UNIVERSITY OF CALGARY. The Role of PIK3CA in Cisplatin Resistance of Cervical Cancer. Cole Merry A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

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1 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

2 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 PIK3CA- E545K, and was not reversed with the use of the PI3K inhibitor GDC The cisplatin resistant cell line showed sensitivity to GDC-0941, suggesting PI3K inhibitors as an alternative to cisplatin. ii

3 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

4 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/ /02/2016 iv

5 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 Cervical cancer Cervical cancer is a significant global health problem Human papilloma virus (HPV) plays a large role in the development of cervical cancer Radiation and cisplatin are currently the standard treatments for locally advanced cervical cancer, given concurrently as chemoradiotherapy (CRT) DNA damage from Ionizing Radiation (IR) Cisplatin damage to DNA Cisplatin combined with radiation shows increased efficacy in killing cancer cells Several repair pathways are involved in the repair of cisplatin and IR damage to cells DNA damage response to IR involves PI3K related kinases NHEJ HR NER (global NER and TCR) Fanconi Anemia pathway MMR BER Phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks) PI3Ks are a large group of receptor associated lipid kinases that regulate important growth, metabolism and survival pathways Active AKT has many nuclear and cytosolic targets that control cell growth, cell death, and metabolism Mutations leading to up-regulation of PI3K/AKT/mTOR signalling have been implicated in tumour progression and resistance to drug therapy Recent clinical studies have identified PI3K as a novel prognostic marker for clinical outcome in cervical cancer patients PI3K inhibitors have been used in vitro and in vivo to increase sensitivity to radiation and cisplatin Cisplatin resistance Several mechanisms of cisplatin resistance have been identified Potential mechanistic links between PI3K and cisplatin or radiation resistance Hypothesis Objectives...27 v

6 CHAPTER TWO: MATERIALS AND METHODS Cell Culture Cell Proliferation Assay Cell Lysis SDS-PAGE and Western Blots of WCEs Ionizing Radiation (IR) and Drug Treatment of Cells Clonogenic Survival Assays (CSAs) Alamar Blue (AB) Cell Survival Assays...37 CHAPTER THREE: RESULTS Cervical cancer cell lines vary in growth rate. PIK3CA-E545K status does not necessarily correlate with faster growth rate in a cell line panel 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 Cervical cancer cell lines with PIK3CA-E545K do not show significant resistance to IR compared with PIK3CA-wt cervical cell lines Cisplatin treatment showed no effect on cervical cancer cell lines initially using both clonogenic survival and Alamar Blue cell survival assays 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 CaSki cells are relatively more resistant to cisplatin than other cervical cancer cell lines using clonogenic survival assays Serum starvation results in decreased RSK pt359 in HeLa cells and increased RSK pt359 in CaSki cells GDC-0941 treatment results in decreased AKT ps473 in CaSki cells but not HeLa cells 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 CHAPTER FOUR: DISCUSSION Conclusions Cervical cancer cell lines display diverse growth characteristics and expression profiles PIK3CA-E545K cells are not radiation resistant CaSki cells are resistant to cisplatin Serum starvation had distinct effects on CaSki and HeLa cells, and did not reliably reduce levels of phospho-akt or phospho-rsk GDC-0941 treatment decreased phospho-akt levels in CaSki cells GDC-0941 is an effective single agent against CaSki cells, but does not reverse cisplatin resistance in CaSki cells Other findings and future experiments Working hypothesis Considerations Survival Assays Drug formulation...63 vi

7 4.3.3 Western Blots Antibodies 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

8 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 Table 3. Information on HeLa, SiHa, CaSki, and Me-180 cell lines obtained from ATCC viii

9 List of Figures and Illustrations Figure 1. Suppression of p53 and prb by HPV E6 and E7 proteins, respectively Figure 2. Repair of DSBs by NHEJ or HR Figure 3. Cisplatin structure and mechanism of adduct and crosslink damage to DNA Figure 4. NER pathway through TCR or GGR Figure 5. FA pathway core components and DDR proteins that interact with FA pathway to repair Interstrand crosslinks Figure 6. Mechanism of MMR Figure 7. BER pathway Figure 8. Domains of p110, p85, AKT and p90 RSK and kinase activity of p110 on PIP Figure 9. Downstream signalling of PI3K. AKT is a major regulator of PI3K signalling Figure 10. Prognosis for 5-year survival of cervical cancer patients based on PIK3CA status (wt or mutant) Figure 11. Growth rates of cervical cell lines Figure 12A. Expression levels of PI3K markers in Hela (H), CaSki (C), SiHa (S), and Me- 180 (M) cell lines Figure 12B. Expression levels of PI3K markers in Hela (H), CaSki (C), SiHa (S), and Me- 180 (M) cell lines 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 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 Figure 15. Effect of clinical, 154mM saline, and DMSO formulated cisplatin on HeLa cell viability using Alamar Blue viability assays 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 ix

10 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) Figure 18. Effect of serum starvation on AKT and RSK activity of HeLa (H) and CaSki (C) cells Figure 19. Treatment of CaSki cells with GDC Figure 20. Treatment of HeLa cells with GDC Figure 21. Clonogenic survival of HeLa (wt PIK3CA), and CaSki (wt/e545k PIK3CA), cervical cancer cell lines following combined treatment with cisplatin and GDC x

11 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

12 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

13 Chapter One: Introduction 1.1 Cervical cancer 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 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

14 oncogenic properties but hasn t been as well studied in human HPV carcinogenesis 6. 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 replication 8. 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 cycle 9. 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

15 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 replication 10. 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 genome 11, 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 virus 12. 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 understood 13. Cervical cell lines used in our study all contain integrated genome(s) of HPV (Table 3). 3

16 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

17 1.2 Radiation and cisplatin are currently the standard treatments for locally advanced cervical cancer, given concurrently as chemoradiotherapy (CRT) 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 phases 14 (see sections &1.3.3) 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

18 and transcription. Major types of repair for cisplatin-induced damage include nucleotide excision repair (NER), homologous recombination (HR), and the Fanconi Anemia (FA) pathways 15. 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 ) 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 repair

19 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 al

20 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 1.3 Several repair pathways are involved in the repair of cisplatin and IR damage to cells 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 repair 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) occurs 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 HR 24,25. RPA is displaced by BRCA2, allowing Rad51 to bind 9

22 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 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

23 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, 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

24 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 DSB 27. 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 Andrea

25 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 lesion 29. 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

26 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, Jiricny

27 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 death

28 Figure 7. BER pathway. Reprinted by permission from Taylor and Francis Group: Critical Reviews in Biochemistry and Molecular Biology, Baute and Depicker Phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks) 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

29 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-3,4,5- triphosphate (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 PDK2 32,33. Nonphosphorylated AKT remains inactive and these two phosphorylation events leave AKT in its active form. 17

30 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

31 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

32 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, Martelli

33 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 mutations Class I p110γ and p110δ subunits facilitate cell-cell interactions/motility and the maintenance of immune cells 39,40. 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 patients 41. 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 p Upregulated AKT activity could abrogate this temporary p53 stabilization and reduce the effects of cisplatin, potentially contributing to resistance. 21

34 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 CRT 45. 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 PIK3CA 45. 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 SCC 11,46. 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 vivo 37,47,48. PIK3CA- E545K is suggested to interrupt the inhibitory interaction of p85 SH2 domains on the kinase domain of p 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 patients 45,46, 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

35 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 chemoradiotherapy 45, Page 418, with permission from Elsevier. 23

36 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 inhibitor 51, has been shown to increase the efficacy of cisplatin treatment of ovarian cancer xenografts in nude mice 52. The PI3K and DNA-PKcs inhibitor 53 LY can also cause cervical cancer cell lines to be more sensitive to radiation. In both CaSki and Hela cells, pre-treatment with LY had a synergistic effect with radiation to reduce clonogenic survival and cell growth 54. Herzog et al. (2013) demonstrated, using mouse xenograft and syngeneic models, that the PI3K/mTOR inhibitor PF has anti-tumourigenic effects in vivo and that the inhibitor can be effective when combined with radiation treatment 55. These effects exhibited by PF 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 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 uptake Cisplatin resistance 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

37 cisplatin resistance 58. 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 resistance 15. Since MMR and BER mediate cisplatin cytotoxicity, downregulation of MMR genes is associated with cisplatin resistance 16. 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 cells 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 SiHa 62. 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

38 FOXO3, and FOXO4 to prevent transcription of genes that promote apoptosis and cell cycle arrest 63. Recent studies have shown the role of another gain of function mutation PIK3CA- H1047R in promoting stem-like multipotency and heterogeneity in tumour development in mouse breast cancer models 64,65. Cancer stem cells (CSCs) are a controversial topic, but studies have shown a link between multipotency and therapy resistance 66,67. 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 therapy 68. 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 stage 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

39 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

40 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

41 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 #SH ) and 1% (50U/mL) Penicillin-Streptomycin (PS) (Thermo Fisher # ). 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 # ), 1% (w/v) Sodium Pyruvate (Thermo Fisher # ), 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 # ) 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

42 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

43 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

44 Elmer #ORT2655). Next, they were exposed to Super RX-N Fuji Medical X-ray film (Fujifilm # ) 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

45 Table 1. Conditions and parameters of antibodies used for Western blot analysis in this study. Antigen (target) Host species primary Company Catalogue # (dilution of primary) Overnight (dilution of secondary) time Ku80 Mouse Abcam Ab3107 1:3000 1: min ps473 AKT Rabbit Santa Cruz sc-7985-r 1:1000 1: hr pt308 AKT Rabbit Santa Cruz sc r 1:500 1: hr AKT Mouse Santa Cruz sc :1000 1: hr PTEN Rabbit Cell signalling :1000 1: hr p85 Rabbit Millipore :2000 1: hr p110α Mouse Mariona Graupera Garcia* Non commercial (Graupera lab) 1:1000 1: hr p110β Rabbit Cell signalling :1000 1: hr pt359 p90rsk Rabbit Cell signalling :500 1: hr ps380 p90rsk Rabbit Cell signalling :500 1: hr p90rsk Rabbit Cell signalling :1000 1: hr Secondary to Goat Bio-Rad rabbit Secondary to mouse Goat Bio-Rad *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 -20 o C. 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

46 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 o C. Drugs were diluted from stock to the final concentrations using the appropriate cell media. 34

47 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 5.8 nm, 76 nm, 0.51 μm, 1.3 μm GDC-0941 p110α/δ 3 nm Pictilisib p110β,p110γ 11 nm, 75 nm n/a Phase I and II For NSCLC and Breast cancer CAL-101 Idelalisib p110δ,γ,β,α 2.5, 89, 565, 820 nm Phase II MCL and CLL LY p110α/δ/β 0.50, 0.57, 0.97 nm n/a TG p110γ/δ 83,235 nm Phase I myocardial infarction Xl-147 p110α/δ/γ/β 39,36,33,383 nm Phase I (general) NVP-BKM120 (135) PF (PKI-587) p110α/β/δ/γ 99,166,116,262 nm I and II multiple including HNSCC and Squamous cervical cancer PI3Kα, PI3Kγ and mtor 0.4 nm, 5.4 nm and 1.6 nm PF 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 Phase I and II Healthy, Breast, NSCLC Ovarian, Colorectal and Endometrial Cancer Phase I (cancer) 35

48 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 Colcount TM (Oxford Optronix) counting machine and software (version ). 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 GDC- 0941, 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

49 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

50 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 PIK3CA- E545K, 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 HPV PIK3CA Origin HeLa type 18 wt Cervix adenocarcinoma SiHa type 16 wt Cervix squamous CaSki type 16 (highly amplified) and possibly 18 E545K heterozygous Me-180 type 39 E545K heterozygous Cervix squamous metastasized (small intestine) Cervix squamous metastasized (omentum) 38

51 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 < 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

52 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

53 Figure 12A. Expression levels of PI3K markers in Hela (H), CaSki (C), SiHa (S), and Me- 180 (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

54 Figure 12B. Expression levels of PI3K markers in Hela (H), CaSki (C), SiHa (S), and Me- 180 (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

55 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

56 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

57 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 ). 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

58 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.

59 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.

60 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 = (CaSki to Me-180), p= (CaSki to SiHa), p < (CaSki to HeLa). **p < (CaSki to all). ***p < 2.0 x (CaSki to all). 48

61 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

62 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. Phospho- RSK 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

63 Figure 19. Treatment of CaSki cells with GDC 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

64 Figure 20. Treatment of HeLa cells with GDC 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

65 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

66 Figure 21. Clonogenic survival of HeLa (wt PIK3CA), and CaSki (wt/e545k PIK3CA), cervical cancer cell lines following combined treatment with cisplatin and GDC 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