 Working hypothesis
While cisplatin resistance was found in one of the PIK3CA-E545K cell lines in the panel, this
characteristic was not universally seen in both PIK3CA-E545K cell lines. Furthermore, cisplatin
resistance seen in CaSki cells was not overcome with the use of PI3K inhibitor GDC-0941,
61
despite GDC-0941 being an effective single agent. Collectively, these findings suggest that
PIK3CA may play a role in promoting effectors of cisplatin resistance, although it is not clear
whether resistance can be overcome inhibition of the PI3K pathway. Many effectors of cisplatin
resistance are known (Chapter 1), however the mechanism(s) of PIK3CA associated cisplatin
resistance remain(s) to be understood.
4.3 Considerations
4.3.1 Survival Assays
The choice of survival assay is a major consideration for selecting drugs. In this study, drugs
were initially screened over a large concentration range using AB assays, while CSAs were used
to determine a dose dependant response to drug and radiation within a small dose range. AB
uses a reagent that is reduced by the electron transport to indicate metabolic activity76. This
assay measures the overall metabolic health of thousands of cells in a single well after long
exposure to a drug, and gives a good general idea of the cytotoxic potential of a drug77. CSAs
detect colonies larger than 50 cells and therefore measures the number of cells that can survive
and undergo at least 5 cycles of division78,79. Therefore, CSAs measure the potential of
individual cells to survive treatment and generate an expanded clonal population. Results of this
study demonstrated the differences between the two assays, cisplatin resistance in CaSki cells
was only seen using CSAs but not using the AB assay. Furthermore, SiHa cells showed higher
survival after cisplatin treatment than CaSki in the AB assays (Figure 16), which was not seen
using CSAs. SiHa cells formed colonies very poorly, even on the control plates, suggesting that
these cells may not be able to tolerate plating at low confluence (Figures 13 & 17). These two
assays measure different properties with AB assay being a great screening technique, and CSAs
62
being useful for determining effects on cellular reproduction in a limited dose range and with
combined treatment using multiple agents. Importantly, while CSAs are a popular in vitro
method for measuring cell survival, their usefulness is limited to cell types that tolerate low
confluence well.
4.3.2 Drug formulation
This study also highlighted the importance of vehicle choice for formulating drugs in cell
culture. Findings on cisplatin formulation agreed with the recent publication from Hall et al
(2014) that found inactivation of cisplatin when made up as a stock solution in DMSO80. Much
of the existing literature using cisplatin is not specific about the preparation of the drug.
Additionally, many sources report using very high, physiologically unlikely, concentrations of
cisplatin over 10 µM. Even in cell lines relatively resistance to cisplatin, concentrations less than
5 µM should be sufficient to elicit a significant response, using both CSAs and AB (Figures 15,
16 &17). In order to properly assess the characteristics of cisplatin, formulation of the drug must
not interfere with its biological activity. Results of this study demonstrated that cisplatin made
up in an aqueous 1 mM stock containing 1X PBS with 154 mM NaCl is equally effective at
reducing cell survival in culture as clinical cisplatin obtained from the pharmacy. Future studies
using cisplatin should be cautious of properly formulating and storing cisplatin. Drug vehicle
information needs to be documented and communicated in an accurate and transparent way.
4.3.3 Western Blots
Western blots were quantified using the band closest to the predicted size. Phosphorylation
events have the potential to shift bands from their predicted size by altering the charge and
molecular weight. This is an important consideration for the AKT probes which showed
63
multiple bands on each blot (Figures 12, 18, 19, &20). In particular, the AKT pT308 marker
showed a band slightly higher than the predicted size. This band was quantified, however the
results are inconclusive and the marker did not respond predictably to either serum starvation or
GDC-0941 treatment. Human error in gel loading may have contributed to error in
quantification (Figure 12A lanes 10-12; Figure 18, Lanes 9 & 10: Figure Figure 19, Lane 9).
Additionally, low expression of certain markers lead to very large differences in quantification.
For example AKT pT308 levels appeared to be very high in the quantification HeLa cells in
Figure 12B, but this was most likely an artifact arising from very low levels of total AKT in
HeLa cells compared to the other cell lines (Figure 12B, lanes 1,5,9). Moreover low levels of
RSK pT359 and pS380 in pre-starved CaSki cells, compared to starved CaSki cells gave rise to a
large apparent increase of over 100-fold. Again, this is most likely an artifact due to the almost
undetectable levels of phospho-RSK in the pre-starved cells.
4.3.4 Antibodies
In addition to the loading issues mentioned above, antibody specificity may be an issue with
western blots. Both phospho-AKT antibodies picked up multiple bands on my Western blots.
While our group did not independently verify the specificity of these antibodies, this would be
useful in strengthening our findings. This could be done by immunoprecipitation (IP) pulldown
with a verified total antibody, and probing the IP samples using the phospho-antibodies. Mass
spec analysis of the predicated band size of IP samples could further verify if AKT is running at
the predicted band size. For verifying specificity of phospho-antibodies, cell lysates can be
treated with lambda phosphatase before Western blot analysis, and compared to untreated
samples. Another approach of antibody verification could be to knockdown AKT using siRNA
64
or CRISPR gene editing, and observe which band corresponds with AKT being knocked out, or
knocked down.
4.4 PI3K inhibitors as therapeutics
This study has shown the potential of PI3K as a valid target in treating cervical cancer. Thus, the
potential for PI3K inhibitors as effective agents against mutant PI3K should be further assessed.
Additionally, isoform-specific and pan-PI3K inhibitors, and their combination with other
inhibitors, cytotoxic agents or radiation, should be investigated further. For example, studies
have demonstrated effectiveness in combining PI3K inhibitors with PARP inhibitors in triple
negative breast cancer81,82. Although PI3K inhibitors in clinical trials have had challenges with
efficacy as single agents, they could still be a viable option for combination therapy with other
agents. While E545K is the hotspot mutation that was focused on during this study because of
its high occurrence in previous patient studies, there are many other mutations in PIK3CA as well
as other proteins in the PI3K pathway implicated in cancer progression and development. The
mechanisms of other mutations in PIK3CA have also been described. Some mutations such as
H1047R show distinct phenotypical differences than E545K. In addition to changes in gene
amplification and in the expression and localization of PI3K proteins, it would be worthwhile to
further assess patient survival in relation to specific mutations in PIK3CA and determine whether
distinct PIK3CA mutations act through similar or different mechanisms. These factors combined
could drastically alter the response to various PI3K inhibitors, and may in the future have
implications for inhibitor effectiveness. This study has shown a potential benefit of using GDC0941 on a PIK3CA-E545K expressing cervical cancer cell line resistant to cisplatin. However,
the applicability and clinical effectiveness of these drugs in cervical cancer treatment regimens
remains to be assessed. Our group will be continuing our study in an orthotopic xenograft mouse
65
model to better understand effects of PIK3CA on metastatic potential in vivo and to investigate
effects of PI3K inhibitors on cancer cell metastasis and survival in vivo.
This study highlights the complexity of signalling in cancer. The discovery of new mechanisms
of cross talk is shedding light on the highly dynamic, complex, and redundant nature of cancer
signalling. Cancer signalling pathways, such as PI3K, have many redundancies and can interact
with parallel networks. In order to overcome potential challenges posed by these redundancies,
novel treatment will need to involve more complex but effective combinations of targeted cancer
therapy that can inhibit one or multiple networks of cancer signalling. Benefits of combined
and/or sequential therapies using a more targeted approach with multiple agents are increasingly
being looked at. Pan-inhibitors such as GDC-0941 may prove useful in shutting down cervical
cancer signalling networks; however, they will need to be combined effectively with other drugs
to have clinically relevant effects that benefit patients. Isoform-specific inhibitors may prove
even more useful in targeting specific mutations, and may be better tolerated at a therapeutic
dose, but will require more research and confidence in predictive biomarker identification. High
throughput techniques will be useful in the future of studying cancer signalling, allowing
researchers and physicians to identify relevant networks and better predict individual patient
response to anti-cancer therapies based on biochemical characteristics of the individual’s cancer.
Thus, additional knowledge about cancer drug resistance, signalling networks, and inhibitors
could open the door for highly specific and novel therapy regimens using personalized medicine
to maximize the synergy and effectiveness of anti-cancer agents.
66
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APPENDIX A: SUPPLEMENTARY FIGURES
72
73
74
APPENDIX B: FIGURE FROM MANUSCRIPT BY DR. ARJUMAND WANI
(PUBLICATION PENDING)
Manuscript title: Phosphatidyl inositol-3 kinase (PIK3CA) E545K mutation confers
cisplatin resistance and a migratory phenotype in cervical cancer cell lines.
Wani Arjumand1,2, Cole D. Merry1,2, Chen Wang1,2, Elias Saba1,2, John B. McIntyre2,3,
Shujuan Fang1,2, Prafull Ghatage4, Corinne M. Doll2,4 and Susan P. Lees-Miller1,2,4.
1. Department of Biochemistry and Molecular Biology, University of Calgary; 2. Arnie
Charbonneau Cancer Institute, Calgary, Alberta; 3. Translational Laboratories, Tom
Baker Cancer Centre, Calgary, Alberta; 4. Department of Oncology, University of Calgary.
Figure 2. HeLa cells expressing PIK3CA-E545K are resistant to cisplatin.
75
Figure 2 legend.
A. HeLa-A5 cells (shRNA depletion of PIK3CA), A5 cells transfected with shRNA
resistant PIK3CA-WT (A5-WT), and A5 cells transfected with shRNA-resistant PIK3CAE545K
(A5-E545K) were grown under standard conditions (5% serum). Whole cell
extracts were generated and aliquots run on SDS PAGE. Western blots were probed with
antibodies to p110α and β, p85, Akt, Akt-pS473, PTEN, Myc (to detect stably
incorporated Myc-tagged PIK3CA) and Ku80 (loading control).
B. A5 (shRNA depletion of PIK3CA in HeLa cells) [open circles], A5 cells transfected with
shRNA resistant PIK3CA WT type (A5-WT) [closed circles], and A5 cells transfected with
shRNA-resistant PIK3CA-E545K (A5-E545K) [closed squares] were either untreated (0), or
treated with different doses of IR (1, 2, 4 and 6 Gy). Clonogenic survival assays were carried out
as described in Figure 1. No statistically significant differences were observed between the three
cell lines at any dose tested.
C. A5, A5-WT, and A5-E545K cells as in panel B were seeded on 6 cm plates and 24
hours later treated with either control PBS (containing 154 mM NaCl) or cisplatin
(formulated in PBS) at 0.1, 0.5, 1 or 2 μM, respectively. Cisplatin was removed after 24
hours and replaced with fresh media. Clonogenic survival assays were carried out as
above. At 0.1, 0.5, 1.0 and 2 μM cisplatin, the p values for A5 compared to A5-E545K
were 0.0288, 0.0004, 0.0016 and 0.0396, respectively. p values of <0.05 were
considered statistically significant and are indicated by the asterisks.
D. A5-WT cells (open and closed circles) and A5-E545K cells (open and closed
squares) were seeded on 6 cm plates and 24 hours later either mock treated with PBS
(open symbols and dashed lines) or treated with cisplatin (formulated in PBS) at 1 μM
(closed symbols, solid lines). Cisplatin was removed after 24 hours and replaced with
fresh media, then cells were either unirradiated (0) or irradiated at 1, 2, 4, and 6 Gy,
respectively. After 14 days, the plates were fixed, stained and colonies were counted
and analyzed as above. At 1, 2, 4 and 6 Gy plus cisplatin data points, the p values for
A5-WT compared to A5-E545K were <0.0001, 0.0005, 0.00045, and 0.023, respectively. p
values of <0.05 were considered statistically significant and indicated by
the asterisks.
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translation should be credited as follows:
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Our Ref: DE/IBMG/P7557
25 May 2016
Dear Cole Merry,
Material requested: Figure 1 - Joke Baute & Anne Depicker (2008) Base Excision Repair
and its Role in Maintaining Genome Stability, Critical Reviews in Biochemistry and
Molecular Biology, 43:4, 239-276
Thank you for your correspondence requesting permission to reproduce the above mentioned
material from our Journal in your printed thesis entitled ‘The role of PIK3CA in Cisplatin
Resistance of Cervical Cancer’ and to be posted in your university’s repository – University of
Calgary.
We will be pleased to grant entirely free permission on the condition that you acknowledge the
original source of publication and insert a reference to the Journal’s web site:
www.tandfonline.com
Please note that this licence does not allow you to post our content on any third party websites or
repositories.
107
Thank you for your interest in our Journal.
Yours sincerely
Debbie East.
Debbie East– Permissions & Licence Administrator - Journals.
Routledge, Taylor & Francis Group.
3 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN, UK.
Web: www.tandfonline.com
Taylor & Francis is a trading name of Informa UK Limited,
registered in England under no. 1072954
From: Cole Merry
Sent: 22 April 2016 15:05
To: Academic UK Non Rightslink
Subject: ibmg20:Base Excision Repair and its Role in Maintaining Genome Stability
Permissions Request
Contact name: Cole Merry
108
Street address:
Town: Calgary, A.B.
Postcode/ZIP code:
Country: Canada
Contact telephone number:
Contact email address:
Article title: Base Excision Repair and its Role in Maintaining Genome Stability
Article DOI: 10.1080/10409230802309905
Author name: Joke Baute, Anne Depicker
Journal title: Critical Reviews in Biochemistry and Molecular Biology
Volume number: 43
Issue number: 4
Year of publication: 2008
Page number(s): 38
Are you the sole author/editor of the new publication?: Yes
Are you requesting the full article?: No
If no, please supply extract and include number of word: Figure
If no, please supply details of figure/table: Figure 1. Schematic BER pathway
Name of publisher of new publication: University of Calgary (Online vault for thesis)
Title of new publication: The role of PIK3CA in cisplatin resistance of cervical cancer
Course pack: No
Number of Students:
Is print: No
109
Electronic: Yes
E-reserve: Yes
Period of use: Perpetual
Short loan library?: No
Thesis: Yes
To be reprinted in a new publication?: No
In print format: No
In ebook format?: No
ISBN:
Languages:
Distribution quantity:
Retail price:
Additional comments: I am requesting this schematic figure for re-use in my MSc Thesis.
Thanks
110
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UNIVERSITY OF CALGARY PIK3CA by