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Targeting Akt signaling in breast cancer : expression of phosphorylated Akt in breast tumors and the… Kucab, Jill 2004

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T A R G E T I N G A K T SIGNALING IN B R E A S T C A N C E R : E X P R E S S I O N O F P H O S P H O R Y L A T E D A K T IN B R E A S T T U M O R S A N D T H E E F F I C A C Y OF C E L E C O X I B A N A L O G U E S A S POTENTIAL INHIBITORS O F A K T ACTIVATION  by JILL K U C A B B . S c , North Carolina State University  A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF S C I E N C E in THE FACULTY OF GRADUATE STUDIES (Department of Experimental Medicine)  T H E UNIVERSITY O F BRITISH C O L U M B I A December 2004  © Jill Erin Kucab, 2004  1  ABSTRACT  Constitutive activation of Akt, due to overexpression of receptor tyrosine kinases (RTKs) or loss of phosphatase and tensin homologue ( P T E N ) , contributes to the development of breast cancer and confers resistance to conventional therapies. Therefore, the Akt signaling pathway is an attractive target for therapeutic intervention. Utilizing tumor tissue microarrays we show that 5 8 % (225/390) of breast cancers express moderate to high levels of activated Akt (P-Akt), compared with 35% (9/26) of normal breast tissues. Additionally we find that P-Akt expression in primary breast cancer is significantly correlated with the expression of three R T K s , human epidermal growth factor receptor-2 (HER-2), insulin like growth factor receptor-1 (IGF-1R) and ephrin receptor EphA2, as well as integrin-linked kinase (ILK) and the transcription factors growth factor independence 1 (GFI-1) and Y box-binding protein-1 (YB-1). Further, we examined the potential of three celecoxib analogues for the treatment of breast cancers expressing P-Akt.  W e found that all three analogues, OSU-03008,  OSU-03012, and OSU-03013, were able to disrupt Akt signaling in the M D A - M B - 4 5 3 breast cancer cell line, which overexpresses H E R - 2 and has very high P-Akt levels. Treating the cells for two hours with the compounds inhibited Akt phosphorylation and kinase activity, as well as its downstream signaling through glycogen synthase kinase3|3 (GSK3-6), at concentrations well below that of celecoxib (^10 u.M).  Disruption of Akt  phosphorylation by O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 was followed by an induction of apoptosis after 12 to 24 hours, whereas OSU-03008 did not cause cell death.  When  tested against a panel of three other breast cancer cell lines, O S U - 0 3 0 1 2 and O S U -  iii 03013 (but not OSU-03008) were found to reduce viability in cell lines which did not constitutively express P-Akt (MDA-MB-231, MCF-7). Overexpression of constitutively activated Akt in the MDA-MB-453 or MCF-7 cells was not able to rescue cells from the cytotoxicity of OSU-03012 or OSU-03013. These data suggest that the celecoxib analogues are potentially useful for inhibiting Akt signaling in breast cancer, although it appears that the cytotoxic effects of OSU-03012 and OSU-03013 occur through additional targets. These inhibitors could hopefully be used in combination with other j  therapies to bypass Akt-mediated drug resistance.  iv  TABLE OF CONTENTS  Page ABSTRACT  Chapter 1  TABLE OF CONTENTS  iv  LIST O F T A B L E S  vii  LIST O F F I G U R E S  viii  LIST O F A B B R E V I A T I O N S  x  INTRODUCTION  1  1.1 1.2  1 1 2 2 4  1.3 1.4 1.5 1.6 1.7 1.8 Chapter 2  ii  Cancer of the Breast: Frequency, Prognosis and Origin Standard Treatments for Breast Cancer 1.2.1 Chemotherapy 1.2.2 Hormone Therapy Molecular Therapeutics: Targeting Receptor Tyrosine Kinases The PI3K7 P-Akt Pathway: Receptor Signaling Convergence Point The Role of Akt in Cancer Development, Progression, and Drug Resistance Expression of P-Akt in Primary Tumors Akt as a Molecular Therapeutic Target: Current Advances in Drug Development Thesis Objectives  MATERIALS AND METHODS 2.1 Tumor Tissue Microarray Construction and Patient Information 2.2 Normal Tissues 2.3 P-Akt Immunohistochemistry 2.3.1 Validation of the P-Akt r473 IHC-Specific Antibody 2.3.2 Protocol for P-Akt Immunohistochemistry 2.4 Immunohistochemistry for Other Proteins on the T M A 2.5 Scoring of P-Akt Staining 2.6 Statistical Analysis of IHC Data 2.7 Cell Culture Conditions 2.8 Cell Lysis and Protein Extraction 2.8.1 Whole Cell Lysis Se  6 11 12 12 15 18 18 18 20 20 21 22 22 22 23 23 23  V  2.8.2 Cytoplasmic and Nuclear Fractionation 2.8.3 Protein Quantification Western Blotting 2.9.1 Preparation of samples 2.9.2 Electrophoresis 2.9.3 Transfer of Separated Proteins to Nitrocellulose Membrane 2.9.4 Primary Antibodies 2.9.5 Detection of Primary Antibody: Protein Complexes Akt Immunoprecipitation and Kinase Assay Drug Preparation Determination of Effects of Celecoxib Analogues on Cell Signaling Cell Viability Analysis Apoptosis Analysis 2.14.1 P A R P Cleavage 2.14.2 Nucleosomal Fragmentation Assay Plasmids Rescue Experiment 2.16.1 Transient Assay 2.16.1.1 Transfection 2.16.1.2 Treatment of Transfected Cells with Analogues to A s s e s s Viability; Analysis of Expression and Activity of Activated Akt Constructs 2.16.2 Stable Assay  24 24 25 25 25 25  RESULTS 3.1 Validation of P-Akt Immunostaining 3.2 Frequency of P-Akt Expression in Tumor and Normal Tissue 3.3 P-Akt Expression is Not Associated with Patient Survival 3.4 Correlation of P-Akt Expression with Other Proteins 3.5 Expression of P-Akt in Breast Cancer Cell Lines 3.6 Analogues of Celecoxib Inhibit Akt Phosphorylation 3.7 Analogues of Celecoxib Inhibit Akt Kinase Activity and Downstream Signaling 3.8 Effect of Analogues on Cell Viability 3.9 OSU-03012 and OSU-03013 Induce Apoptosis 3.10 Cell Confluency Protects Against Analogue-induced Cell Death 3.11 Serum Protects Against Cytotoxic Effects of Celecoxib and Low Doses of Celecoxib Analogues 3.12 Activated Akt Does Not Rescue Cytotoxic Effects of  34 34 34  2.9  2.10 2.11 2.12 2.13 2.14  2.15 2.16  Chapter 3  26 26 27 27 28 28 29 29 30 31 31 31 31 32  33  36 40 40 42 42 46 48 51 51 54  vi Analogues Chapter 4  DISCUSSION 4.1 Introduction 4.2 Expression of Phospho-Akts r473 in Primary Breast Tumors 4.2.1 Frequency of Expression and Relationship to Patient Prognosis 4.2.2 Correlation of P-Akt with the Expression of Other Proteins 4.3 Expression of Phospho-Aktser473 in Normal Breast Tissue 4.4 Targeting Akt Signaling in Anticancer Therapy 4.5 Analogues of Celecoxib A s Inhibitors of Akt Signaling and Cytotoxic Agents 4.5.1 Summary of Inhibitory Effects on Akt Phosphorylation 4.5.2 Summary of Effects on Cell Viability and Apoptosis 4.6 Necessity of P-Akt Inhibition for Induction of Cell Death 4.6.1 Discrepancy Between Akt Inhibition and Cytotoxicity 4.6.2 Attempt to Rescue the Cytotoxic Effects of OSU-03012 and OSU-03013 by Overexpressing Activated Akt 4.7 Other Targets for Analogues of Celecoxib 4.8 Usefulness of the Celecoxib Analogues as a Breast Cancer Therapy 4.9 Therapies for Downstream Targets of Akt: m T O R 4.10 Summary, Conclusions, and Future Work e  58 58 58 59 61 65 66 68 68 69 69 69 71  71 72 74 75  REFERENCES  78  APPENDIX 1 APPENDIX 2  95 99  vii LIST O F T A B L E S  Page Table 1.  Characteristics of the T M A Study Population  19  Table 2.  The Correlation of P-Akts r473 with Other Proteins in Breast Tumors  41  e  viii  LIST OF FIGURES  Page Figure 1.  The PI3K/Akt Signaling Network  Figure 2.  Structure and Design of the Celecoxib Analogues  16  Figure 3.  Validation of the P-Akt r473 Antibody for Immunohistochemistry  35  Figure 4.  Expression of P-Aktser473 in Tumor and Normal Tissues of the Breast  37  Figure 5.  P-Akts r473 is Highly Expressed More Frequently in Breast Cancer Compared with Normal Breast  38  Figure 6.  P-Akts r473 Expression Alone Does Not Predict Poor Prognosis  39  Figure 7.  Levels of P-Akt and HER-2 in a Panel of Breast Cancer Cell Lines  43  Figure 8.  Phosphorylation of Akt is Inhibited by Analogues of Celecoxib  44  Figure 9.  Akt Kinase Activity is Decreased and Downstream Signaling is Inhibited in MDA-MB-453 Cells Treated with Celecoxib Analogues  45  Figure 10.  Assessment of the Effects of Celecoxib Analogues on Cell Viaibilty  49  Figure 11.  Induction of Apoptosis by Celecoxib Analogues  50  Figure 12.  Confluency Protects Cells from Cytotoxic Effects of Celecoxib and Analogues.  52  Figure 13.  Effect of Serum on Efficacy of Celecoxib and Analogues  53  Figure 14.  Transient Overexpression of Activated Akt Does Not Rescue Cells from Cytotoxic Effects of Treatment with OSU-03012 or OSU-03013  56  Se  e  e  7  Figure 15.  Stable Overexpression of Myr-Akt1 Does Not Rescue Cells from Analogues  X  LIST OF ABBREVIATIONS  4E-BP1  eukaryotic initiation factor 4E-binding protein 1  AFX  ALL1 fused gene from chromosome X  Akt  v-akt murine thymoma viral oncogene homolog 1  Akt-DN  dominant negative of Akt  AMPK  AMP-activated protein kinase  API-2  Akt/protein kinase B signaling inhibitor-2  Bcr-Abl  breakpoint cluster region - Abelson murine leukemia viral oncogene homolog  BSA  bovine serum albumin  CDK2  cyclin dependent kinase 2  cDNA  complementary D N A  CHK1  cell-cycle-checkpoint kinase-1  CML  chronic myeloid leukemia  CMV  cytomegalovirus  COX-2  cyclooxygenase-2  DMBA  7, 12-dimethylbenz(a)anthracene  DMSO  dimethyl sulfoxide  DNA  deoxyribonucleic acid  DNA-PK  D N A dependent protein kinase  EGFR  epidermal growth factor receptor  elF4E  eukaryotic initiation factor 4 E  xi ER  estrogen receptor  FBS  fetal bovine serum  FDA  U S Food and Drug Admininstration  FKHR1  forkhead in rhabdomyosarcoma  FOX01-4  forkhead box 01, 02, 03 or 04  GFI-1  growth factor independence 1  GIST  gastrointestinal stromal tumor  GPEC  Genetic Pathology Evaluation Center  GSK-3a/6  glycogen synthase kinase-3a/B  HER1-4  human epidermal growth factor receptor 1,2,3 or 4  Hsp90  heat shock protein 90  IC50  concentration of compound that is required to produce a 50 percent inhibition of enzyme  IGF-I  insulin like growth factor-l  IGF-1R  insulin like growth factor receptor-1  IHC  immunohistochemistry  IKKa  inhibitor kappa B kinase alpha  ILK  integrin-linked kinase  KIT  v-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog  LDS  lithium dodecyl sulfate  MDM2  mouse double minute 2 homolog  MDR-1  multidrug resistance-1  MMP-9  matrix metalloproteinase 9  xii mRNA  messenger R N A  mTOR  mammalian target of rapamycin  MTS  (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H- trazolium)  NFKB  nuclear factor kappa  NSAID  non-steroidal anti-inflammatory drug  NSCLC  non-small cell lung cancer  p70S6K  p70 S6 kinase  P-Akt  phosphorylated Akt  PARP  poly(ADP-ribose) polymerase  PBS  phosphate buffered saline  PDGFR  platelet-derived growth factor receptor  PDK-1  phosphoinositide-dependent kinase 1  PH  pleckstrin homology  PI(3,4,5)P PI(4,5)P  2  3  B  phosphatidylinositol-3,4,5-triphosphate phosphatidylinositol-4,5-bisphosphate  PI3K  phosphatidylinositol 3-kinase  PIAs  phosphatidylinositol analogues  PIN  prostatic intraepithelial neoplasia  PKB  protein kinase B  PKCa PMS  protein kinase C alpha phenazine methosulfate  PR  progesterone receptor  PTEN  phosphatase & tensin homologue deleted on chromosome 10  RAPID  Rapid Access to Preventive Intervention Development  RNA  ribonucleic acid  RTK  receptor tyrosine kinase  SDS-PAGE  sodium dodecyl sulfate polyacrylamide gel electrophoresis  SERM  selective estrogen receptor modulators  siRNA SRC  small interfering R N A symbol for the human gene homologous in sequence to the v-src gene of the Rous sarcoma virus  TBS  tris buffered saline  TCN  tricyclic nucleoside  TMA  tumor tissue microarray  uPA  urokinase plasminogen activator  VEGF  vascular endothelial growth factor  YB-1  Y box-binding protein-1  1  CHAPTER 1 INTRODUCTION  1.1  Cancer of the Breast: Frequency. Prognosis and Origin In the year 2004 over 215,000 women will be diagnosed with breast cancer and  40,000 will die from the d i s e a s e (data from the A m e r i c a n C a n c e r Society; http://www.cancer.org/).  Several subtypes of breast cancer have been identified, with  the majority of cases originating from the epithelial cells of breast ducts and lobules (invasive ductal carcinoma and invasive lobular carcinoma, respectively).  The  development of cancer is a complex process initiated by mutations in, and dysregulation of, genetic material. Mutations may be inherited or may develop during the course of one's life upon exposure to environmental insults or viruses [1].  Not all genetic  mutations will lead to the development of cancer. However, it seems that cellular errors affecting processes such as proliferation, programmed cell death, invasion, or angiogenesis are most likely to generate what are considered cancer cells. Thus, cancers are populations of cells that exhibit self-sufficiency in growth signals, rapidly proliferate, can evade apoptosis, and are, in the worst cases, able to metastasize from the primary site of tumor development [2].  1.2  Standard Treatments for Breast Cancer  If breast cancer is detected early and does not metastasize, a woman can expect a 97% chance of surviving five years post-diagnosis. However, once the cancer has spread outside of the breast to distant organs, such as the lungs, liver or brain, there is only a 2 3 % 5-year survival rate. This point underscores how important it is to eliminate the  2 primary tumor cells, and prevent them from spreading. The standard treatment options for patients include surgery, radiation, chemotherapy and hormone therapy.  1.2.1  Chemotherapy  Chemotherapeutic agents commonly operate by preferentially inducing cell death in rapidly dividing cells.  For example, anthracyclines, such as doxorubicin and  epirubicin, induce apoptosis by preventing D N A synthesis (due to intercalation within DNA strands) and by causing double-strand breaks (stabilization of topoisomerase II in complex with DNA) [3].  Another class of drugs, known as taxanes (e.g., paclitaxel),  functions by stabilizing microtubules. This effect causes improper spindle formation and aberrant mitosis, leading to cell death [4].  Disadvantages of these standard  chemotherapies include the toxic effects on normal cells, leading to hair loss, neurotoxicity (taxanes) [5] and cardiotoxicity (anthracyclines) [6].  Additionally, many  tumors either intrinsically possess or acquire drug resistance. The mechanisms for chemoresistance may include the action of ATP-binding cassette transporters, such as p-glycoprotein, which are responsible for the efflux  of xenobiotics (such as  chemotherapy) from cells [7]. Further, overexpression of anti-apoptotic proteins, such as Bcl2, or hyperactivation of survival signaling via growth factor receptors and Akt (discussed below), can also enable cancer cells to avoid death when confronted with cytotoxic drugs [8].  1.2.2  Hormone Therapy  Beyond the chemotherapies described above, hormone therapy is another standard option for some breast cancer patients, due to the importance of estrogen signaling in breast cancer. Approximately half of all breast cancers express estrogen  3 receptor a (ERa)  [9], whereas only a small percentage of normal mammary epithelial  cells are ERa positive [10]. ERa primarily becomes activated when bound to its ligand, the steroid hormone estrogen. It should be noted that ERa can also be activated in a ligand-independent fashion, through phosphorylation induced by growth factor receptor signaling [11]. In pre-menopausal women estrogen is produced in the ovaries, whereas in post-menopausal women aromatase enzymes synthesize estrogen from androgenic precursors in adipose tissues. It has been shown that prolonged estrogen exposure predisposes women to breast cancer occurrence. This is reflected by the fact that removal of the ovaries in pre-menopausal women decreases breast cancer risk, and high body fat increases this risk in post-menopausal women [12]. Estrogen binding to ERa  exerts its effects by modulating the transcription of genes that stimulate  proliferation and growth. There are two ways in which this can occur. Firstly, ligand binding to ERa leads to the dissociation of ERa from heat shock proteins, subsequent ERa dimerization, and association of these dimers with various cofactors. This complex binds to estrogen response element ( E R E ) sequences in the promoters of affected genes to stimulate or inhibit transcription [11]. Alternatively, estrogen binding to ERa may cause its association with transcription factors such as AP-1 and S p 1 , and actually enhance the binding of these proteins to their respective promoter  recognition  sequences to affect transcription [13], [14]. Since the 1970's, selective estrogen receptor modulators ( S E R M s ) , such as tamoxifen, have been used quite successfully to treat tumors which express the estrogen and/or progesterone receptor (ER or P R ) [15], [16].  Tamoxifen exerts anti-  estrogenic activity in the breast by competitively inhibiting estrogen binding to  ERa,  4  leading to cytotoxic and cytostatic effects in hormone-dependent tumor cells.  More  recently, aromatase inhibitors (e.g., letrozole), which inhibit the synthesis of estrogen, have also proven to be an effective treatment against hormone responsive tumors [17]. Unfortunately, therapies targeting estrogen signaling are only useful for patients which express the E R , and most advanced breast cancer patients that initially responded to tamoxifen will become resistant [18]. Although the mechanism for tamoxifen resistance is unclear, it may be related to 1) loss of E R a expression, 2) changes in expression of E R a cofactors, or 3) crosstalk between E R a and growth factor signaling [11], [19].  1.3  Molecular Therapeutics: Targeting Receptor Tyrosine Kinases Resistance to chemotherapy and anti-estrogen therapy, in part, is associated  with upregulation of growth factor signaling and hyperactive survival networks within tumor cells. In an effort to reduce side effects of cancer therapy and bypass drug resistance, researchers are now focused on designing drugs to specifically target the disrupted cellular pathways of tumors [20].  A s many kinases are commonly  overexpressed in breast cancer and can promote tumor growth, metastasis, and resistance to chemotherapies and endocrine therapies, much effort has been expended in developing inhibitors to these particular molecules [21].  For example, receptor  tyrosine kinases (RTKs) represent a major class of molecules for which small molecule inhibitors have recently been developed. The first clinically successful R T K inhibitor was STI571 (Gleevec, imatinib mesylate), which is active against the proteins Abl, BcrAbl, KIT, and the platelet-derived growth factor receptor ( P D G F R ) . STI571 has proven efficacious in treating patients with chronic myeloid leukemia (CML), as this cancer expresses and is dependent upon the Bcr-Abl fusion protein, [22], and also in treating  5 patients with gastrointestinal stromal tumors (GISTs) which commonly express mutated KIT or P D G F R proteins [23]. Other notable R T K s being targeted for therapy are members of the human epidermal growth factor ( E G F R ) family of transmembrane receptor tyrosine kinases ( E G F R / H E R - 1 , H E R - 2 , H E R - 3 , HER-4) [24], which are expressed to varying degrees in many cancer types. HER-2, for example, is overexpressed in 25-30% of invasive ductal breast carcinomas. When H E R - 2 is overexpressed, it is associated with poor patient prognosis and increased risk of recurrence [25]. Additionally, overexpression of HER-2 in a cancer cell line model, using M C F - 7 cells, leads to increased resistance to chemotherapy [26].  However, a clinically applicable monoclonal antibody targeting  H E R - 2 , called Herceptin, was developed to block the activation of the receptor [27]. This drug achieves a 30% response rate when used as a treatment of metastatic breast cancer, and demonstrates synergistic effect in combination with chemotherapy [28]. Further, targeting the tyrosine kinase domain of E G F R with small molecule inhibitors such as ZD-1839 (Iressa), has proven effective for 10% of non-small cell lung cancer (NSCLC) patients in phase II clinical trials [29].  It is possible that Iressa could benefit  breast cancer patients as well, as it has been shown to inhibit growth of breast cancer cell lines [30]. E G F R is highly expressed in about 2 7 % of primary breast cancers [31], although expression of the receptor alone does not seem to predict sensitivity to Iressa [32]. N S C L C patients whose tumors contain specific mutations around the ATP-binding pocket of the tyrosine kinase domain of E G F R , which leads to hyperactive signaling, appear to have the best clinical response to Iressa [33].  6 Besides the EGF-receptor family, overexpression of another R T K , the insulin like growth factor receptor-1 (IGF-1R) is associated with malignant transformation in vitro and in vivo [34].  Signaling through the IGF-1R is also important for breast cancer  invasion and metastasis [35].  Therefore, drugs are being sought to inhibit this receptor.  It was recently demonstrated that targeting the IGF-1R with a human monoclonal antibody significantly inhibited the growth of breast tumors in vivo [36].  Another  strategy for inhibiting IGF-1R is to use kinase inhibitors. Two such therapies have been developed, NVP-AEW541 [37] and NVP-ADW742 [38] that have both shown efficacy in vivo.  Further studies will determine whether these drugs will be useful for cancer  patients. By combining R T K inhibitors with standard chemotherapy, this removes one avenue for developing cancer drug resistance and hopefully will result in more success in the clinic.  1.4  The PI3K/ P-Akt Pathway: Receptor Signaling Convergence Point R e s e a r c h e r s are also setting their sights on inhibiting the  downstream  intermediates of growth factor receptors. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been shown to be a major convergence point for R T K signaling and is activated by numerous receptors, including IGF-1R and H E R - 2 (see Figure 1) [39]. Actiavted R T K s recruit and activate PI3K. PI3Ks are actually a family of proteins which have been divided into three classes (I, II, and III), based on structure and substrate specificities.  Growth factor R T K s primarily associate with the class IA PI3Ks [40].  Class IA PI3Ks are heterodimers composed of a regulatory (p85) and catalytic (p110) subunit [41].  The src-homology 2 (SH2) domains of the p85 subunit bind phospho-  tyrosine residues on an activated receptor or on associated adaptor proteins and recruit  7  Growth Factor Receptor  PI-3,4,5-P3 PI-4.5-P2  AKT LY294002  KD  l-cr Thr308  ^ | o <«-  ILK, PDK2, DNA-PK, Akt  Ser473  mTOR, p27, GSK-3B, FKHR1  survival, proliferation, growth  Figure 1. The PI3K/Akt S i g n a l i n g Network. This schematic illustrates the activation of the PI3K/Akt pathway by receptor tyrosine kinases (RTKs). Activated RTKs recruit and activate PI3K, which converts phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P ). The PI(3,4,5)P lipids trigger attachment of Akt and PDK-1 to the plasma membrane (PM) by their P H domains. At the P M , PDK-1 phosphorylates threonine 308 on the kinase domain (KD) of Akt. To achieve full activation, Akt must also be phosphorylated in its regulatory domain (RD) at serine 473. This occurs either by ILK, D N A - P K , PDK-2 or autophosphorylation. Phospho-Akt then dissociates from the P M and proceeds to phosphorylate both cytoplasmic and nuclear target proteins, including mTOR, F K H R , G S K - 3 P and p27Kip-i. The PI3K7Akt system is also negatively regulated by the phosphatase P T E N , which converts PI(3,4,5)P lipids to PI(4,5)P . 3  3  2  3  8 the enzyme to the membrane. molecule  At the membrane PI3K phosphorylates the lipid  phosphatidylinositol-4,5-bisphosphate  (PI(4,5)P2),  to  create  phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3) [42] in response to growth factor signals.  The PI(3,4,5)P3 lipids trigger attachment of the protein Akt (also known as  protein kinase B, or P K B ) to the plasma membrane by specifically binding its pleckstrin homology (PH) domain. Akt is a serine/threonine kinase that contains a kinase domain and hydrophobic tail in addition to its P H domain.  O n c e attached to the plasma  membrane, Akt subsequently becomes phosphorylated at two key sites, threonine 308 (in the kinase domain) and serine 473 (in the hydrophobic tail).  Phosphorylation of  serine 473 is required for full activation and has been shown to correlate with Akt kinase activity [43]. Threonine 308 is phosphorylated by phosphoinositide-dependent kinase 1 (PDK1), while the mechanism of phosphorylation at serine 473 is controversial. There are several theories to explain serine 473 phosphorylation, including the action of integrin-linked kinase (ILK) [44], D N A dependent protein kinase ( D N A - P K ) [45], autophosphorylation [46], or an as yet unidentified " P D K 2 " . Once Akt is fully activated (P-Akt) it dissociates from the plasma membrane and proceeds to phosphorylate both cytoplasmic and nuclear target proteins, which will be discussed below. The PI3K/Akt system is also negatively regulated by the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which is responsible for converting PI(3,4,5)P3 lipids to PI(4,5)P [47]. In PTEN-null cells the PI3K/Akt pathway is constitutively activated [48]. 2  Loss of P T E N expression as detected by immunohistochemistry has been reported in approximately 4 0 % of breast cancers [49], [50], [51].  Only one study has found an  association between loss of P T E N and disease-related death in breast cancer [49].  9 However, P T E N mutations in H E R - 2 positive tumors predict resistance to Herceptin treatment [51]. There are three members of the Akt family, Akt1, Akt2 and Akt3. Akt1 is located on chromosome 14q [52], Akt2  is on chromosome 19q [53], and Akt3  maps to  chromosome 1q [54]. They exhibit 80% amino acid sequence homology, share the same key phosphorylation sites (Thr308/309/305 and Ser473/474/472, respectively), and appear to be regulated by similar mechanisms in cell culture [55].  However,  differences in tissue distribution and results from knockout experiments suggest that the isoforms are not redundant in function.  At the level of m R N A , Akt1 and Akt2 are  expressed in most normal tissues [54], whereas Akt3 is predominantly found in the brain and testis, with lower levels in the heart, kidney and placenta [56], [54].  Akt1 knockout  mice exhibit enhanced apoptosis and stunted growth [57].  In contrast, Akt2 knockout  mice display a diabetic phenotype, but normal size [58].  Double Akt1/Akt2 knockout  mice have also been generated [59].  These mice die shortly after birth and display  impeded adipogenesis, as well as deficiencies in growth, muscle cell size and bone development.  A n Akt3 knockout mouse has not been reported to date.  This work  implies that specific targets or functions of the isoforms exist, which cannot be compensated for by the other two.  However, all three isoforms when constitutively  activated can transform normal cells [43], [60], implying that many key substrates are shared. The diverse substrates of the Akt family include proteins that regulate the cell cycle, cell growth, invasion, angiogenesis, translation and, perhaps most importantly, cell survival. Akt phosphorylates and inactivates glycogen synthase kinase-36 ( G S K -  10  36), a protein responsible for controlling proteolysis of cyclin D1 [61]. Thus, activation of Akt allows levels of cyclin D1, a cell cycle promoter, to accumulate in the cell by preventing its degradation.  Akt also regulates the cell cycle inhibitor p27(Kip1) by  phosphorylating its nuclear localization motif and trapping it in the cytoplasm [62], [63]. Phospho-p27(Kip1) can no longer inhibit cyclin dependent kinase-2 (CDK2) in the nucleus, thus allowing cell cycle progression. Another important target of the PI3K/Akt pathway is the mammalian target of rapamycin (mTOR).  Inhibition of PI3K with  LY294002 results in inactivation of m T O R [64] and one study has shown direct phosphorylation of m T O R by Akt [65].  m T O R is responsible for activating p70 S 6  kinase (p70S6K), a translational enhancer, [66] and inhibiting the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a translational repressor [67].  Finally, Akt  mediates cell survival through several other targets. By phosphorylating members of the F O X O subclass of forkhead transcription factors, F O X 0 1 / F K H R 1 (forkhead in rhabdomyosarcoma) [68] and F O X 0 4 / A F X (ALL1 fused gene from chromosome X) [69], Akt prevents their nuclear localization and thus inhibits the transcription of proapoptotic genes, such as F a s ligand. Akt also phosphorylates inhibitor kappa B kinase alpha (IKKa) [70], which indirectly leads to activation and nuclear translocation of nuclear factor kappa B (NFKB), a survival-promoting transcription factor. N F K B appears to play a role in drug resistance by upregulating expression of multidrug resistance-1 (MDR-1) [71].  Additionally, N F K B has been shown to negatively regulate P T E N expression,  creating a feedback loop for Akt activation [72]. Another notable target of Akt is M D M 2 (mouse double minute 2 homolog). nuclear translocation [73].  Phosphorylation of M D M 2 by Akt promotes its  In the nucleus M D M 2 binds to, and promotes the  11 degradation of, p53, a key regulator of apoptosis. Thus, through wide-ranging effectors, the activity of Akt provides a powerful growth and survival advantage.  1.5  The Role of Akt in Cancer Development. Progression, and Drug Resistance Evidence from experimental models suggests that Akt is a key regulator of tumor  development and progression.  Mammary epithelium-specific coexpression of  constitutively active Akt1 and a mutant polyomavirus middle T antigen promotes tumor progression in mice [74].  It has also been shown that constitutive activation of Akt1  transforms NIH3T3 mouse fibroblast cells, which can then form tumors in nude mice [43]. Additionally, Akt promotes cancer cell invasion and metastasis. Overexpression of wild-type Akt1 doubles the rate of HT1080 cell migration and triples their rate of invasion through Matrigel.  The observed increase in migration is correlated with  upregulation of matrix metalloproteinase 9 (MMP-9) production [75].  Another study  showed that the overexpression of Akt2 increased the ability of breast cancer cells to invade by upregulating  61 integrins.  This study went on to show that Akt2-  overexpressing xenografts exhibit increased incidence of metastases when compared with xenografts which developed from control cells expressing the empty vector [76]. Akt is also able to regulate blood vessel formation. Expression of activated Akt in the chorioallantoic membrane of chicken embryo induces angiogenesis [77].  Finally, in  addition to its role in cancer development and migration, Akt promotes the survival of tumor cells when confronted with chemotherapeutics and radiation.  For example, in  breast cancer cells, expression of constitutively active Akt1 reduced the ability of doxorubicin [78] or ionizing radiation [79] to induce apoptosis.  On the other hand,  combination of the PI3K inhibitor, LY294002, or a dominant-negative Akt1 with common  12 chemotherapeutic agents increased the drug-induced apoptosis [78].  T h e s e data  suggest that inhibiting P-Akt signaling could have important therapeutic applications.  1.6  Expression of P-Akt in Primary Tumors In support of P-Akt as a molecular target for therapy, several laboratories report  that P-Akt is expressed in aggressive primary tumors. Akt is activated in a wide range of cancers, including carcinomas of the breast [80], kidney [81], lung [82], colon [83] and prostate [84].  High levels of P-Akt expression are associated with metastatic disease in  renal cell carcinoma patients [81] and are also associated with high-grade, poorly differentiated prostate cancer [84].  In the context of breast cancer, a study of  endocrine-treated breast cancer patients (n = 93) showed that P-Akt1 expression was linked to increased relapse with distant metastasis [80].  Additionally, Akt1 kinase  activity was shown to be significantly increased in approximately 40-50% of tumor samples from breast (19/50 cases), ovary (11/28 cases) and prostate patients (16/30 cases) relative to normal tissue [43].  1.7  Akt as a Molecular Therapeutic Target: Current Advances in Drug Development Currently, there are no Akt inhibitors available to patients.  However, recent  studies inhibiting Akt or Akt phosphorylation in preclinical models have attempted to answer several important questions regarding the utility of such inhibitors in cancer treatment.  It was previously unclear whether inhibition of Akt would itself induce  apoptosis or rather would sensitize cells to apoptosis induced by other agents. It was also unknown whether all cells would respond the same to inhibition of Akt signaling, or whether there would be differences in response based on pathway activation. One way to inhibit Akt specifically was achieved by retroviral expression of dominiant negative  13 Akt (Akt-DN) [85]. Akt-DN inhibited the growth of tumor cell lines and induced apoptosis preferentially in cell lines expressing high levels of P-Akt. The injection of Akt-DNadenovirus into mice containing breast cancer cell (ZR75-1) xenographs lead to 90% inhibition of tumor growth.  This group also showed that Akt-DN does not cause  apoptosis in normal cells, presumably because all of the normal cell lines they used express low levels of P-Akt. Other studies have recently reported the development or identification of small molecule inhibitors to inhibit Akt signaling. Castillo et al. described the synthesis and biological analysis of phosphatidylinositol analogues (PIAs) used to specifically inhibit Akt activation [86].  These PIAs were originally developed by  modeling the interaction of phosphatidylinositol 3,4 bisphosphate with the P H domain of Akt. Subsequent modifications to the inositol ring improved stability and showed even greater potency for inhibiting Akt phosphorylation (IC o < 5 \xM) than the first generation 5  analogues [87], [88].  In cell culture PIAs inhibit Akt phosphorylation and downstream  signaling targets within 2 hours. Treatment for 24 hours results in 40 to 80% apoptosis induction in cell lines in which Akt is highly activated, whereas cells with low levels of Akt activation show only 10 to 20% apoptosis. PIAs may not be useful in vivo, however, because of potential poor bioavailability and toxicity [89]. A novel Akt inhibitor was also recently identified from the National Cancer Institute Diversity Set, and is promising based on pre-clinical evidence [90]. This inhibitor, termed API-2 (Akt/protein kinase B signaling inhibitor-2), is a tricyclic nucleoside that, again, selectively inhibits cancer cells that express high levels of activated Akt.  Anti-cancer activity of A P I - 2 was  demonstrated both in cell culture and in an animal model.  API-2 was identified  previously as an anticancer agent that inhibits DNA synthesis, and was designated T C N  14 (tricyclic nucleoside) [91]. A P I - 2 / T C N was used in phase I and II clinical trials where high doses resulted in side effects such as hepatotoxicity, thrombocytopenia, and hyperglycemia, which suggest that it may have limited clinical use [92], [93].  It is  unclear if these side effects are related to inhibition of Akt signaling or alternative mechanisms of API-2 action. A s a final example, the non-steroidal anti-inflammatory drug (NSAID) celecoxib has been shown to inhibit Akt phosphorylation and induce apoptosis in prostate cancer cells [94]. In addition to its effect on Akt phosphorylation, the known tolerability of celecoxib in patients made it a superior lead compound for the development of more potent analogues. Celecoxib is a cyclooxygenase-2 (COX-2) inhibitor traditionally used to treat the symptoms of patients with osteoarthritis and rheumatoid arthritis because of its antiinflammatory properties [95]. It was further shown that celecoxib is chemopreventative against colon cancer [96] and was approved by the F D A for the treatment of patients with familial adenomatous polyposis [97].  In a rat model of breast cancer, celecoxib  treatment can both prevent and regress the formation of tumors induced by 7, 12dimethylbenz(a)anthracene (DMBA) [98], [99]. The effectiveness of celecoxib against cancer was initially attributed to the anti-apoptotic roles of C O X - 2 . Celecoxib induces the mitochondrial apoptosis pathway [100], however, apoptosis can be induced in cells that do not express C O X - 2 , indicating C O X - 2 independent mechanisms for cell death [94].  Several targets were proposed, including Akt [94].  Phosphorylation of Akt is  downregulated in response to celecoxib treatment, which has been attributed to, at least in part, the inhibition of PDK-1 activity [101]. However, the doses of celecoxib which inhibit Akt phosphorylation in cell lines [50 to 100 u,M] are well above the 2 u.M serum  15 concentrations achievable in patients [102].  Nevertheless, the observation that  celecoxib inhibited P-Akt set the course for the development of analogues that optimally disrupt this pathway at lower concentrations. Through structure-activity analysis, Zhu et al. determined the structural features of celecoxib necessary for C O X - 2 inhibition and for apoptosis induction [103].  By  manipulating the 4-methylphenyl moiety of celecoxib, and maintaining the pyrazole ring, they created a series of celecoxib analogues (see Figure 2). The new analogues have no C O X - 2 inhibitory ability, increased PDK-1 inhibition, and improved apoptosis induction [104]. In particular, the compounds designated OSU-03012 and OSU-03013 could inhibit PDK-1 activity in vitro with an IC o of 2 to 5 u . M , 5  and inhibit  Akt  phosphorylation at doses less than 10 u.M in cell culture. Upon longer exposure, these inhibitors were shown to induce apoptosis in P C - 3 prostate cancer cells. A s a part of the Rapid A c c e s s to Preventive Intervention Development (RAPID) program at the National Cancer Institute, (http://www3.cancer.gov/prevention/rapid/). a panel of 60 cancer cell lines was screened for response to OSU-03012 and OSU-03013. They determined that the compounds were potent inhibitors of tumor cell growth with an average IC5o= ~1 to 2 uM  The characterization of the celecoxib analogues thus far indicates that they could  be very useful for treating many types of cancer.  1.8  Thesis Objectives  Aim 1: Expression Profiling of P-Akt in Normal and Cancer Tissues of the Breast Significance Although the key role of Akt in cancer aggression has been definitively shown in experimental models, the expression and activity of the P-Akt signaling network in  16  B.  Analogue Design Strategy A  fc  N-N  N-N  "^scyiH,  ^ S O ^ J H ,  OSU02067  rs.  N-N  - lS °t o»  c  A  9n  B  k>  N-N  b_«^  OSU03008  N-N  cv^v 0  OSU03012  N-N  by;  OSU03013  Figure 2. Structure and Design of the C e l e c o x i b A n a l o g u e s . (A) The structure of the parent compound, celecoxib, is shown. This molecule includes three notable features; (1) the heterocyclic pyrazole ring with a tri-fluoromethyl moiety, (2) a sulfonamide group and (3) a terminal phenyl ring. (B) An initial strategy to increase the cytotoxic capability of celecoxib generated OSU-02067. This compound has a large hydrophobic, tricyclic aromatic ring replacing the terminal phenyl ring of celecoxib. (C) The latest strategy to further optimize apoptosis induction and strengthen PDK-1 inhibition, lead to the creation of OSU-03008, OSU-03012 and OSU-03013. The sulfonamide moiety of OSU-02067 was replaced with a series of heteroatom-rich functional groups to potentially increase hydrogen bonding of the compounds within the kinase pocket of P D K - 1 .  17 primary breast tumors is not well characterized. Additionally, the expression of P-Akt in normal breast tissue has not been explored. Experimental Design In order to better understand the function of activated Akt in breast cancer, we evaluated its expression on a tissue microarray containing tumor samples from 481 breast cancer patients.  We also assessed P-Akt in normal breast tissues to help  establish the potential significance of its expression in tumors. Moreover, we examined the association of P-Akt expression with 30 other proteins previously immunostained on the same T M A series.  Aim 2: Characterization of Small Molecule Inhibitors to the Akt Signaling Pathway Significance Pre-clinical models targeting P-Akt signaling clearly show that tumor cells depend on this pathway for survival and drug-resistance. However, there are currently no inhibitors to P-Akt available for treating patients. Experimental Design The effectiveness of celecoxib analogues was evaluated for the treatment of breast cancer. Three compounds, OSU-03008, O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 , were examined for their ability to inhibit Akt phosphorylation and decrease viability in breast cancer cells. The necessity for P-Akt inhibition, in regards to efficacy of analogue treatment, was also determined. Additionally, the effects of serum concentration and cell confluency during analogue treatment were analyzed.  18  CHAPTER 2 MATERIALS AND METHODS  2.1  Tumor Tissue Microarrav Construction and Patient Information For construction of the TMA, 481 primary breast cancer samples were obtained  from archival cases at Vancouver General Hospital dating between 1974 and 1995. Patient information and tumor pathology are summarized in Table 1. Tumor samples were taken prior to initiation of cancer treatment, formalin-fixed and embedded in paraffin [105].  A hematoxylin and eosin stained section of each tumor block was used  to define and mark representative tumor regions.  This section served as a guide for  the selection of two 0.6 mm punches from each original tumor block which were transferred to three composite recipient array blocks using a Tissue Micro Arrayer (Beecher Instruments; Sliver Springs, MD) as previously described [105]. Serial 4 u,mthick sections of the arrays were cut using a Leica microtome (Leica Microsystems; Nussloch, Germany) and mounted onto charged polylysine-coated glass slides. This work was done at the Genetic Pathology Evaluation Centre ( G P E C ) (Vancouver, British Columbia).  2.2  Normal Breast Tissues Twenty-six samples of normal breast tissue were obtained from patients  undergoing reduction mammoplasty at Vancouver General Hospital from 2000 to 2001. The tissue was formalin-fixed and paraffin-embedded. Serial 4 jxm-thick sections were cut from each tissue block.  One section of each sample was stained with hematoxylin  and eosin and then assessed by a pathologist (N. Makretsov) to ensure presence of  19 Characteristics  Data  Correlation to P-Akt (Chi-Square)  Diagnostic Age, yr Mean Median Range  61.01 63.07 59.03 0.957  Lymph Node Status, n (%) Negative Positive Unknown  266 (60.7) 126 (28.8) 46 (10.5) 0.170  ER" status, n (%) 93 (21.2) 216 (49.3) 129 (29.4)  Negative Positive Unknown  0.276  Tumor Grade, n (%) 1 2 3  94 (21.5) 236 (53.9) 108 (24.6)  <= 5mm <= 1cm <= 2cm > 2cm Unknown  4(2.2) 64 (14.6) 142 (32.4) 147 (33.6) 81  Histology, n (%) in situ carcinoma IDC , NOS IDC, variants ILC IDC and ILC  16 (3.6) 353 (80.5) 24 (5.5) 43 (10) 2 (0.4)  0.900  Tumor Size, n (%)  b  0.143  c  d  Total Follow-up, yr 14.47 15.4 20.34  Mean Median Range P-Akt Expression, n (%)  43 (11.0) 122 (31.3) 120 (30.8) 105 (26.9) 48  0 1 2 3 unscorable  Table 1. Characteristics of the TMA Study Population. Listed are the known characteristics of patients, and their tumors, for the 438 cases of invasive breast carcinoma included on the TMAs. The correlation of P-Akt to the clinicopathologic features was determined by Chi-Square analysis (p values less than 0.05 are considered significant). E R , estrogen receptor; I D C , invasive ductal carcinoma; N O S , not otherwise specified; I L C , invasive lobular carcinoma. a  b  d  c  20 normal breast epithelium.  Ages of patients and general pathology are described in  Figure 5.  2.3  P-Akt Immunohistochemistry 2.3.1  Validation of the P-Aktw7* IHC-Specific Antibody  Two controls were performed to verify the specificity of the phospho-Akt (S473) IHC specific antibody (Cat#9277; Cell Signaling Technology (CST); Beverly, MA). For the first control, P-Akt was induced in T47D cells and analyzed by both western blot and immunohistochemistry. To induce P-Akt T47D cells were first grown to 90% confluence in normal growth medium and then grown in medium without F B S overnight (refer to section 2.7).  The next day the cells were treated with or without IGF-I (100 ng/ml)  (Diagnostic Systems Laboratories, Inc.; Webster, TX) for 30 minutes. IGF-I is known to stimulate the phosphorylation of Akt on serine 473 [106].  Initially, the expression and  localization of P-Akt were verified by western blot. The treated T47Ds were lysed to separate cytoplasmic and nuclear proteins (see section 2.8). Protein extracts (100 u,g each) were subjected to S D S - P A G E and transferred to a nitrocellulose membrane (see section 2.9).  The membrane was probed with phospho- and total-Akt  primary  antibodies ( C S T Cat#9271 and #9272, respectively). Next, the T47D cells treated with and without IGF-I were examined by immunohistochemistry.  The treated cells were  pelleted by gentle centrifugation (1000 rpm, 5 minutes) and then fixed in 10% formalin for 20 minutes. The fixed cells were mixed with warm (37°C) Histogel (Richard-Allan Scientific; Kalamazoo, Ml) and then placed on ice to form a solid clot. The clots were paraffin-embedded in the Pathology lab at Vancouver General Hospital, sectioned 4 \im thick, mounted onto charged polylysine-coated slides, and processed by IHC (section  21 2.3.2). A s a final control, the IHC-Specific Phospho-Akt antibody was incubated for two hours on ice with its specific blocking peptide (Cat#1140; C S T ) , prior to the application of the antibody for immunohistochemistry on a section of breast cancer tissue (obtained from C S T ) .  2.3.2  Protocol for P-Akt Immunohistochemistry  This protocol was used to detect P-Akt (S473) expression in the T M A s , normal breast tissue, and T47D cell blocks. Sections of each were first deparafinized in xylene and rehydrated in graded ethanol solutions.  Antigen retrieval was achieved by  incubating the slides for 30 minutes in 10 mM citrate buffer pH 6.0 (Appendix 1a) at 90°C in a vegetable steamer. Endogenous peroxidases were quenched by incubating the sections for 10 minutes in 3% hydrogen peroxide diluted in water.  Prior to  application of the primary antibody, non-specific interactions were blocked for 30 minutes using a non-serum (0.25% casein in P B S ) blocking reagent (Cat# X0909; D A K O , Denmark), followed by twenty minutes with an avidin/biotin blocking solution (DAKO). The primary antibody (Phospho-Akt S473 IHC Specific, Cat# 9277; C S T ) was diluted 1:250 with a 1% bovine serum albumin solution in T B S (Appendix 1b), applied to the slides and incubated overnight at 4°C. For signal amplification, we then used the L S A B + System (DAKO), which involved incubation with a biotinylated secondary antibody (30 minutes) followed by peroxidase-labelled streptavidin treatment (30 minutes). Signal was visualized by addition of NovaRed substrate-chromogen solution (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin. Additionally, a negative control reaction with no primary antibody was performed for each T M A slide, normal breast section or cell block, in parallel.  22  2.4  Immunohistochemistry for Other Proteins on the TMA Immunohistochemistry for 30 other proteins was also performed on the T M A s  using methods with minor modifications to the above P-Akt IHC protocol.  Antibody  details, including dilutions and antigen retrieval methods, for the other proteins are described on the website: www.gpec.ubc.ca. and in Appendix 2.  2.5  Scoring of P-Akt Staining Two pathologists and a basic scientist arrived at a consensus score for each core  on the T M A and normal breast section. The scoring system for P-Akt expression was as follows: 0) negative, 1) weak, 2) moderate and 3) high staining intensity.  Cores on  the T M A with staining that could not be interpreted (i.e., no tumor tissue present) were scored as "X". In the occasional instance of heterogeneous staining, the overall score was determined based on 7 5 % or more of epithelial cells expressing the same level of P-Akt. In any case where duplicate T M A cores had conflicting scores, the higher score was taken. Raw scores were entered into a standardized electronic spreadsheet (Excel for Windows, Microsoft, Redmond, WA).  2.6  Statistical Analysis of IHC Data Data was processed using Deconvoluter software designed for management of  T M A data [107], and then analyzed with the S P S S for Windows statistical software package ( S P S S version 11; S P S S , Chicago, IL). For some analyses P-Akt scores were grouped as low (0 or 1) versus high (2 or 3) expression to create binary categories. Univariate analysis between proteins on the T M A was calculated by Spearman correlation coefficient and Fisher's Exact Test using S P S S . Data was considered significant when p < 0.05. We did not use the Bonferroni multicomparison test because  23 this aspect of the project was simply a hypothesis-generating exercise. The difference in P-Akt expression between normal and tumor breast tissue was calculated using ChiSquare analysis. The Kaplan-Meier method was used for survival analysis, and significance of differences between survival curves was assessed by the log-rank test. 2.7  Cell Culture Conditions MDA-MB-453, MDA-MB-231, MCF-7 and T47D breast cancer cells were  obtained from ATCC.  The 184htrt preneoplastic breast cell line and their growth  conditions were described previously [106]. The MDA-MB-453, MCF-7 and T47D cells were grown in RPMI-1640 without phenol red (Cat# 11835, Invitrogen), supplemented with 5% fetal bovine serum (FBS), and 100 units/ml of penicillin and streptomycin (Invitrogen; Carlsbad, CA). The MDA-MB-231 cells were grown in DMEM (Cat# 11995, Invitrogen), also supplemented with 5% fetal bovine serum and antibiotics. All cells were grown at 37°C with 5% CO2. Growth medium was replaced every three days and stock plates were trypsinized and replated 1:10 once a week.  Cell numbers for  experimental plating were based on maintaining the same confluency between cell lines. Thus, cell lines of smaller size (MDA-MB-453 and T47D) were plated at higher number than larger cell types (MDA-MB-231, MCF-7, 184htrt) to achieve the same final confluency. 2.8  Cell Lysis and Protein Extraction 2.8.1  Whole Cell Lysis  Cells were harvested by scraping, rinsed in ice-cold P B S (Appendix 1c), and pelleted by centrifuging at 3000 rpm for 5 minutes. The pellets were resuspended in four to five packed cell volumes of whole cell lysis buffer (Appendix 1d). Cells were  24 incubated in the lysis buffer for 30-45 minutes on ice, with finger-vortexing every 10 minutes. The lysates were sonicated (amplitude = 40 watts; cycle = 1 second; 5 cycles per 100 u.l volume) and then clarified by centrifuging for 10 minutes at 12000 rpm, 4°C. The supernatants were transferred to a fresh microtube, quantified (section 2.8.3), and stored at -80°C.  2.8.2  Cytoplasmic and Nuclear Fractionation  Cells were harvested and pelleted as described in section 2.8.1. To extract cytoplasmic proteins, cells were resuspended in 4-6 packed cell volumes of cytoplasmic extraction buffer (Appendix 1e), incubated for 8 minutes on ice, and then passed through a 22 gauge needle 10 times. Nuclei were pelleted by centrifuging the lysate at 10000 rpm for 2 minutes at 4°C, and the supernatant (the cytoplasmic fraction) was transferred to a fresh tube.  The pelleted nuclei were rinsed in a W a s h Buffer B  (Appendix 1f) and then resuspended in one packed cell volume of nuclear extraction buffer (Appendix 1g). The nuclei were incubated on ice for 30 minutes, ruptured by passing through a 26 gauge needle 10 times, and then incubated for another 10 minutes on ice. Nuclear debris was pelleted by centrifuging for 20 minutes at 13000 rpm at 4°C. Both fractions were quantified as described in section 2.8.3 and stored at -80°C.  2.8.3  Protein Quantification  Proteins were quantified using the Bio-Rad Protein A s s a y (Cat#500-0006; BioR a d , Hercules, C A ) .  Standard curves were created by 1) incubating increasing  amounts of bovine serum albumin (1 pg, 2 \ig, 5 u,g, 10 u.g, 20 ng) in 1 ml of dye reagent for 20 minutes, 2) reading the absorbance of each standard at 595 nm with a  25 spectrophotometer  and 3) plotting the absorbance values versus the  protein  concentration and performing linear regression. 1-2 \x\ of cell protein extract were then incubated with 1 ml of dye reagent for 20 minutes and absorbance values read at 595 nm were used to extrapolate concentrations from the standard curve.  Quantifications  were performed in at least duplicate.  2.9  Western Blotting 2.9.1  Preparation of samples  A n amount of 50-100 jxg of whole cell, cytoplasmic, or nuclear extracts was combined 3:1 with 4 X L D S sample buffer (Cat# NP0008, Invitrogen; Appendix 1h) with 2-mercaptoethanol and boiled for 5 minutes, to denature the proteins.  2.9.2  Electrophoresis  Proteins were separated using the S D S - P A G E gel and buffer system. Denatured samples and 5 uJ of prestained broad range standards (Cat#161-0318; Bio-Rad) were loaded onto a 12% acrylamide gel submerged in running buffer (running  buffer:  Appendix 1i, stacking gel: Appendix 1j, separating gel: Appendix 1k). Samples were compressed in the stacking gel using 70 Volts of current and then separated using 120 Volts of current for 1.5 to 2.5 hours.  2.9.3  Transfer of Separated Proteins to Nitrocellulose Membrane  Proteins were transferred from the separating gel to a 0.45 \im  pore-size  nitrocellulose membrane using the Mini Trans-Blot® Electrophoretic Transfer Cell (BioRad). First, gels and membranes were pre-equilibrated in transfer buffer (Appendix 11) for 15 minutes. Transfers occurred for 1 hour at 110 Volts in chilled transfer buffer. After transfer, blots were stained with Ponceau-S (0.1% in 5% acetic acid solution; Cat#  26 P3504, Sigma) to inspect for proper separation and transfer of proteins and to confirm even loading of samples.  2.9.4  Primary Antibodies  Nonspecific protein binding sites on the nitrocellulose membranes were blocked with 5% bovine serum albumin in P B S with 0.1% Tween (Appendix 1c) for one hour at room temperature before addition of primary antibodies.  Antibodies against the  following proteins were used to probe the western blots: Actin (goat polyclonal; Cat# sc1616;  Santa Cruz Biotechnology, Inc., C A ) , total Akt (rabbit polyclonal; Cat# 9272,  CST), phospho-Akt er473 (rabbit polyclonal; Cat# 9271, C S T ) , phospho-Akt 308 (rabbit S  thr  polyclonal; Cat# 9275, C S T ) , total 4 E - B P 1 (rabbit polyclonal; Cat# 9452, C S T ) , phospho-4E-BP1 (Ser65) (rabbit polyclonal; Cat# 9451, C S T ) , phospho-GSK-3a/B (rabbit polyclonal; Cat# 9331, C S T ) , total G S K - 3 p (mouse monoclonal; Cat# sc-7291, Santa C r u z Biotechnology), H E R - 2 (rabbit polyclonal; Cat# ab-2428, A b e a m ; Cambridge, UK), and cleaved P A R P (Asp214) (rabbit polyclonal; Cat# 9541, C S T ) . All primary antibodies were diluted 1:1000 in 5% B S A (in phosphate buffer solution with 0.1% Tween), with the exception of anti-Actin (1:500), and were incubated with the membrane overnight on a rocking platform at 4°C.  2.9.5  Detection of Primary Antibody: Protein Complexes  After incubation with primary antibody, membranes were washed three times in P B S with 0.1% Tween. In order to visualize the protein/antibody complexes, an antimouse/rabbit/or goat IgG horseradish peroxidase-linked antibody  (Amersham;  Piscataway, NJ) was next incubated with the membranes. Secondary antibody was diluted 1:7500-10000 in 5% non-fat milk in P B S with 0.1% Tween.  Membranes were  27 incubated with the secondary antibody for 1 to 1.5 hours at 4°C on a rocking platform. The nitrocellulose was again washed three times with P B S with 0.1% Tween before subjection to the enhanced chemiluminescence (ECL) detection system (Amersham) and exposure to film.  2.10 Akt Immunoprecipitation and Kinase Assay Nonradioactive Akt kinase assays were performed as described by Castillo et al. [86], with the following modifications to the protocol.  Briefly, 500 \ig of whole cell  extracted protein (section 2.8.1), were immunoprecipitated with the 5 G 3 pan-Akt antibody (mouse monoclonal; Cat# 2966, C S T ) , rotating overnight at 4°C in a 500 \i\ volume of whole cell lysis buffer. A s a negative control, 500 u,g of protein was incubated with normal mouse IgG (Upstate) in place of 5 G 3 antibody.  The following day the  antibody:protein complexes were incubated with protein G-coated agarose beads (Cat# P4691; Sigma), rotating for 4 hours at 4°C. The beads were washed two times in whole cell lysis buffer, one time in Sato wash buffer (Appendix 1m) [108] and two times in kinase assay buffer (Appendix 1n). To assay for Akt kinase activity, the beads were incubated for 30 minutes at 30°C in kinase assay buffer with 1 uxj of recombinant G S K - 3 protein (CST) and 200 \iM A T P . 4X L D S sample buffer with 6-mercaptoethanol was added to stop the reaction. The assays were boiled for 5 minutes, spun at 10000 rpm for 1 minute to pellet the beads, and then the supernatants were frozen at -20°C. For further analysis, one third of each reaction was separated on a 12% acrylamide gel and western blotted (section 2.9).  2.11  Drug Preparation  28 LY294002 was dissolved in D M S O to a stock concentration of 30 m M . Celecoxib was obtained from Pharmacia and was dissolved in D M S O to a stock concentration of 40 mM. The analogues of celecoxib, OSU-03008, O S U - 0 3 0 1 2 and OSU-03013, were synthesized at Ohio State University in the laboratory of Dr. Ching-Shi Chen (Figure 2) [104]. The analogues were also dissolved in D M S O to a stock concentration for each of 10 m M . All drugs were stored at - 2 0 ° C in single use aliquots. For the treatment of cells, the drugs were mixed in warm medium before applying to the monolayers. Appropriate treatment concentrations for the celecoxib analogues were determined based on the work of Zhu et al. (Zhu, 2004).  2.12  Determination of Effects of Celecoxib Analogues on Cell Signaling For signaling studies, cells were plated to 6 0 % confluence on 100 mm dishes.  To achieve 6 0 % confluence, the following number of cells were plated: MDA-MB-453, 5x10 ; T47D, 3x10 ; M C F - 7 , 2.25x10 ; M D A - M B - 2 3 1 , 2 x 1 0 6  6  6  6  Cells were then treated  with the inhibitors in RPMI-1640 medium supplemented with 5% F B S for two hours and harvested by scraping. Drugs were used at the following concentrations: LY294002, (30 nM); celecoxib, (50 and 75 nM); OSU-03008, OSU-03012, OSU-03013, (5 and 10  nM). 2.13  Cell Viability Analysis To determine effects of drugs on cell viability, cells were plated to 6 0 %  confluence on 96 well plates. To achieve 60% confluence, the following number of cells were plated: M D A - M B - 4 5 3 , 3x10 ; T 4 7 D , 2 x 1 0 ; M C F - 7 , 1.5x10 ; M D A - M B - 2 3 1 , 4  4  4  1.25x10 ; 184htrt, 2.5x10 . To test the effect of confluency on drug treatment, the MDA4  4  MB-453 cells were plated at the following confluencies: 6 0 % (3x10 cells); 80% (4x10 4  4  29 cells); 100% (5x10 cells); >100% (6x10 cells). Cells were treated with the inhibitors in 4  4  RPMI-1640 medium supplemented with either 0.1% F B S or 5 % F B S for 24 hours. Drugs were used at the following concentrations: LY294002 (30 u,M); celecoxib, (25, 50, 75 or 100 fiM); OSU-03008 (5, 10, or 20 uM); OSU-03012 and O S U - 0 3 0 1 3 (2.5, 5, 7.5, or 10 |xM). Treatments were in quadruplicate. After 24 hours the cells were subjected to the CellTiter 96® Aqueous NonRadioactive Cell Proliferation A s s a y (Promega).  This a s s a y involves adding a  tetrazolium compound, M T S (Appendix 1p), and an electron coupling reagent, P M S (Appendix 1q), to treated cells. Cells that are metabolically active will bioreduce M T S (by dehydrogenase enzymes) into a formazan product. soluble in cell culture  This formazan product is  medium and has an a b s o r b a n c e of 490 nm.  Thus,  spectrophotometrically-measured formazan is directly proportional to the number of metabolically active cells. For a 96 well plate, 2.0 ml of M T S was mixed with 100 uJ of P M S . This solution was then added to 9.6 ml of RPMI-1640 (without F B S or antibiotics) and 120 [i\ of the mixture was applied to each well. The cells were given between 40 and 120 minutes to metabolize the M T S and then the plate was read at 490 nm. The average absorbance for each treatment was background subtracted and then divided by the absorbance of control cells (treated with DMSO) to calculate "% cell viability".  2.14  Apoptosis Analysis 2.14.1 PARP Cleavage Poly(ADP-ribose) polymerase ( P A R P ) , an enzyme involved in D N A repair in  response to stress, is cleaved by caspase-3 during the execution of apoptosis.  In  human cells it is cleaved between Asp214 and Gly215, creating a 24 kDa and 89 kDa  30 cleavage product.  To measure the effect of celecoxib and its analogues on the  cleavage of P A R P , the M D A - M B - 4 5 3 cells were plated to 6 0 % confluency (7.5x10  5  cells) on 6 well plates. When confluency was an additional variable for drug efficacy, the cells were also plated at 80% confluence (1.0x10 cells) and 100% confluence 6  (1.25x10 cells). The next day cells were treated in growth medium for 12 or 24 hours 6  with the following: D M S O , LY294002 (30 u.M), celecoxib (50 or 100 u.M), OSU-03008 (10 or 20 nM), OSU-03012 (5, 7.5 or 10 u.M), O S U - 0 3 0 1 3 (5, 7.5, or 10 nM).  After  treatment both attached and floating cells were harvested. Cell lysates were prepared for each treatment using the lysis buffer included in the Cell Death Detection E L I S A kit (see section 2.14.2).  P L U S  Each lysate (50 ng) was analyzed by western blot using a  cleaved P A R P antibody (see section 2.9).  2.14.2  Nucleosomal Fragmentation Assay  In addition to P A R P cleavage, endogenous endonucleases are activated during apoptosis. These endonucleases cleave double stranded D N A at the exposed regions between nucleosomes. This yields fragments of D N A complexed with histones H2A, H2B, H3, and H4. These fragments can be found in the cytoplasm of apoptosing cells prior to the later event of plasma membrane destruction. ELISA  P L U S  The Cell Death Detection  assay (Roche; Penzberg, Germany) measures this release of fragmented  nucleosomes into the cytoplasm during apoptosis. The same fresh, unfrozen extracts used to measure P A R P cleavage (section 2.14.1) were used to measure nucleosome fragmentation, in duplicate. Each sample (5 ng total protein) was diluted to 20 \i\ in lysis buffer and processed according to the manufacturer's instructions.  In brief, nucleosomes in the cytoplasmic extracts are  31 bound to anti-histone-biotin and anti-DNA-POD antibodies on a streptavidin-coated 96 well plate. This creates a sandwich of streptavidin:biotin:histones:DNA:POD. Thus, the amount of nucleosomes in the cytoplasmic extract can be measured photometrically by reacting the peroxidase of P O D (present in the sandwich) with an A B T S substrate. This reaction is read at 405 nm and unreacted A B T S is read at 490 nm. For each sample, the absorbance at 490 nm is subtracted from that at 405 nm. The resulting number is divided by the amount measured in the D M S O control cells, to show the fold increase in cytoplasmic nucleosome enrichment.  2.15  Plasmids The myr-Akt1 plasmid and its corresponding p U S E a m p (+) empty vector were  obtained from Upstate (Cat# 21-151). The myr-Akt1 construct contains mouse Akt1 c D N A with the myristoylation sequence of c-src at the 5' end and a Myc-His tag at the 3' end of the sequence, all under control of a C M V promoter. The DD-Akt1 construct was a gift from Dr. James Woodgett (Ontario Cancer Institute, Toronto). It consists of Akt1 cDNA, with the Thr308 and Ser473 sites mutated to aspartic acid residues, inserted into the pcDNA3 vector from Invitrogen.  Both the myr-Akt1 and DD-Akt1 constructs contain  a neomycin resistance gene for selection purposes.  2.16  Rescue Experiment 2.16.1 Transient Assay 2.16.1.1  Transfection  Optimized transfection conditions were used for each cell line.  MDA-MB-453  cells were plated on 6 well dishes at low density (7.5x10 cells). M C F 7 cells were 5  plated on 6 well dishes at high density (5.0x10 cells). 5  The following day, cells were  32 transfected in duplicate wells, with either empty vector (EV), DD-Akt1, or myr-Akt1 (section 2.15) using Lipofectamine™ 2000 (Invitrogen) according to manufacturer's instructions. In brief, 3 u.g of each plasmid was added to 200 u.l of RPMI 1640, without F B S or antibiotics (Tube A).  In a second tube (Tube B) 9 u.l of Lipofectamine™ 2000  was added to 200 \i\ of RPM11640, without F B S or antibiotics. The contents of Tubes A and B were combined (Tube C) and incubated at room temperature for 20 minutes. Another 600 \x\ of serum free RPMI 1640 was added to Tube C . The cells were rinsed with serum free media and then the DNA:Lipofectamine complexes (Tube C) were added to the cells dropwise. After a 6 hour incubation at 37°C, the complexes were removed from the cells, replaced with normal growth medium, and cells were allowed to recover overnight.  2.16.1.2  Treatment of Transfected Cells with Analogues to Assess Viability; Analysis of Expression and Activity of Activated Akt Constructs  The next day, 18 hours post transfection, cells were counted and replated to 96 well dishes at 6 0 % density (see section 2.13), with the remaining cells replated on a new 6 well dish. Cells were allowed to attach overnight.  O n the 96 well plate, cells  transfected with either empty vector, DD-Akt1, or myr-Akt1  were treated in  quadruplicate for 24 hours with the following drugs diluted in growth medium: D M S O , LY294002 (30 u,M), OSU-03012 (5, 7.5 or 10 u.M), O S U - 0 3 0 1 3 (5, 7.5, or 10 u.M). Viability of treated cells was determined using the M T S assay (see section 2.13). Cells which had been replated to the 6 well dishes were harvested to determine expression and activity of the transfected constructs.  Whole cell extracts of harvested cells  (section 2.8.1) were western blotted and probed for P-Akt expression (section 2.9).  33 Additionally, these extracts were subjected to an Akt kinase assay to verify levels of Akt activity in the transfected cells (section 2.10). 2.16.2  Stable A s s a y  MDA-MB-453 and M C F - 7 cells were transfected as described in section 2.16.1.1. The next day transfected cells were trypsinized and replated in normal growth media containing neomycin (600 ng/ml for MDA-MB-453 cells; 400 u.g/ml for M C F - 7 cells). Cells were selected for resistance to neomycin over the course of two weeks. Neomycin resistant pooled clones were harvested and assessed for expression of DDAkt1 and myr-Akt1, as described in section 2.16.1.2. Cells stably expressing activated Akt or empty vector were plated to 96 well dishes and treated as described in section 2.16.1.2.  34  CHAPTER 3 RESULTS  3.1  Validation of P-Akt Immunostaining To determine whether the P-Akt antibody was specific, we first treated T47D cells  with and without IGF-I for 30 minutes and then analyzed the cells by both western blot and immunohistochemistry.  IGF-I stimulated the phosphorylation of Akt, as detected by  western blot, where P-Akt was predominantly cytoplasmic (Figure 3A, lanes 1 and 2) though a small amount trafficked into the nuclear fraction upon IGF-I treatment (Figure 3A, lanes 3 and 4). The cells were fractionated to confirm localization that might be observed in tissues. Next, by immunohistochemistry, using the IHC-specific antibody, the T47D cells treated with IGF-I showed a significant increase in P-Akt staining compared with untreated cells (Figure 3B). The staining was noted to be predominantly cytoplasmic with some membranous stain. A s a final control, we preincubated the IHCspecific P-Akt antibody with its competing peptide and reduced immunohistochemical staining to that of background levels on a section of breast cancer tissue (Figure 3C). These controls show that the antibody was specific to P-Akt.  3.2  Frequency of P-Akt Expression in Tumor and Normal Tissue Of the 481 cases on the array, 438 contained invasive carcinoma and the  characteristics of these patients are described in Table 1. Following P-Akt staining, a total of 390/438 invasive carcinoma cases were interpretable for P-Akt expression and were the only c a s e s considered in this study.  P-Akt expression was  cytoplasmic, although nuclear staining was visible in a few cases. P-Akt was  primarily  35  cytoplasmic SF +IGF  nuclear SF +IGF  T-AKT  B.  serum starved/ -IGF-1  serum starved/ +IGF-1  no competing peptide  + competing peptide  Figure 3. Validation of the P - A k t S e r 4 7 3 A n t i b o d y for Immunohistochemistry.  (A) The expression of P-Akt was first analyzed by western blot of T47D cells stimulated with and without IGF-I (SF (serum free) indicates no addition of IGF-I). Localization of P-Akt is shown by nuclear and cytoplasmic fractionation. (B) Using an IHC-specific antibody, P-Akt expression was detected by immunohistochemistry of formalin-fixed, paraffin-embedded T47D cells stimulated with and without IGF-I. (C) Finally, by preincubating the IHC-specific P-Akt antibody with a competing peptide, staining of breast cancer tissue is reduced to background level.  36 predominantly expressed in epithelial cells and also was noted in endothelial cells, but was not expressed in the stroma.  The distribution of P-Akt expression in the 390  tumors was: (no staining) 43/390 (11%), (weak) 122/390 (31%), (moderate) 120/390 (31%), and (strong) 105/390 (27%) (Figure 4A-D, respectively). Thus, 58% (225/390) of the breast cancers on this array expressed moderate to high levels of P-Akt. A s shown in Figure 4 E , some cores on the T M A contained normal ducts alongside cancer tissue. In this particular example, the cancer cells expressed high levels (score 3) of P-Akt whereas the normal ducts expressed low levels (score 1) of PAkt.  To extend this initial observation, we immunostained 26 normal breast tissue  samples for P-Akt. A s seen in the tumor tissue, P-Akt was predominantly expressed in epithelial cells. The stroma was consistently negative for P-Akt, while endothelial cells (Figure 4H) were found to express P-Akt at weak to moderate levels. W e found the normal tissue distribution to be: (negative) 6/26 (23.1%), (weak) 11/26 (42.3%), (moderate) 4/26 (15.4%), and (strong) 5/26 (19.2%) (Figure 4 F - G ) .  Thus, only 35%  (9/26) of the normal breast samples expressed moderate to high levels of P-Akt. The difference between P-Akt expression in normal versus tumor tissue was statistically significant (p «s 0.025) by Chi-Square analysis (Figure 5).  3.3  P-Akt Expression is Not Associated with Patient Survival W e hypothesized that patients with tumors that expressed high levels of P-Akt  would have poor survival. However, we found that there was not a significant difference (p =0.5839) in overall survival between patients that expressed high levels of P-Akt versus those which expressed low levels of the activated protein (Figure 6). We also evaluated the relationship of P-Akt expression on the T M A to other clinicopathologic  37  Figure 4. E x p r e s s i o n of P-AktSer473 in Tumor and Normal T i s s u e s of the Breast. (A-D) On the tumor tissue microarray, P-Akt staining of invasive ductal breast carcinomas (IDC) ranged in intensity from 0 (no stain), 1 (weak), 2 (medium), to 3 (strong stain) respectively. (E) Normal ducts (dashed arrow) adjacent to the IDC (solid arrow) expressed appreciably less P-Akt. (F) Likewise, P-Akt staining was weak when whole sections of normal breast tissue were stained. (G) Examples of intense P-Akt staining in normal breast tissue were also observed. (H) It was also noted that P-Akt was present in endothelial cells. Pictures were taken at a magnification of 200X (A-G) or 400X (H).  38  AGE (yr)  ID  P-Akt Score  19 21 22 24 26 26 27 28 29 30 30 31 34 36 37 38 38 40 42 42 42 42 46 46 57 59  91-01 206-00 150-00 35-00 174-00 185-00 95-00 191-00 108-01 201-00 109-01 151-00 105-00 220-00 99-00 168-00 110-01 51-01 36-00 71-00 232-00 148-01 170-00 274-00 72-01 175-01  1 0 3 2 3 1 1 1 2 0 3 1 1 0 0 1 3 0 1 2 1 3 1 0 1 2  Ductal Epithelium Pathology normal normal normal normal adenosis FCD normal normal normal normal adenosis normal adenosis normal normal - w/ duct adenosis FCD normal adenosis normal FCD FCD FCD FCD FCD FCD  mean: 35 median: 35  B. Distribution of P-Akt Expression in Normal and Tumor Tissues SO 70  58  60 50  • Low P-Akt  40  • High P-Akt  30 20 10 0  Chi S q u a r e Analysts Tumor Low (0,1)  165  Hiqh ( 2 . 3 ) Total  225 390  Normal 17  Total  9  182 234  26  416  p = 0.025  Figure 5. P-AktSer473 is Highly E x p r e s s e d More Frequently in Breast Cancer C o m p a r e d with Normal Breast. (A) The range and frequency of P-AktSer473 expression in normal breast tissue is listed along with ductal pathology and age of each patient (FCD: fibrocystic disease). (B) Frequency of low (score 0 or 1) or high (score 2 or 3) P-Akt expression in normal and tumor breast samples is shown by bar graph. PAkt is highly expressed in 58% of tumors and only 35% of normal breast samples. This difference in expression was determined to be significant by Chi-Square analysis, as shown.  39  Figure 6. P-AktSer473 Expression Alone Does Not Predict Poor Prognosis. Kaplan-Meier analysis was used to assess the relationship of P-Akt expression in breast tumors with patient prognosis. The differences in survival were not statistically significant (p=0.5839). score ofO (green curve); score of 1 (pink curve); score of 2 (yellow curve); score of 3 (grey curve)  40 variables, such as grade, lymph node status and histology, and found no significant correlations (Table 1). 3.4  Correlation of P - A k t E x p r e s s i o n with Other Proteins We next determined what proteins are coordinately expressed with P-Akt in  primary breast cancer by comparing it to the expression profiles of 30 other proteins. These 30 proteins, listed in Table 2, were previously examined on the same breast cancer T M A series for other studies within, or in collaboration with, G P E C (Vancouver, British Columbia).  W e determined that the expression of P-Akt was significantly  correlated with the expression of six proteins, by univariate analysis (Table 2).  Of the  six proteins, three were tyrosine kinase receptors (IGF-1R (p=0.000), H E R - 2 (p=0.012), EphA2 (p=0.001)). We also discovered that the expression of ILK (p=0.004) correlated with P-Akt expression.  Lastly, the expression of two transcription factors, GFI-1  (p=0.016) and YB-1 (p=0.004), were positively correlated with expression of P-Akt. 3.5  E x p r e s s i o n of P-Akt in Breast C a n c e r C e l l L i n e s A panel of breast cancer cell lines was screened for levels of P-Akt (S473 and  T308) and total Akt expression during exponential growth (Figure 7). A s the effects of the analogues were analyzed in 5% F B S , the levels of P-Akt were a s s e s s e d accordingly. The four breast cancer cell lines express similar levels of total Akt protein, whereas the 184htrt preneoplastic breast epithelial cells express very little Akt. Although all cell lines were grown in the presence of serum, which contains many growth factors that should activate Akt, only the M D A - M B - 4 5 3 and T47D cell lines expressed high levels of P-Akt in this condition.  It should be noted, however, that the  184htrts, MDA-MB-231 s and M C F - 7 s can be induced to express higher levels of P-Akt  41  * *  lit *  *  *  Protein  Spearman Correlation  F i s h e r ' s Exact T e s t  N u m b e r of Patients  CA9  -0.085  0.192  310  CD3  -0.004  1.000  298  CD20  -0.012  0.306  315  CD43  -0.025  0.795  297  CD68  -0.051  0.351  317  Chromogranin  -0.005  1.000  319  Clusterin  -0.090  0.103  320  Cox-2  -0.051  0.816  333  Cyclin E  0.053  0.443  280  E2F1  0.085  0.190  302  E-cadherin  0.053  0.323  335  EPHA2  0.201  0.001  299  ER  0.107  0.170  309  GFI-1  0.144  0.016  292  HER2  0.139  0.012  344  HSP27  -0.058  0.146  291  IGF-1  0.060  0.274  266  IGF-1R  0.265  0.000  327  IGFBP-2  -0.091  0.238  292  IGFBP-5  -0.049  0.387  290  ILK  0.171  0.004  299  Ki-67  -0.046  0.621  308  MEP21  0.019  1.000  260  NSE  0.054  0.296  320  p53  -0.030  0.751  296  PR  0.054  0.314  336  Survivin  0.067  0.717  318  YB-1  0.159  0.004  373  Table 2. The Correlation of P-Akt  Ser  473  with Other Proteins in Breast Tumors.  P-  A k t e x p r e s s i o n w a s c o m p a r e d with t h e e x p r e s s i o n o f 3 0 other proteins i m m u n o s t a i n e d o n t h e s a m e b r e a s t T M A s e r i e s ( c o l u m n 1).  Protein correlation w a s determined b y  S p e a r m a n ' s c a l c u l a t i o n ( c o l u m n 2) a n d s i g n i f i c a n c e w a s a s s e s s e d b y F i s h e r ' s E x a c t Test (column 3). Negative correlations represent inverse relationships. d e s i g n a t e s c o r r e l a t i o n s that a r e significant.  A n asterisk  " N u m b e r o f p a t i e n t s " ( c o l u m n 4) i n d i c a t e s  the total n u m b e r of patients that w e r e s c o r a b l e f o r both P-Akt a n d t h e c o m p a r e d protein.  42 when stimulated with supplemented IGF-I [106], [109]. O n the other hand, both the M D A - M B - 4 5 3 and T47D cells can maintain Akt phosphorylation in serum starved conditions [78], [110]. The reason for high Akt activation in the M D A - M B - 4 5 3 s and T47Ds may be accounted for by the expression of the H E R - 2 receptor in these cell lines (see Figure 7).  Overexpression of H E R - 2 can lead to constitutive activation of the  PI3K/Akt pathway [78]. Additionally, M D A - M B - 4 5 3 cells express low levels of P T E N compared with MDA-MB-231, M C F - 7 , and T47D cells [78], [110].  3.6  Analogues of Celecoxib Inhibit Akt Phosphorylation The M D A - M B - 4 5 3 cells, which express high levels of P-Akt in log-growing  conditions, provided a useful model system in which to characterize the effects of the celecoxib analogues on Akt signaling. After treatment for two hours in 5% serum, O S U 03008, OSU-03012, and OSU-03013 all inhibited Akt phosphorylation at a concentration of 10 |xM, similarly to the PI3K inhibitor, LY294002 (30 u.M) (Figure 8, top four panels). Both S e r 4 7 3 and Thr308 phosphorylation were affected.  There w a s slight  downregulation of P-Akt using 5 u,M of the analogues, and 50 JAM and 75 nM of the parent compound, celecoxib. It is important to note that studies which found complete inhibition of Akt phosphorylation using 50 \iM of celecoxib were done in serum free conditions [94]. W e also examined the effects of the analogues, celecoxib and LY294002 at 2 hours in another cell line, T47D (Figure 8, bottom four panels). The inhibition of Akt phosphorylation by the analogues in the T47Ds w a s more robust, perhaps because there is less basal P-Akt in those cells compared with the MDA-MB-453s.  3.7  Analogues of Celecoxib Inhibit Akt Kinase Activity and Downstream Signaling  43  f  &  #£f  0 Phospho-Akt  S473  Phospho-Akt  T308  Total-Akt  Total-Akt (longer exposure)  HER-2  Actin  Figure 7. Levels of P-Akt a n d H E R - 2 in a Panel of Breast C a n c e r Cell L i n e s . Four breast cancer cell lines (MDA-MB-231, M C F - 7 , T47D, MDA-MB-453) and a preneoplastic breast cell line (184htrt) were analyzed for constitutive P-Akt expression during exponential growth. These levels were also compared to expression of total Akt and the receptor tyrosine kinase, HER-2. A longer exposure for total Akt shows a small amount of this protein in the 184htrt cells. Actin was used as a loading control.  44  S  3 O CO 1  o Q  CM O O •<*  cn  CM >-  I  co  o o co o  C/>  O  © 1-  w3  oo  CN  o o co o 3 CO  O  O CO  o  Z>  o  o  5  3 O  5  3 O  m  2  3  o CO o  o CO o  o CO o  x O  .a 'x o  {/)  w O  =5  a O  O  o  o  o  o £ ca  Phospho-Akt  S473  Phospho-Akt  T308  Total Akt Actin  Phospho-Akt  S473  Phospho-Akt  T308  Total Akt Actin  Figure 8. P h o s p h o r y l a t i o n of A k t is Inhibited by A n a l o g u e s of C e l e c o x i b . Both MDA-MB-453 and T47D cells were treated for 2 hours with the indicated drugs in media containing 5% fetal bovine serum. Phosphorylation of Akt on Thr308 and Ser473, in addition to total levels of Akt, was assessed by western blotting whole cell lysates. Actin was used as a loading control.  45  Phospho-GSK3a/p Total-GSKa/p  Phospho-4EBP1 Total-4EBP1  Actin  Phospho-GSK Total-GSK  Total Akt Input  Figure 9. A k t K i n a s e Activity is Decreased and Downstream S i g n a l i n g is Inhibited in M D A - M B - 4 5 3 C e l l s Treated with C e l e c o x i b A n a l o g u e s . (A) The phosphorylation status of a direct (GSK-3p) and indirect (4E-BP1) substrate of Akt was analyzed by western blot, using protein extracts from MDA-MB-453 cells treated for 2 hours with the indicated compounds. The blot was reprobed for total GSK-3p\ total 4 E - B P 1 , and Actin proteins. (B) Additionally, kinase activity of Akt was found to be attenuated after MDAMB-453 cells were treated with celecoxib analogues (10 p M each) or the PI3K inhibitor (30 pM) for two hours. Akt was immunoprecipitated from treated cells and subjected to an in vitro nonradioactive kinase assay, using recombinant GSK-3{3 as a substrate. Levels of total recombinant GSK-3p\ phosphorylated recombinant GSK-3p\ and immunoprecipitated Akt were detected by western blot.  46 To confirm that the inhibition of Akt phosphorylation actually corresponded to suppression of the Akt signaling network, downstream targets of P-Akt were also examined in MDA-MB-453 cells treated for two hours with the compounds. Each of the three analogues, using 10 \iM, and LY294002 (30 nM) inhibited phosphorylation of G S K - 3 a / 6 (Figure 9A). O S U - 0 3 0 1 3 at 5 u.M had moderate success in inhibiting this target as well. Although the effect of celecoxib was modest on P-Akt (Figure 8), the phosphorylation of G S K - 3 a / 6 is quite attenuated by the parent compound, suggesting some difference in mechanism of drug action. The effect of analogue treatment on 4 E - B P 1 phosphorylation, however, was quite modest even at the 10 \x,M dose.  LY294002, on the other hand, completely  abolished phosphorylation, as detected using the Ser65 phospho-specific 4 E - B P 1 antibody.  Reprobing the western blot with a total 4 E - B P 1 antibody resulted in three  detectable bands in the D M S O control sample, indicative of the various phosphorylation states of 4 E - B P 1 .  Of all the samples treated with signaling inhibitors, only the  L Y 2 9 4 0 0 2 treated sample displayed the complete downward band shift of a hypophosphorylated 4E-BP1 protein. The kinase activity of Akt w a s measured to verify that inhibition of Akt phosphorylation at Thr308 and Ser473 correlates with loss of activity of the enzyme. Immunoprecipitated Akt from MDA-MB-453 cells treated for two hours with each of the analogues (10 u,M) was indeed inhibited in its ability to phosphorylate recombinant G S K - 3 (Figure 9B), albeit not completely. Similar inhibition was observed with LY294002 treatment.  3.8  Effect of Analogues on Cell Viability  47 W e have shown that the celecoxib analogues inhibit Akt phosphorylation after two hours of treatment. W e next wanted to assess the effect of the drugs upon longer exposure, as Zhu et al. showed cytotoxic effects of OSU-03012 and O S U - 0 3 0 1 3 in the P C - 3 cells within 24 to 72 hours of treatment [104]. Increasing doses of celecoxib and the analogues were added to the MDA-MB-453 cells for 24 hours.  The treated cells  were then subjected to an M T S assay. This assay does not distinguish between live and dead cells, it simply measures metabolic activity in the present cells. In this report M T S results are referred to as "cell viability" measurements, bearing in mind that a reading may be lower because growth has slowed or metabolic activity has decreased, not necessarily because the cells are dead. A s shown in Figure 10, 24 hour treatment with the PI3K inhibitor LY294002 resulted in approximately 63(±3)% viable cells. Celecoxib decreased viability at doses of 75 and 100 \iM to 55(±8)% and 14(±5)%, respectively. Interestingly, OSU-03008, at doses as high as 20 u M , only reduced cell viability by 15(±3)% of the D M S O control. However, both O S U - 0 3 0 1 2 and OSU-03013 had a robust effect at doses of 7.5 and 10 u.M, resulting in less than 2 0 % viable cells. These concentrations of the analogues are 10-fold lower than doses of celecoxib required to have similar effects. Further, as little as 5 u.M of O S U - 0 3 0 1 3 was able to inhibit cell growth by 53(±10)%. The effects of the analogues were also examined in three other breast cancer cell lines; MDA-MB-231, M C F - 7 and T47D (Figure 10). Results of treating the panel of cells were similar to those of the M D A - M B - 4 5 3 cells, with the following exceptions. Treatment with LY294002 affected viability of the T47D cells (as it did the M D A - M B 453s) but not the MDA-MB-231 s or M C F - 7 s , in keeping with others who found that  48 LY294002 preferentially kills cells with high levels of P-Akt [78]. OSU-03008 was most effective in the T47D cells, resulting in a 21 (±2)% reduction in M T S metabolism at 20 uM. The T47Ds were also the most sensitive cell line to OSU-03012 and OSU-03013 treatment, responding to a low dose (5 u.M) of O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 by 44(±14)% and 70(±14)%, respectively. M C F - 7 cells, on the other hand, were the least sensitive to low doses of O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 , showing only a 35(±3)% reduction in viability with 7.5 \iM of OSU-03012 and 25(±10)% reduction with 5 JAM of OSU-03013.  In general, the M D A - M B - 4 5 3 and T47D cells were the most sensitive to  low doses (5 and 7.5 u,M) of OSU-03012, but all four cell lines responded similarly to the 10 u.M dose of both OSU-03012 and OSU-03013. 3.9  OSU-03012 and OSU-03013 Induce A p o p t o s i s Treatment of the MDA-MB-453 cells with 7.5 to 10 piM of the celecoxib analogues  OSU-03012 and OSU-03013 for 24 hours resulted in a >80% reduction of viable cells by the M T S assay. These cells also show visual characteristics of apoptosis, such as cell rounding and lifting from the plate and membrane blebbing. Taking into account these observations, we hypothesized that OSU-03012 and O S U - 0 3 0 1 3 induce apoptosis in breast cancer cells.  M D A - M B - 4 5 3 cells were treated with increasing doses of the  analogues for 12 and 24 hours. Comparisons were also made to treatment with the parent compound as well as treatment with LY294002. Extracts from the treated cells were then analyzed for P A R P cleavage in addition to release of fragmented DNA. Compared with DMSO-treated control cells, treatment with LY294002, celecoxib (100 u.M), OSU-03012 (7.5 and 10 u.M), and OSU-03013 (7.5 and 10 u.M) all induced P A R P cleavage by 12 hours, which continued to occur through the 24 hour timepoint (Figure  49  MDA-MB-453  <£>  &  <#  50  75  100  Celecoxib  5  10  20  2.5  OSU03008  5  7.5  10  2.5  OSU03012  5  7 5  10  OSU03013  MCF7  flu III I I  ^  ,0  ,0-  50  75  H  B  ,  i  11  J:I  m ml 100  Celecoxib  5  1 II  10  20  OSU03008  2.5  5  7.5  m 10  is 2.5  OSU03012  5  7.5  10  OSU03013  MDA-MB-231  ,X>  #  & c#"  50  75  100  Celecoxib  5  10  20  2.5  OSU03008  5  7.5  10  2.5  OSU03012  5  7.5  10  OSU03013  T47D  eP <JP  & of*  5  50  75  100  Celecoxib  5  10  20  OSU03008  2 5  5  7.5  OSU03012  10  2.5  5  7.5  OSU03013  10  Figure 10. Assessment of the Effects of Celecoxib Analogues on Cell Viaibilty.  Four breast cancer cell lines, MDA-MB-453, MCF-7, MDA-MB-231 and T47D, were treated with the indicated compounds for 24 hours in medium containing 5% fetal bovine serum. Cell viability was measured using the MTS Proliferation Assay. Shown in the bar graphs are the percentages of viable cells after treatment compared with the DMSO control.  50  Celecoxib A.  DMSO  LY  50uM 100uM  OSU-03008  OSU-03012  OSU-03013  10uM 20uM 5uM 7.5uM 10uM 5uM 7.5uM 10uM  cleaved PARP Actin  cleaved PARP Actin  Figure 11. Induction of A p o p t o s i s by C e l e c o x i b A n a l o g u e s . (A) P A R P cleavage increased substantially after MDA-MB-453 cells were treated for 12 to 24 hours with OSU-03012 [7.5 or 10 uM], OSU-03013 [7.5 or 10 u.M], Celecoxib [100 \iM], and LY294002 [30 u.M] as detected by western blot. Blots were reprobed for actin as a loading control. (B) In parallel to P A R P cleavage, nucleosomal fragmentation also increased when MDA-MB-453 cells were treated as in (A). Release of nucleosomal fragments into the cytoplasm was assessed using a Cell Death Detection ELISA.  51 11A).  Low dose celecoxib (50 u.M), all doses of O S U - 0 3 0 0 8 , and low dose (5 uM)  OSU-03012 and O S U - 0 3 0 1 3 failed to induce P A R P cleavage. A similar pattern was observed when nucleosomal fragmentation was analyzed (Figure 11B).  Thus, two  methods confirm the induction of apoptosis by celecoxib, OSU-03012, OSU-03013 and LY294002 in the MDA-MB-453 cells.  3.10  Cell Confluency Protects Against Analogue-induced Ceil Death It was observed that when cells were plated more densely, the cytotoxic effects  of the analogues were abrogated. To investigate this further, increasing densities of MDA-MB-453 cells were plated and then treated with the inhibitors for 24 hours. These cells were analyzed for M T S metabolism, P A R P cleavage and P-Akt expression. By the MTS assay, more confluent cells were protected from 100 fxM of celecoxib and 10 nM of both OSU-03012 and OSU-03013 (Figure 12A). In addition, P A R P cleavage was more pronounced in the less confluent cells after treatment (Figure 12B). Higher levels of P A R P cleavage could be induced in confluent cells by increasing the dose of O S U 03013 to 15 u.M (data not shown). Notably, increased cell density did not change the effect of LY294002 on cell viability, as measured by the M T S assay, but P A R P cleavage was inhibited by confluency.  Further, it was noted that basal P-Akt expression was  increased in more confluent cells (Figure 12B). Expression of actin, as assessed by reprobing the western blots used to detect cleaved P A R P and P-Akt, showed equal loading of protein samples.  3.11  Serum Protects Against Cytotoxic Effects of Celecoxib and Low Doses of Celecoxib Analogues We also considered the fact that 5% serum in our treatment medium may protect  against the effects of the analogues. This phenomenon was noted for celecoxib by  52  B. Cleaved PARP 60% confluent  P-Akt (S473) Actin Cleaved PARP  80% confluent  P-Akt (S473) Actin Cleaved PARP  100% confluent  P-Akt (S473) Actin  Figure 12. C o n f l u e n c y Protects Cells from Cytotoxic Effects of C e l e c o x i b a n d A n a l o g u e s . MDA-MB-453 cells were plated at increasing densities (60% to >100% confluent) and then treated with the indicated compounds for 24 hours. (A) Cell viability in response to treatments was assessed by M T S assay. Increased confluency protected against high dose celecoxib [100 uM] and high dose OSU-03012 and OSU-03013 [10 u.M]. (B) Cleavage of P A R P and inhibition of P-Akt was assessed by western blot. Blots were probed with actin as a loading control. Increased cell density inhibited the ability of the analogues to induce apoptosis or attenuate Akt phosphorylation.  53  (3 5% serum • 0.1% serum  MCF7 cells  DMSO  LY  [25uM] [50uM] [75nM] [100uM] [SuM]  Celecoxib  [10uM] [20uM] [2.5uM]  OSU-03008  [5uM]  [7.5uM] [10uM] [2.5uM]  OSU-03012  [5i.M]  [7.5i,M] [10uM]  OSU-03013  Figure 13. Effect of Serum o n Efficacy of C e l e c o x i b and A n a l o g u e s . MDA-MB-453 and M C F - 7 cells were treated with the indicated compounds for 24 hours in medium containing either 0.1% or 5% fetal bovine serum. Cell viability was measured by the M T S proliferation assay. Strong serum protection against celecoxib treatment was noted for both cell lines, except at 100 u.M. Low serum conditions (0.1%) slightly increased the sensitivity of the MDA-MB-453 cells to all doses of OSU-03008 and [5 u.M] of O S U 03013. There was a considerable increase in response of the MDA-MB-453 cells to [5 u.M] of OSU-03012 in low serum. The M C F - 7 cells were much more sensitive in low serum media to all doses of OSU-03008, to 5 and 7.5 u.M of OSU-03012, and to 5 uM of OSU-03013. The highest dose [10 u.M] of OSU-03012 and OSU-03013 was not protected by 5% serum.  54 Levitt, et al. [111].  Thus M D A - M B - 4 5 3 cells were again treated with the signaling  inhibitors in medium containing only 0.1% serum, monitored for cell viability, and compared to the results seen in 5% serum conditions. Doses of celecoxib that were <10% effective in 5% serum (i.e., 50 \xM) resulted in 100% loss of cell viability when serum was deprived (Figure 13). The effects of OSU-03008 and OSU-03013 were only slightly improved upon serum withdrawal, whereas the 5 \iM dose of O S U - 0 3 0 1 2 became 30(±17)% more effective in serum-deprived medium. This experiment was also repeated in the M C F - 7 breast cancer cell line, where similar results were measured (Figure 13). Notably, 5% serum provided the M C F - 7 cells with more protection against the effects of OSU-03008, OSU-03012, and OSU-03013 than that seen in the M D A - M B 453 cells. Serum did not protect the MDA-MB-453 cells from LY294002. However, in serum-deprived conditions, the M C F - 7 cells became 12(±3)% more sensitive to LY294002.  3.12  Activated Akt Does Not Rescue Cytotoxic Effects of Analogues In an attempt to show whether the inhibition of Akt phosphorylation was a  necessary mechanism for the cytotoxic effects of the celecoxib analogues rather than a secondary result, rescue experiments were performed. Zhu, et al. observed a 20-30%) rescue of cell viability when P C - 3 cells transiently expressing constitutively activated Akt were treated with OSU-03012 [104]. Therefore, M D A - M B - 4 5 3 and M C F - 7 cells were transiently transfected with either of two constitutively activated Akt constructs. MyrAkt1 contains the myristoylation tag of c-src, which leads to anchoring of Akt to the membrane independently of PI3K activity [112]. DD-Akt1 is an Akt construct where both Ser473 and Thr308 are mutated to aspartate residues, which mimics phosphorylation  55 [74], [113].  Western blot analysis of transfected cells shows that cells expressing  activated Akt constructs indeed have higher levels of P-Aktser473 than vector-expressing cells (Figure 14A). The phospho-myr-Akt1 is larger than wild type phospho-Akt due to a Myc-His tag.  Additionally, kinase a s s a y s of immunoprecipitated Akt from cells  transfected with either myr-Akt1 or DD-Akt1 show that these constructs are highly active in their ability to phosphorylate recombinant G S K - 3 , proving the functional capacity of the constructs (Fig 14A). However, transient expression of neither DD-Akt1 nor myrAkt1 was able to rescue MDA-MB-453 cells from the cytotoxic effects of OSU-03012 or OSU-03013 (Figure 14B). A n attempt was also made to generate MDA-MB-453 and M C F - 7 cell lines stably overexpressing DD-Akt1 or myr-Akt1. W e were unable to maintain stable expression of DD-Akt1 in either cell line, and also could not retain myr-Akt1 expression in the M C F - 7 cells. M D A - M B - 4 5 3 cells stably expressing myr-Akt1 were established (Figure 15A), although expression was lost after six passages. While the cells were expressing myrAkt1 (passage 3) they were treated with O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 for 24 hours alongside M D A - M B - 4 5 3 control cells expressing an empty vector.  There was no  difference in viability of cells expressing an empty vector versus those with constitutively activated Akt (Figure 15B).  56  MDA-MB-453  A .  MCF-7  M  M MYR-AM1  Whole Cell Extracts  WT P-Akt/ DD-Akt1  Kinase Assay  Phospho-GSK3 (recombinant)  M 1 2 3 4 5 6 Ponceau S  B.  MDA-MB-453 cells  DMSO OSU-03012  OSU-03013  empty vector HDD-Aktl •Myr-Aktl  M C F 7 cells  Ir  DMSO  [5nM]  I  l i f e .  [7.5„M]  OSU-03012  [10nM]  [S^M]  [7SnM]  [10 M] M  OSU-03013  Figure 14. O v e r e x p r e s s i o n of Activated A k t Does Not R e s c u e Cells from Cytotoxic Effects of Treatment with OSU-03012 or OSU-03013. MDA-MB-453 and M C F - 7 cells were transiently transfected with either DD-Akt1 (D), myr-Akt1 (M), or an empty vector control (V). (A) Expression of transgene was confirmed by western blot using a phosphoAkt antibody. Both DD-Akt1 and myr-Akt1 have much higher levels of P-Akt expression compared with control cells. (Note: myr-Akt1 is larger than wild type(WT)- and DD- Akt due to a Myc-His tag). Equal loading was assessed by staining the blot with Ponceau S (respective lanes are labeled 1-6; M = size marker; 120,100, 55, 38, 29, 20 kDa (top blue band to bottom blue band)). Additionally, activity of Akt was confirmed by immunoprecipitation of Akt from each sample followed by a kinase assay, using recombinant G S K - 3 as a substrate. Phospho- recombinant G S K - 3 was detected by western blot. (B) Transiently transfected cells were treated for 24 hours with the indicated compounds and cell viability was subsequently measured by MTS assay.  57  Ponceau S  B.  MDA-MB-453 ceils 100 90 80 70 60  n empty vector • myr-Aktl  50 40 30  n  20 10 0  DMSO  LY  [5u.M] [7.5u.M] [10u.M] OSU-03012  [5(xM]  [7.5u.M] [lOu-M]  OSU-03013  Figure 15. Stable O v e r e x p r e s s i o n of Myr-Akt1 D o e s Not R e s c u e C e l l s from C e l e c o x i b A n a l o g u e s . MDA-MB-453 cells stably expressing myr-Akt1 or empty vector were generated as described in Materials and Methods. (A) Expression of myrAktl construct in cell lysates was verified by examining phosphorylated Akt on a western blot. Myr-Akt1 is larger than wild type (WT) Akt due to a Myc-His tag. Equal loading was assessed by staining the blot with Ponceau S (M = size marker; 120,100, 55, 38, 29, 20 kDa (top blue band to bottom blue band)). (B) Stably transfected cells were treated for 24 hours with the indicated compounds and cell viability was subsequently measured by M T S assay. Bars in graph represent the percentage of viable cells remaining in each treatment group compared with D M S O control.  58  CHAPTER 4 DISCUSSION  4.1  Introduction Our laboratory has spent the last several years researching the role of receptor  tyrosine kinases and Akt in breast cancer. We have shown that signaling through the IGF-1 R/P-Akt axis is responsible for upregulating the expression of molecules important for invasion and angiogenesis, such as urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF) [114], [106]. Further, the expression of IGF1R and u P A in primary breast cancer was found to be associated with poor survival [115].  The potential for therapeutic intervention of this pathway has also been  examined. Dunn et al. found that inhibition of IGF-1 R in tumor-bearing mice, using a dominant-negative mutant of the receptor, significantly decreases metastases [116]. Additionally, Nielsen et al. used the heat shock protein 90 (Hsp90) inhibitor, 17allylamino geldanamycin, to degrade the IGF-1 R and inhibit downstream signaling through Akt in breast cancer cells [115]. The project reported herein continues in the vein of our prior research. W e determined the frequency and level of Akt activation in 390 breast tumors, and assessed coordinate expression of P-Akt with other signaling molecules, including R T K s , in the same tumors. Further, we explored the potential for using analogues of the anti-inflammatory drug celecoxib as inhibitors of Akt phosphorylation and therefore as a novel treatment for breast cancer.  4.2  Expression of Phospho-Aktw73 in Primary Breast Tumors  59  4.2.1  Frequency of Expression and Relationship to Patient Prognosis  By screening tumor tissue arrays containing samples from 390 breast cancer patients, we determined that a majority (58%) of breast tumors express moderate to high levels of activated Akt.  This frequency is in accordance with other studies, in  various cancer types (50-66% in breast cancer [117], [80], [62], 5 7 % in ovarian carcinomas [118], 59% in pancreatic cancer [119], 50% in N S C L C [120]. Considering this data, a high percentage of patients could potentially benefit from inhibition of the Akt pathway. Additionally, we hypothesized that women whose tumors expressed activated Akt would have poor survival.  However, our data indicate that P-Akt status is not a  prognostic marker for breast cancer. There have been conflicting reports as to whether P-Akt expression is prognostic or not.  In breast cancer, at least three studies have  indicated that P-Akt does not correlate with survival [121], [122], [117].  However,  another study indicated that P-Akt predicted distant recurrence and shorter disease-free survival in endocrine treated breast cancer patients [80].  Further, a study of node-  negative breast cancers found P-Akt status to be correlated with decreased overall survival [123]. The differing results extend to other cancer types as well.  Although P-  Akt is found in high gleason grade prostate cancers and predicts poor outcome [84], [124], another study showed that P-Akt was associated with reduced rate of disease progression in lung cancer and actually predicted sensitivity to Iressa [120]. These differences could be accounted for based on variations in immunohistochemical  60 methods, antibodies, tissue fixation or scoring, but may be associated with the variety of treatment options used to treat the patients. The lack of prognostic value of P-Akt expression for the patients in our study does not discount a role for Akt activation in tumorigenesis or in cancer progression. Phospho-Akt levels can be modulated in many ways and can change during the progression of a tumor.  Measuring the phosphorylated state of a protein is a  "snapshot" of a dynamic system of signaling, similarly, T M A s only capture one moment in the progression of many tumors.  Therefore, detection of high P-Akt levels does not  imply that the pathway is constitutively activated in those tumors, just as detection of low levels does not mean that the tumors will not later activate P-Akt.  Firstly, the  patients in our study were not treated with a standardized chemotherapeutic regimen. A s P-Akt may provide resistance to some treatments and not others, a study examining the relationship of activated Akt to the survival of women given the same therapies may provide more insight.  Secondly, the tumor samples on our array were taken prior to  initiation of therapy. P-Akt levels might change once the therapy begins. For example, the development of drug resistance in experimental models suggests that Akt phosphorylation is upregulated in the acquisition of this phenotype [125]. Tumor cells that remain in the body throughout chemotherapy may be forced to develop new mutations activating an anti-apoptotic pathway in order to survive.  A final important  consideration could be that Akt signaling becomes activated in metastatic cells. Patients rarely die from a primary breast tumor, but rather from the resulting metastases.  Akt activation prevents anoikis [126] and has been shown to be  upregulated in metastatic variants of GI101A human breast cancer cells [127]. A s the  61 cases on our T M A were all primary cancers, a future array containing both primary and metastatic tumors, if possible, could prove or disprove this theory.  4.2.2  Correlation of P-Akt with the Expression of Other Proteins  The T M A s we employed for this study were previously immunostained for 30 other proteins (by us and others, see Appendix 2). This allowed us to explore the network of proteins coordinately expressed with P-Akt, some of which may be involved in the activation of Akt and/or may be a consequence of its signaling. By univariate analysis we found that activated Akt was positively correlated (significantly) with the expression profiles of six other proteins; H E R - 2 , IGF-1 R, EphA2, ILK, Y box-binding protein-1 (YB-1), and growth factor independence 1 (GFI-1).  Bearing in mind that  association does not necessarily imply causation, we developed two theories to explain this data. First, it is possible that the seven proteins are regulated by a common upstream pathway or common phase of the cell cycle, resulting in their coordinate expression in these breast tumors, but without direct links to one another. Secondly, perhaps coordinate expression does indicate regulatory or functional links between PAkt and the other molecules. The second theory was given merit by the fact that coexpression of H E R - 2 , IGF1R and ILK with P-Akt in primary tumors corroborates previous data in experimental models, showing that expression of these molecules can activate the Akt signaling pathway.  For example, M C F - 7 breast cancer cells engineered to overexpress H E R - 2  exhibit an increase in Akt kinase activity [26]. Cell lines that endogenously overexpress H E R - 2 also exhibit upregulated P-Akt [128]. Similarly, stimulation of the IGF-1 R with IGF-I rapidly activates Akt [106] and transgenic mice overexpressing IGF-1 R in the  62 heart have a hyperactivated Akt pathway [129].  Finally, a role for ILK in Ser473  phosphorylation of Akt was demonstrated by ILK knock-out, using either R N A interference or the Cre-Lox system. Cells or mice lacking ILK expression showed a significant decrease in P-Akt and also Akt kinase activity [130]. In addition to in vitro evidence, our finding that activation of Akt is correlated with H E R - 2 expression in primary breast tumors is also supported by previous reports [123]. However, our study is the first to demonstrate a link in primary breast cancer between IGF-1R and ILK with P-Akt.  Despite the significant correlations we observe between P-Akt and HER-2, IGF-  1R and ILK, they are not complete correlations. Meaning, some tumors with P-Akt were negative for ILK, and some tumors positive for IGF-1R and H E R - 2 were negative for PAkt.  Thus, based on our data, the relationships between these molecules are not  necessarily linear, but rather more complex. A s there is evidence to explain some of the correlations with P-Akt, perhaps the T M A s could serve as a hypothesis-generating tool for exploring novel signaling relationships.  In this study, activated Akt was also significantly correlated with the  expression of the R T K EphA2, as well as the transcription factors GFI-1 and Y B - 1 . Several characteristics of these molecules make functional or regulatory relationships with P-Akt a logical scenario. EphA2 is upregulated in 4 0 % of breast cancers and has been shown to impart tumorigenic and metastatic potential when overexpressed in M C F - 1 0 A normal breast epithelial cells [131]. The mechanism of EphA2 function in cancer remains unclear, and appears to be dually involved in cellxell adhesion and repulsion. For example, overexpression of EphA2 mediates cell:cell contact repulsion, and allows for survival independent of basement membrane attachments [132]. A s Akt  63 is also involved in anchorage independent growth, it is possible that E p h A 2 overexpression may positively regulate the activation of Akt, creating a survival signal for tumor cells and promoting cell migration. The relationship between P-Akt and the expression of the transcription factors GFI-1 and YB-1 also has interesting implications. Although the function of GFI-1 in the malignant transformation of T-cells and lymphoma progression is well established, GFI-1 has never before been studied in the context of breast cancer.  In T-cells, GFI-1 operates as a transcriptional repressor of Sax, which  correlates with suppression of apoptosis [133]. GFI-1 also regulates the expression of cell-cycle factors cMyc and P21 in myeloid cell lines [134]. YB-1 expression, on the other hand, has been previously examined in breast cancer patients and is related to poor prognosis [135]. This protein has diverse functions in both the cytoplasm, where it binds to R N A [136], and in the nucleus where it acts as a transcription factor [137] and regulates m R N A transport [138]. Overexpression of YB-1 in HBL100 pre-neoplastic breast epithelial cells promotes their resistance to doxorubicin [139]. One hypothesis to explain the observed correlations on the T M A is that perhaps P-Akt signals upregulate the expression of these two transcription factors, as a novel mechanism for resisting drug-induced apoptosis or promoting tumor growth.  It is also possible that the  relationships are inverted, where GFI-1 or YB-1 signaling indirectly upregulates Akt phosphorylation.  Further, the possibility exists that the activation of Akt leads to  upregulation of the R T K s H E R - 2 , IGF-1 R or EphA2. Future research could determine whether the correlations observed on the T M A relate to novel, functional arms of the Akt signaling network.  64 This study highlights the power of analyzing signaling networks in a large number of patients, which sharply contrasts the traditional method of measuring individual molecules of a signaling cascade in cell culture. Molecular profiling of tumors by cDNA, protein, or tissue microarray technologies, presents many opportunities for translational research [140]. A s a clinical application, profiling makes it possible to 1) classify tumor types for diagnosis by identifying tumor-specific markers, 2) predict response to therapy or 3) project the likelihood of metastasis. From a research standpoint the ability to discern global expression patterns allows us to 1) comprehend the network of molecular interactions occurring within a cell and/or tumor, 2) decipher the differences between normal and cancerous cells and 3) rationally design therapies that exploit signaling pathways which cancer cells depend on for survival and metastasis. The ability to analyze signaling networks in primary tumors was demonstrated by Choe, et al., who determined the expression of multiple molecules of the PI3K pathway in glioblastoma using T M A s [141].  Their study was able to validate, in patients, relationships between  P-Akt and the expression of molecules previously studied only in in vitro models (phospho-FKHR and phospho-mTOR). Similar to the Choe study, our work used T M A s to further explore the P-Akt signaling axis in patients. However, because we examined a total of 30 different proteins, most of which had no prior connection with P-Akt, we were able to identify potential novel relationships in this pathway.  Hopefully, using  T M A s to establish patient profiles for expression levels of particular growth/survival pathway proteins (i.e., RTKs and P-Akt) will aid in the development of tailored therapies. For example, identifying patients which overexpress P-Akt s e e m s to predict positive lung cancer response to Iressa [120].  65  4.3  Expression of  Phospho-Aktsnr^  in Normal Breast Tissue  In addition to determining the level of P-Akt in breast tumors, we also examined this protein in a set (n=26) of normal breast tissues. W e found that the frequency of Akt activation in normal breast tissue is significantly lower than that seen in breast tumors (58% in tumor versus 35% in normal).  However, the range of expression in normal  tissue was similar to that of the tumors, with some tissues expressing no P-Akt (score 0) and some tissues expressing very high levels (score 3) of P-Akt. The expression of P-Akt in normal tissue is not surprising. Akt is phosphorylated in hippocampal neurons to facilitate survival [142], in skeletal muscle during exercise [143], and in cardiomyocytes where it regulates cell size and contractile function [144]. The range of P-Akt expression in the normal breast, however, leads to questions regarding its regulation and necessity in mammary tissue.  There has not been much  attention placed on normal breast cells and Akt in the research community, thus its role and regulation can only be speculated upon. One important question is whether the expression of P-Akt represents normal function of the Akt signaling pathway, or is actually representative of an adverse event. Although we did not find P-Akt expression to be prognostic of poor survival on the breast T M A , we did not rule out a role for P-Akt in cancer development, most likely in combination with another mutation. In fact many studies have shown that constitutive P-Akt is transforming and causes mammary tumor formation when coexpressed with H E R - 2 or mutant polyomavirus middle T antigen [145], [74]. However, 9 of 26 (1 in 2.8) women in our study had moderate to high levels of P-Akt, and only 1 in 7 women will develop breast cancer in their lifetime. Therefore, statistically speaking, it is more likely that the levels of P-Akt we observed are due to  66 normal processes of the mammary gland.  This apparently is not due to age or  fibrocystic disease, as we observed a relatively random distribution of P-Akt among those variables in our study (Figure 5). Normal fluctuations in hormones which can activate Akt, such as estrogen and progesterone, or growth factors, such as insulin or leptin, may be responsible for variations in P-Akt [146], [147]. For example, changes in ductal proliferation during menstrual cycling are associated with variations in estrogen and progesterone secretions from the ovary, which could lead to increases in Akt phosphorylation. Thus, it is still unclear whether high P-Akt levels in our sample of 26 normal breast tissues is indicative of healthy mammary function or represents patients at high risk for developing breast cancer. By analyzing these same normal tissues for P T E N mutation or overexpressed R T K s , we could rule in or out adverse mechanisms for Akt activation.  4.4  Targeting Akt Signaling in Anticancer Therapy Perhaps the most important question is, since P-Akt can be expressed in both  normal and tumor tissues at high levels, will inhibiting Akt in cancers result in deleterious side effects? The current dogma for Akt phosphorylation is that 1) in a normal setting, P-Akt increases and decreases in response to extra- and intra-cellular signals in a regulated manner and 2) in a mutant cell, the P-Akt signal can also be modulated, but it is often constitutive because of overexpressed R T K s or P T E N mutation.  Thus, without stimulation, a normal cell would have little P-Akt, whereas a  cancer cell might express very high levels. For example, the preneoplastic breast cell line, 184htrt, expresses undetectable P-Akt in log growing conditions, suggesting that it does not require P-Akt for normal growth.  However, upon addition of IGF-I, one can  67 achieve a spike in Akt phosphorylation [106]. The 184htrt cells use Akt to transmit specific signals at particular times, but they don't necessarily depend on these signals for survival. Could the requirement for P-Akt be what makes a cell sensitive to its inhibition? The key role for Akt in survival signaling entertains the thought that any inhibition of this molecule in cells will result in certain death. How can a cell survive without it? It might be hypothesized that cells with low levels of P-Akt would be the most sensitive to Akt inhibition, since the target would be destroyed quickly; whereas cells with higher levels of P-Akt would be more resistant, as there is more target to inhibit.  But the  results of several studies using either LY294002 or more specific inhibitors of Akt phosphorylation, suggest that it is actually the cells with the most P-Akt that are most sensitive [85], [86].  Cells with low or undetectable levels of P-Akt barely respond to  inhibitors of the pathway.  During the development of cancer, there is a selection for  mutations in pathways protecting cells from programmed cell death (including p53, Bcl2, Akt). These same mutations may simply be crutches which, if removed, leave the tumor cells particularly vulnerable to death.  Drugs targeting Akt signaling are attractive  because they attack cancer cells at a point of resistance to apoptosis. The recognized dependency of many tumors (albeit, not all) on P-Akt, is hopefully not shared by the normal tissues of the body.  Current studies with potential Akt pathway inhibitors  suggest that this could indeed be the case [85], [90]. Thus, inhibitors of Akt signaling should sensitize tumors which overexpress P-Akt to standard chemotherapies, without affecting normal cells or tumors cells which do not depend on the Akt pathway for survival.  68  4.5  Analogues of Celecoxib As Inhibitors of Akt Signaling and Cytotoxic Agents 4.5.1  Summary of Inhibitory Effects on Akt Phosphorylation  Although much work has been done developing new inhibitors to Akt signaling, currently there are still no therapies that can be given to patients. Because celecoxib is already used clinically and is known to cause few side effects, this was a superb lead compound for new inhibitors of Akt signaling. Unfortunately, it was determined that while celecoxib can inhibit Akt phosphorylation, this only occurs at superphysiological concentrations. To potentially identify a new treatment for breast cancers dependent on P-Akt signaling, we characterized a new class of small molecule inhibitors derived from celecoxib.  The three inhibitors, OSU-03008, O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 have the  ability to inhibit PDK-1 kinase activity in vitro, with an  IC o 5  reported of 16  u,M,  5  LIM  and  2 u , M , respectively [104]. By treating breast cancer cells with the analogues for 2 hours, we found that, at 10 u . M , all of the inhibitors were able to significantly reduce Thr308 phosphorylation of Akt, the target site of P D K - 1 . Additionally, the phosphorylation of Ser473 was abrogated. The inhibition of Ser473 phosphorylation indicated one of three things: 1) PDK-1 plays a role in Ser473 phosphorylation, 2) Ser473 phosphorylation requires T308 to also be phosphorylated or 3) the analogues act on molecules other than P D K - 1 . This point will be discussed further below. The inhibition of Thr308 and Ser473 phosphorylation also corresponded to decreased Akt kinase activity against recombinant G S K - 3 6 substrate.  Further, in treated cells we were able to show that  phosphorylation of the direct Akt substrate, G S K - 3 6 , was strongly attenuated. Phosphorylation of the indirect Akt substrate 4 E - B P 1 (which is a direct substrate of mTOR) was only somewhat downregulated, however, indicating that other pathways  69 which inactivate 4E-BP1 (and activate mTOR) are unaffected by the analogues. These data suggest that the Akt signaling pathway is functionally inhibited by the three analogues, albeit not completely. On the other hand, the parent compound celecoxib had a very mild inhibitory effect on P-Akt, despite the much higher doses used.  4.5.2  Summary of Effects on Cell Viability and Apoptosis  W e also examined the effect of the analogues on cell viability. For these assays we treated the cells for 24 hours, and observed that O S U - 0 3 0 1 2 , O S U - 0 3 0 1 3 , high dose celecoxib and LY294002 all had a negative effect on cell viability, based on an M T S cell proliferation assay. The cells responded in a dose-dependent manner to these drugs, again with the analogues potent at much lower doses than celecoxib. To determine the mechanism for a decrease in cell viability, we examined P A R P cleavage and nucleosomal fragmentation, classic results of apoptosis. W e found that doses of 7.5 u.M and 10 nM, for OSU-03012 and OSU-03013, as well as LY294002 treatment, induced programmed cell death. The decrease in cell viability seen with the 5 nM dose of OSU-03012 and OSU-03013 was not due to apoptosis (at least in the in the M D A MB-453 cells), but could indicate cell cycle arrest.  Although O S U - 0 3 0 0 8 does not  induce apoptosis at the doses and times examined in this study, this compound does inhibit Akt phosphorylation.  Future work could analyze the effects of long term  treatment (more than 24 hours) with OSU-03008, to determine if apoptosis is eventually induced, specifically in cell lines overexpressing P-Akt.  4.6  Necessity of P-Akt Inhibition for Induction of Cell Death 4.6.1  Discrepancy Between Akt Inhibition and Cytotoxicity  70 A discrepancy arises, however, between the ability of the drugs to induce apoptosis and their ability to inhibit Akt phosphorylation. For example, OSU-03008 is equally effective at inhibiting Akt phosphorylation as O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 , however it has a minimal effect on cell viability. W e wondered if perhaps this compound is less stable than the other two analogues. Meaning, perhaps the effects of O S U 03008 are similar to the other compounds at 2 hours, but longer time points might show a relapse in Akt phosphorylation. If OSU-03008 cannot maintain inhibition of P-Akt, it could allow the MDA-MB-453 cells to recover, whereas a sustained inactivation of P-Akt by O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 would not allow the cells to recover and apoptosis could occur. However, when P-Akt levels are compared after 24 hours between the treatment groups, no difference is detected, despite the increase in P A R P cleavage in the OSU-03012 and OSU-03013 treated cells (Figure 12B). Another discrepancy is that although LY294002 completely inhibits Akt phosphorylation at 2 hours and induces apoptosis at 12 to 24 hours, 60% of cells remain viable after 24 hours.  This suggests  that cell death initiated by the inhibition of PI3K and Akt phosphorylation should not be complete by 24 hours. However, the 10 u.M dose of both OSU-03012 and OSU-03013, the dose required to best inhibit Akt phosphorylation, results in almost no remaining viable cells. These observations introduced the idea that the cytotoxic properties of O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 might be due to other targets not related to Akt phosphorylation. This theory seems even more likely considering the fact that O S U 03012 and O S U - 0 3 0 1 3 kill M D A - M B - 4 5 3 , T 4 7 D , M C F - 7 and M D A - M B - 2 3 1 cells similarly. A s has been shown in other studies, cells expressing the most constitutive PAkt levels should be the cells most sensitive to its inhibition. In fact, in our experiments  71 the M D A - M B - 4 5 3 and T47D cells were the most responsive to LY294002 treatment, showing proof of principle. Thus if OSU-03012 and OSU-03013 were specific inhibitors of the Akt signaling pathway, M D A - M B - 4 5 3 and T 4 7 D cells should have been preferentially sensitive over the M C F - 7 and MDA-MB-231 cells.  4.6.2  Attempt to Rescue the Cytotoxic Effects of OSU-03012 and OSU03013 by Overexpressing Activated Akt  Our data seemed to indicate that inhibition of Akt phosphorylation is not the only function of the celecoxib analogues. To further explore the question of Akt inhibition as a necessary or sufficient target for analogue-induced apoptosis, a rescue experiment was performed.  Transient transfection of activated Akt has been shown to rescue  cytotoxic effects of drugs which act by inhibiting Akt signaling. For example, transient overexpression of myr-Akt1 decreased apoptosis induced by U C N - 0 1 , a drug that inhibits P D K - 1 [148].  Further, transient transfection of myr-Akt1 also decreased  apoptosis induced by phosphatidylinositol ether lipid analogues, which inhibit Akt activation [86]. In our rescue experiment, however, transient transfection with either of two constitutively activated Akt constructs did not rescue the M D A - M B - 4 5 3 or M C F - 7 cells. There w a s not even a partial rescue, which was apparently found in the P C - 3 cells by our collaborators [104]. Additionally, we did not observe a rescue when MDAMB-453 cells stably overexpressing myr-Akt1 were treated with the celecoxib analogues (compared with empty vector expressing cells).  This data further suggests that  although Akt phosphorylation is inhibited by OSU-03012 and OSU-03013, this is not the primary mechanism for inducing cell death.  4.7  Other Targets for Analogues of Celecoxib  72 Based on our data, several possibilities arise, including: 1) Targets of P D K - 1 , other than P-Akt, mediate the pro-apoptotic response initiated by PDK-1 inhibition, 2) The OSU-03012 and OSU-03013 analogues target other proteins in addition to P D K - 1 . The first scenario s e e m s unlikely considering that expression of PDK-1 antisense oligonucleotides in human glioblastoma cells (which express mutant PTEN) only results in 10% apoptosis induction [149].  It would be an important experiment to express  antisense or s i R N A to PDK-1 in our breast cancer cell line panel and observe effects on cell viability, in order to determine if selective inhibition of PDK-1 mediates apoptosis. Further, it is quite possible that OSU-03012 and O S U - 0 3 0 1 3 have additional cellular targets, which causes cell death independently of P-Akt signaling. Such targets would likely be common to all of the cell lines tested in this study, as there were only slight cell-type specific responses to the analogues. Perhaps the structure of the celecoxib analogues allows them to bind to kinase domains of multiple proteins, particularly those with kinase pockets similar to that of P D K - 1 .  Drugs that potently inhibit PDK-1 have  previously been shown to inhibit the activity of other kinases. staurosporine and UCN-01 both strongly inhibit P D K - 1 .  For example,  However, when screened  against a panel of 29 different kinases, these drugs also inhibit the activity of cell-cyclecheckpoint kinase-1 (CHK1), protein kinase C a ( P K C a ) , AMP-activated protein kinase (AMPK), cyclin-dependent kinase 2 (CDK2/cyclinA), and others with similar potency [150].  Further screening of O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 , perhaps against such a  kinase panel as used by Komander, et al., is necessary to determine their effects on other kinases and signaling pathways.  4.8  Usefulness of the Celecoxib Analogues as a Breast Cancer Therapy  73 W e have found that the celecoxib analogues OSU-03008, OSU-03012 and O S U 03013 have the ability to inhibit Akt phosphorylation in breast cancer cells, including cells which overexpress H E R - 2 . Thus, it is conceivable that these agents could be used in combination with other therapies that are susceptible to drug resistance due to upregulated Akt signaling. For example, H E R - 2 overexpressing tumors often either do not initially respond to Herceptin treatment or later develop resistance. At this point, the underlying mechanism for recalcitrance to Herceptin is not understood. One possible explanation for resistance is expression of high levels of P-Akt, possibly through IGF-1R stimulation [151].  This idea is supported by a study showing that transfection of  constitutively activated Akt into BT-474 cells, a cell line known to be sensitive to HER-2 inhibition, rendered the cells insensitive to Herceptin treatment [152]. Thus, it would appear that inhibiting P-Akt could improve responsiveness to Herceptin.  This is  consistent with another report showing that inhibiting P-Akt with LY294002 enhances the cytotoxic effect of Herceptin [78].  Further, inhibiting P-Akt with LY294002 or a  dominant negative inhibitor to the p85 subunit of PI3K also prevents the anchorage independent growth of breast cancer cell lines that overexpress H E R - 2 , such as the MDA-MB-453 cells [153]. The ability of the celecoxib analogues OSU-03012 and O S U 03013 to inhibit P-Akt and ultimately kill Herceptin-resistant MDA-MB-453 cells suggests their potential clinical utility. This is timely given a recent report showing that the less potent parent compound, celecoxib, did not benefit patients with H E R - 2 overexpressing tumors resistant to Herceptin [154]. The analogues could feasibly be used to treat any tumor that is drug resistant due to hyperactivation of the Akt signaling network.  Future  experiments in cell culture using O S U - 0 3 0 0 8 , O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 in  74 combination with other chemotherapeutic agents, or with Herceptin, could determine the potential synergistic effects.  It is also important to begin preclinical testing of the  analogues in a mouse model of breast cancer. Determining the bioavailability of the compounds, as well as their capacity to inhibit Akt phosphorylation in tumors or xenographs in vivo are key objectives for future work.  4.9  Therapies for Downstream Targets of Akt: mTOR Another mechanism for treating tumors with hyperactivated Akt signaling is to  target the downstream effectors of P-Akt, rather than the upstream regulators or Akt itself. In an example of this theory coming to fruition, inhibitors to the protein mTOR are already in Phase l-lll trials, exhibiting some success against a range of tumor types [155], [156]. Regulation of mTOR is complex and not fully understood, but it is linked to activity of the PI3K/Akt pathway either through direct phosphorylation by Akt on Ser2448 [65], or through Akt's inactivation of the tuberous sclerosis complex [157], [158], an inhibitor of mTOR. Regardless of the route, activation of Akt is associated with activation of m T O R [159], which is responsible for phosphorylating the further downstream proteins, ribosomal S 6 kinase and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Phosphorylated 4E-BP1 dissociates from eukaryotic initiation factor 4 E (elF4E), an R N A cap-binding protein, allowing the formation of the e l F 4 F complex. This complex is responsible for initiating translation of m R N A s such as cyclin D1 [160], in turn regulating cell proliferation and cell cycle [161].  m T O R was first found to be  inhibited by rapamycin, an anti-fungal, immunosuppressive agent isolated from the bacteria Streptomyces  hygroscopicus  [162].  However, b e c a u s e of stability and  solubility issues, analogues of rapamycin were recently developed that can be delivered  75 orally or intravenously, namely CCI-779 and RAD-001. Rapamycin and its analogues have been shown to chemosensitize tumors dependent on P-Akt/mTOR signaling. For example, combining rapamycin with doxorubicin results in complete remission of mouse lymphomas which express constitutively activated Akt [163].  This observation is  particularly noteworthy since these lymphomas were resistant to treatment with chemotherapy alone. Other studies have also shown that sensitivity to mTOR inhibition is dependent on an activated Akt pathway, such as through loss of P T E N [164], [165]. Further, in a mouse model of prostate cancer, mTOR inhibition with RAD-001 was able to completely reverse a prostatic intraepithelial neoplasia (PIN) phenotype generated by overexpressing activated Akt1 in the ventral prostate [166]. This work suggests that, as with other inhibitors to P-Akt signaling, m T O R inhibition is very useful for cancer intervention, but predominantly for cancers which overexpress and are dependent on this pathway. Although the integral role of mTOR in mediating P-Akt signals is now quite apparent, it remains to be seen whether inhibitors to other downstream P-Akt effectors would be quite as efficacious.  4.10  Summary. Conclusions, and Future Work In this study we have determined that Akt is moderately to highly activated in  58% of breast carcinomas. In contrast, only 3 5 % of normal breast tissues express moderate or high levels of P-Akt. Thus, Akt is activated more frequently in cancerous versus normal breast tissue.  Future objectives to expand this study include 1)  Determine level of P T E N mutation and R T K activation in coordination with P-Akt expression in tumors, 2) A s s e s s whether coordinate expression of P-Akt with either mutant P T E N or overexpressed/ hyperactivated R T K s is related to poor survival, 3)  76 Obtain matched samples of primary breast tumors with metastases to determine whether P-Akt levels change during cancer spread. Additionally, we found that levels of P-Akt in breast cancer are significantly correlated with the expression of H E R - 2 , IGF-1R, EphA2, ILK, GFI-1 and Y B - 1 .  Future  work will determine the significance of these associations by 1) Overexpressing activated Akt or dominant negative Akt in cell culture and measuring changes in expression of the associated proteins, 2) Modulating the expression of the associated proteins and assessing changes in Akt phosphorylation, or 3) Determining if these proteins are associated on T M A s at other laboratories. W e also characterized analogues of celecoxib for their usefulness in treating breast cancer. The three compounds, OSU-03008, OSU-03012, and OSU-03013, were able to inhibit Akt phosphorylation within 2 hours of treatment in cell culture.  Two  compounds, O S U - 0 3 0 1 2 and O S U - 0 3 0 1 3 , induced apoptosis 12 to 24 hours after treatment, whereas the compound O S U - 0 3 0 0 8 did not cause cell death.  All three  analogues could be used in the presence of serum, whereas the parent compound, celecoxib, was severely inhibited by 5% serum. However, the ability of the analogues to inhibit Akt phosphorylation or induce apoptosis appears to be limited by cell density. W e further show that the mechanism for apoptosis induction is not dependent on inhibition of Akt phosphorylation as evidenced by: A) O S U - 0 3 0 1 2 and OSU-03013 kill breast cancer cell lines with low and high levels of P-Akt similarly, and B) Overexpression of activated Akt does not rescue cells from analogue induced cytotoxicity.  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Phosphate Buffered Saline (PBS) 8 g NaCl 0.2 g KCI 1.44 g N a H P 0 0.24 g K H P 0 deionized water to 1000 ml adjust pH to 7.4 2  2  1d.  4  4  Whole Cell Lysis Buffer 20 mM Tris (pH 7.5) 150 mM NaCl 1 mM EDTA 1 mM E G T A 1%Triton-X100 Protease Inhibitors: 2.5 mM Na-pyrophosphate 1 mM 6-Glycerophosphate 1 mM N a V 0 1 (xg/ml leupeptin 1 mM P M S F 3  1e.  4  Buffer A (Cytoplasmic Extraction Buffer)  96 10 mM HEPES (pH7.9) 1.5 mM MgCI 10 mM KCI 1 mM EDTA 0.1% NP40 2  Protease Inhibitors (see whole cell lysis buffer) 1f.  Buffer B (Nuclear Pre-extraction Wash Buffer) 10 mM Tris-Hcl (pH 7.2) 2 mM MgCI 2  Protease Inhibitors (see whole cell lysis buffer) 1g.  Buffer C (Nuclear Extraction Buffer) 0.42 M NaCl 20 mM HEPES (pH 7.9) 1.5 mM MgCI 20% glycerol Protease Inhibitors (see whole cell lysis buffer) L P S Sample Buffer. 4X 4.00 g Glycerol 0.682 g Tris Base 0.666 g Tris HCI 0.800 g Lithium Dodecyl Sulfate 0.006 g EDTA 0.75 ml of 1% solution Serva Blue G250 0.25 ml of 1% solution Phenol Red Ultrapure Water to 10 ml 2  1h.  Add 10 u.l of B-mercaptoethanol to 90 \i\ of 4X buffer before adding to protein samples. 1i.  10X Electrode Running Buffer 30.3 g Tris Base 144.0 g Glycine 10.0 g SDS deionized water to 1000 ml  To make 1X buffer: Dilute 50 ml of 10X stock in 450 ml deionized water 1j-  Stacking Gel for SDS-PAGE  97 4 % acrylamide/bis 0.125 M Tris pH 6.8 0.1% S D S 0.05% ammonium persulfate 0.1% T E M E D 1k.  Separating Gel for SDS-PAGE 12% acrylamide/bis 0.375 M Tris pH 8.8 0.1%) S D S 0.05%o ammonium persulfate 0.05% T E M E D  11.  10X Transfer Buffer 140 g Glycine 30.3 g Tris Base deionized water to 1000 ml  To make 1X buffer (20% methanol): Dilute 100 ml of 10X Transfer Buffer in 700 ml deionized water and 200 ml of methanol. Chill before use. 1m.  SATO Wash Buffer 20 mM Tris-HCl (pH 7.5) 0.2% NP40 10% glycerol 1 mM EDTA 1.5 mM MgCI 137 mM NaCl 2  1n.  Akt Kinase Assay Buffer 25 mM Tris-HCl (pH 7.5) 5 mM B-Glycerophosphate 2 m M DTT 0.1 mM N a V 0 10 mM MgCI 3  4  2  1o.  Dulbecco's Phosphate Buffered Saline (DPBS) 0.2 g KCI 8.0 g NaCl 0.2 g K H 2 P 0 4 1.15 g N a 2 H P 0 4  98 deionized water to 1000 ml adjust pH to 7.35 add the following and mix thoroughly: 100 mg M g C I 2 « 6 H 2 0 133 mg CaCI2 • 2 H 2 0  1p.  MTS Reagent  (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)  Protect from light during preparation and storage. Add 42 mg M T S powder to 21 ml D P B S (section 5.1.15) Mix for 15 minutes. Adjust pH to 6.0-6.5. Filter sterilize through 0.2 \im filter. Store at -20°C. (stock concentration = 2 mg/ml)  1q.  PMS Reagent  (phenazine methosulfate)  Protect from light during preparation and storage. Dissolve 92 mg P M S powder to 100 ml D P B S (section 5.1.15). Filter sterilize through 0.2 u.m filter. Store at -20°C. (stock concentration = 0.92 mg/ml)  99  Appendix 2: Antibody and Protocol Information for TMA Immunohistochemistry  Protein  Clone and Source  Dilution  Antiqen R e t r i e v a l  Staininq Pattern  Akt/ Protein Kinase B  Cell Signaling Technologies, 9277; Beverly, MA  1 :250  Steam 30min Citrate Buffer (pH=6.0)  cytoplasmic, occasionally nuclear  carbonic anhydrase IX (CA9)  1 :50  Steam 30min Citrate Buffer (pH=6.0)  membranous  CD3  Chia, S. K. et al. J Clin Oncol. 2001 Aug 15; 19(16) :3660-8. Novocastra; United Kingdom  1 :600  Decloak 5 min, EDTA  stromal T-cell  CD20  DAKO; Denmark  1 :500  0.059? pronase  stromal B-cell  CD43  MTI, RVR Sci  1 :10  Decloak 5min TRS  stromal T-cell  CD68  DAKO; Denmark  1 :800  Decloak 5min TRS  stromal macrophages  Chromogranin A  LC2H10Biogenex; San Rmon, CA  1 :1000  None  cytoplasmic  Clustering beta isoform  Santa Cruz Biotechnology, SC-6420 goat polyclonal Santa Cruj, CA CX229, Cayman Chemical; Ann Arbor, Ml  1 :500  Steam 30min Citrate Buffer (pH=6.0)  cytoplasmic  1 :100  Steam 20min Citrate Buffer (pH=6.0)  cytoplasmic  Cyclin E  CYE05, Neomarker; Fremont, CA  1 :300  Microwave 30min Citrate Buffer (pH=6.0)  nuclear  E2F1  KH95, Neomarker; Fremoont, CA  1 :200  Microwave 15m in Citrate Buffer (pH=6.0)  nuclear  E-cadherin  HECD-1, Zymed/lntermedico; Markham, CA  1 :150  Decloak 5min TRS  cytoplasmic  EphA2  1 MOO B208; Med immune Dr. Michale Kinch, Gaithersburg, MD)  None  membranous  Estrogen Receptor (ER)  DAKO; Denmark  1 :100  Steam 20min EDTA  nuclear  Growth Factor Independence-1 (GFI-1)  Dr. Lee Grimes, mouse monoclonal Institute for Cellular Therapeutics, Louisville, KV DAKO, A485; Denmark  1 :20  Steam 30min Citrate Buffer (pH=6.0)  nuclear  1 :500  Steam 20min, TRS  membranous  Steam 30min Citrate Buffer (pH=6.0)  membranous  cyclo-oxygenase 2 (Cox-2)  HER2/neu  Heat Shock Protein 27 (HSP27)  Novocastra; United Kingdom  1 :800  100 Appendix 2: (continued)  Protein  Clone and Source  Dilution  Antigen R e t r i e v a l  Staining Pattern  Insulin-like Growth Factor 1 (IGF-0  Santa Cruz Biotechnology, SC-713; Santa Cruz, CA  1 :IOOO  Steam 30min Citrate Buffer (pH=6.0)  cytoplasmic  Insulin-like Growth Factor-1 Receptor (IGF-1R)  Cell Signaling Technologies, 3D21; Beverly, MA  1 :200  Steam 30min Citrate Buffer (pH=6.0)  membranous and cytoplasmic  1 :200  Steam 30min Citrate Buffer (pH=6.0)  cytoplasmic  1 :300  Steam 30min Citrate Buffer (pH=6.0)  cytoplasmic  1 MOO  Steam 30min Citrate Buffer CpH=6.0)  cytoplasmic  1 MOO  Pressure cooker in MV, 2min nuclear  Insulin-like Growth Factor Binding Protein-2 Santa Cruz Biotechnology, goat polyclonal; (IGFBP-2) Santa Cruz, CA Insulin-like Growth Factor Binding Protein-5 Santa Cruz Biotechnology, goat polyclonal; (IGFBP-5) Santa Cruz, CA Cell Signaling Technologies, Integrin-linked Kinase rabbit polyclonal; (ILK) Beverly, MA MM1, Novocastra; United Kingdom Ki-67  cytoplasmic  Neuron-specific Enolase (NSE)  DAKO; Denmark  1 MOO  None  p53  DO-7, DAKO; Denmark  1 :400  Pressure cooker in MV, 2min nuclear  Podocalyxin (MEP-21)  3D3, Dr. David Kershaw University of Michigan Ann Arbor, Ml DAKO; Denmark  1 :80  Steam 30min Citrate Buffer (pH=6.0)  cytoplasmic, nuclear, membranous  1 MOO  Steam 20min EDTA  nuclear  Santa Cruz Biotechnology, rabbit polyclonal, SC-10811; Santa Cruz, CA Dr. Nelson Prostate Center VGH University of British Columbia Vancouver, BC  t :200  Steam 30min Citrate Buffer (pH=6.0)  cytoplasmic, nuclear  1 :2000  Steam 30rnin Citrate Buffer (pH=6.0)  cytoplasmic, some nuclear  Progesterone Receptor PR survivin  Y-box Binding Protein 1 (YB-0  

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