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YB-1 is a downstream component of the PI3K/Akt signaling pathway and regulates EGFR in breast carcinoma… Wu, Chia-Cheng Joyce 2006

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YB-1 IS A DOWNSTREAM COMPONENT OF THEJTOK/AKT SIGNALING PATHWAY AND REGULATESJEGFR IN BREAST CARCINOMA: A MECHANISM FOR BREAST CANCER GROWTH by CHIA-CHENG JOYCE WU B.Sc. (Biochemistry and Molecular Biology, Hons), The University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL FULLFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA August 2006 ©Chia-Cheng Joyce Wu, 2006 A B S T R A C T High level of activated or phosphorylated serine/threonine kinase Akt is commonly observed in aggressive breast cancer. Hence, the Akt signaling pathway has become a popular target for therapeutic interventions. Previously our lab discovered that phosphorylated Akt is co-expressed with a transcription/translation factor called the Y-box Binding Protein-1 (YB-1) and that Akt phosphorylates YB-1 at Serl02 in vitro. It was also found that Y B - 1 , but not the YB-1 mutant in which Ser 102 is mutated to alanine (YB-1A102), enhanced breast cancer cell growth. Since YB-1 was originally isolated as a D N A binding protein that interacts with the regulatory elements of the epidermal growth factor receptor (EGFR), we addressed the possibility that phosphorylated YB-1 stimulates breast cancer cell growth by up-regulating EGFR expression. First, we demonstrated that YB-1 was phosphorylated through the PI3K/Akt pathway ex vivo by immunoprecipitation and western blotting. We then illustrated that loss of Ser 102 affects the nuclear translocation of Y B - 1 , implying that Akt may also regulate YB-1 nuclear trafficking. By exogenously expressing YB-1 or YB-1 A l 0 2 in breast cancer cell lines we showed that YB-1 but not YB-1 A l 02 induced the levels of EGFR m R N A and protein. It was revealed that YB-1 bound to the -2 kb of the EGFR promoter by chromatin immunoprecipitation. Interestingly, disruption of Ser 102 prevented YB-1 from interacting with the first 1 kb of the EGFR promoter, indicating that binding to this region by YB-1 is regulated by the PI3K/Akt signaling. We then demonstrated that mutation of Ser 102 perturbed YB-1 from activating the -1 kb of the EGFR promoter by luciferase reporter assays. Finally, since activation of Akt depends on 3-phosphoinositide-dependent protein kinase-1 (PDK1), we examined the potential of a PDK1 inhibitor, OSU-03012, to inhibit the functions of Y B - 1 . It was found that OSU-03012 blocked YB-1 nuclear translocation and binding to the -1 kb promoter of EGFR. Together these results suggested that activation of the PI3K/Akt pathway leads to phosphorylation of YB-1 at Seri 02, resulting in up-regulation of EGFR gene expression. Here we propose that Akt, YB-1, and EGFR may all function in the same pathway to promote breast cancer cell growth. iv T A B L E O F C O N T E N T S A B S T R A C T i i T A B L E OF CONTENTS iv LIST OF FIGURES viii ABBREVIATIONS ix A C K N O W L E D G E M E N T xiii DEDICATION xiv C H A P T E R 1 I N T R O D U C T I O N 1 1.1 Cancer of the Breast 1 1.1.1 Frequency and Origin 1 1.1.2 Classification of Breast Cancer 2 1.1.3 Standard Treatment of Breast Cancer 3 1.2 The PI3K7Akt Signaling Pathway 5 1.2.1 The Signaling Cascade 5 1.2.2 The Functions of Akt 7 1.2.3 The Oncogenic Property of Akt 10 1.2.4 Akt as a Molecular Therapeutic Target for Breast Cancer 11 1.3 The Y-box Binding Protein-1 (YB-1) 14 1.3.1 Gene Family and Structure 15 1.3.2 Protein Structure and Function 16 1.3.3 The Pleiotropic Functions of YB-1 18 YB-1 in Translation 18 YB-1 in Transcription 19 YB-1 in D N A Repair 21 YB-1 in Drug Resistance 22 V 1.3.4 The Oncogenic Property of YB-1 23 1.3.5 YB-1 and Breast Cancer 24 1.4 The Epidermal Growth Factor Receptor (EGFR) 25 1.4.1 Gene Family and Structure 25. 1.4.2 Protein Structure and Function 26 1.4.3 The Oncogenic Property of EGFR 27 1.4.4 EGFR and Breast Cancer 28 1.5 Thesis Hypothesis and Objectives 29 C H A P T E R 2 M A T E R I A L A N D M E T H O D S 31 2.1 Plasmids 31 2.2 Maintenance of Cell Culture 31 2.3 Growth Factor or Drug Treatment 32 2.3.1 Activation of PBK/Akt Pathway by IGF-1 in MCF-7 32 2.3.2 Inhibition of PBK/Akt Pathway by OSU-03012 in MDA-MB-453 and MDA-MB-468 33 2.4 Cell Lysis and Protein Fractionation 33 2.4.1 Whole Cell Lysis 33 2.4.2 Cytoplasmic and Nuclear Fractionation 34 2.4.3 Protein Quantification 34 2.5 Immunoprecipitation 35 2.6 Western Blotting 36 2.6.1 Electrophoresis 36 2.6.2 Transfer of Separated Proteins to Nitrocellulose Membrane 36 2.6.3 Primary Antibody Incubation and Detection .... 36 vi 2.7 Immunofluorescence 37 2.8 Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) 38 2.9 Chromatin Immunoprecipitation(ChlP) 38 2.9.1 ChIP in MCF-7 Expressing Flag:EV, Flag:YB-l,orFlag:YB-lA102 38 2.9.2 ChIP in MDA-MB-468 39 2.9.3 Polymerase Chain Reaction (PCR) 41 2.10 Transfection 42 2.10.1 Transfection in HEK293T Cells 42 2.10.2 Transfection in MDA-MB-468 Cells 42 2.11 Luciferase Reporter Assay 43 CHAPTER 3 RESULTS 44 3.1 YB-1 is a Downstream Component of PI3K/Akt Cascade 44 3.1.1 YB-1 is Phosphorylated at Ser 102 via PI3K/Akt Signaling ex vivo 44 3.1.2 Disruption of Seri 02 Suppresses YB-1 Nuclear Translocation 47 3.2 Activated YB-1 Induces EGFR Expression 50 3.2.1 YB-1 Promotes EGFR mRNA and Protein Expression 50 3.2.2 YB-1 Interacts with the Promoter of EGFR 50 3.2.3 YB-1 Activates the Promoter of EGFR 56 3.3 OSU-03012 Inhibits the Functions of YB-1 58 3.3.1 OSU-03012 Blocks YB-1 Nuclear Translocation 58 3.3.2 OSU-03012 Prevents YB-1 from Binding to the EGFR Promoter 59 vii C H A P T E R 4 D I S C U S S I O N 64 4.1 Phosphorylation of YB-1 by PBK/Akt 65 4.1.1 Potential Proteins that Phosphorylate YB-1 via P B K Signaling 65 4.1.2 Potential Proteins that Phosphorylate YB-1 via MAPK Signaling 68 4.2 The Role ofSerl02 in the Regulation of YB-1 by Akt 69 4.2.1 Ser 102 and Nuclear Translocation 69 4.2.2 Ser 102 and DNA Binding 72 4.3 Regulation of EGFR Gene Expression by YB-1 : 73 4.4 The Role of Akt, YB-1, and EGFR in Breast Cancer 77 4.5 Targeting YB-1 in Breast Cancer 79 4.6 Conclusion and Future Direction 81 R E F E R E N C E S 85 A P P E N D I X BUFFERS AND REAGENTS 116 viii LIST OF FIGURES Figure 1 The PI3K7Akt Signaling Pathway 6 . Figure 2 The Structure and Functions of YB-1 17 Figure 3 Characterization of the Cell Lines Used in the Study 45 Figure 4 YB-1 was Serine Phosphorylated via the PBK/Akt Signaling Cascade ... 46 Figure 5 Loss of Ser 102 Suppressed Nuclear Translocation of YB-1 48 Figure 6 YB-1, but not YB-1 Al02, induced EGFR Expression 51 Figure 7 The First 2 kb of the EGFR Promoter 53 Figure 8 Validation of the Primers and Conditions for Chromatin-immunoprecipitation 54 Figure 9 YB-1, but not YB-1A102, Interacted with the -1 kb of the EGFR Promoter 55 Figure 10 Disruption of Ser 102 Inhibited YB-1 to Activate EGFR Transcription .... 57 Figure 11 OSU-03012 Inhibited YB-1 Nuclear Translocation 60 Figure 12 OSU-03012 Prevented YB-1 to Bind to the -1 kb of the EGFR Promote . 63 Figure 13 Putative Proteins that Phosphorylate YB-1 via the PBK or the MAPK Signaling Pathway 67 Figure 14 Potential Transcription Factors that Regulate the -2 kb of the EGFR Promoter Predicted by CONSITE 76 Figure 15 YB-1 Up-regulates EGFR Expression and Enhances Breast Cancer Cell Growth via the PBK/Akt Signaling Cascade 82 ix ABBREVIATIONS AF-1 Activation function-1 AF-2 Activation function-2 AFX ALL 1 fused gene from chromosome X A l Aromatase inhibitor BAD BCL2-antagonist of cell death CDK Cyclin-cyclin-dependent kinase COX-2 Cyclooxygenase-2 CRS Cytoplasmic retention site CSD Cold shock domain CTD C-terminal domain DbpA DNA binding protein A DbpB DNA binding protein B DbpC DNA binding protein C DMSO Dimethyl Sulphonite DNA-PK DNA-dependent protein kinase 4E-BP1 4E-binding protein 1 EGF Epidermal growth factor Egf-1 Early growth response factor-1 EGFR Epidermal growth factor receptor ELB Egg lysis buffer ER Estrogen receptor ERDBP-1 EGF-responsive DNA-binding protein-1 ERK1 Extracellular-signal-regulated kinase 1 ES cell Embryonic stem cell ETR EGFR transcriptional repressor FBS Fetal bovine serum FKHR Forkhead in rhabdomyosarcoma GCF GC factor GSK3 Glycogen synthase kinase 3 GSK3P Glycogen synthase kinase-3p HB-EGF Heparin-binding EGF HIF-1 a Hypoxia inducible factor-1 HIV-I Human T-cell lymphotropic virus type I HTLV-I Human T-cell lymphotropic virus type I IGF-1 Insulin-like growth factor-1 IGF-1R Insulin-like growth factor-1 receptor IKK IKB kinase ILK Integrin-linked kinase INF-y Interferon y Jak IFN-y- Janus kinase JCV JC virus LARII Luciferase assay reagent II MAPK Mitogen-activated protein kinase MDR-1 Multidrug resistance 1 MHC Major histocompatibility complex MMP-2 Matrix metalloproteinase 2 MMP-12 Matrix metalloproteinase 12 mRNP Messenger ribonucleoprotein particles MRP 1 Multidrug resistance-related protein 1 MSH2 MutS homologue 2 MVP Major vault protein NF-KB Nuclear factor kappa B NLS Nuclear localization signal NSCLC Non-small-cell lung cancer NSEP-1 Nuclease-sensitive element protein-1 pAkt Phosphorylated Akt PCNA Proliferating cell nuclear antigen PDK1 3-Phosphoinositide-dependent protein kinase-1 PH Pleckstrin-homology PIC Phosphatase inhibitor cocktail PBK Phosphatidylinositol-3 kinase PIP2 Phosphatidylinositol 4,5-bisphosphate PIP3 Phosphatidylinositol 3,4,5 -trisphosphate PKB Protein kinase B PKC-8 Protein kinase C-epsilon PLB Passive lysis buffer PMA Phorbol 12-myristate 13-acetate PMSF Phenylmethylsulfonyl fluoride pSer-YB-1 Phospho-Ser-YB-1 PTEN Phosphatase and tensin homologue RIPA Radio-immunoprecipitation assay RSK p70 Ribosomal S6 kinase RSV-d Rous sarcoma virus d RTK Receptor tyrosine kinase SERM Selective estrogen receptor modulator SH2 SRC-homology-2 TAE Tris-acetate-EDTA TBP TATA-binding protein TGF-a Transforming growth factor-a TGF-p Transforming growth factor-P TMA Tumor tissue microarray VEGF Vascular endothelial growth factor WRN Werner syndrome protein YB-1 Y-box binding protein-1 X l l l A C K N O W L E D G E M E N T First, I would like to thank Dr. Sandra Dunn, for giving me the opportunity to explore the fun of research. Under your guidance, I have grown so much. You made me realize that intelligence is only minor; it is your hard work, commitment, and passion that distinguish you from other experts in the field. I am grateful to my graduate committee, Dr. Rob Kay, Dr. Michael Kobor, and Dr. Wan Lam, for your invaluable advices. Special thanks to Dr. Michael Kobor, for being inspirational, for being there when I needed help, and for having faith in me. I would also like to thank the chair and the university examiner of my defence, Dr. Wendy Robinson and Dr. Fabio Rossi, for making this experience memorable. I will not forget the members of the Dunn Lab, Michelle, Cathy, Nancy, Goli, Karen, Anna, Jennifer, and Gina. I had so much fun in the lab during the past two years because of you. Michelle and Cathy, we have gone through so much together, and I would not be able to pass the hard times without your support. Nancy, you are my first true Caucasian friend, and I will miss sharing the "exposed" spot with you. Goli, thank you for bringing life and laughter to the lab. Gina, I especially thank you for your help and for making this lab function smoothly. To the University of British Columbia and the Child and Family Research Institute, for providing the resources and the studentship. Finally, to my dearest Peter Kuo, for your love and support. If there could be more than one author for my thesis, you know you are the second author! DEDICATION To m y fami ly, Eric Wu, Janet Lee, and Jelemee Wu For a l l their l ove and support 1 CHAPTER 1 INTRODUCTION Cancers develop through a multi-step process, which reflects the genetic alterations that drive the progressive transformation of normal human cells into highly malignant derivatives (Hanahan and Weinberg 2000). In the United States a total of 556,902 cancer deaths were recorded in 2003 (Jemal et. al. 2006). A study reported by Jemal et. al. also revealed that currently one in four deaths is due to cancer (Jemal et. al. 2006). With statistics like these, it is not surprising that cancer has surpassed heart disease as the leading cause of mortality for Americans under age of 85 since 1999 (Jemal et. al. 2006). 1.1 Cancer of the Breast 1.1.1 Frequency and Origin Among women in North America (Jemal et. al. 2006,, breast carcinoma was estimated to be the most common malignancy and the second leading cause of cancer deaths in women in 2006. This year, 212,920 of American women will be diagnosed with breast cancer and 40,970 will die of it (Jemal et. al. 2006). The statistics for Canadian women are also devastating as 22,300 new breast cancer cases will be expected and 5,300 patients will not survive the disease (www, In these patients, it is not the primary tumor, but metastases at distant sites that are the main cause of deaths. According to the American Cancer Society, patients with localized breast tumors can expect a 98% chance of survival five years post-diagnosis. However, once the cancer has spread to distant sites such as the lungs, liver, or bone, the five-year survival rate drops to as low as 16% ( 2 In breast cancer both genetic and environmental factors, such as age and increased hormone exposure, contribute to the accumulation of mutations in essential genes involved in cell cycle check points, cell survival and growth, and DNA repair (Baselga and Norton 2002). The most common genetic abnormalities in both familial and sporadic breast cancers are loss of heterozygosity (LOH) at multiple loci resulting in mutations in alleles of tumor suppressor genes (Baselga and Norton 2002). Loss of heterozygosity in BRCAl (Hall et. al. 1990, Miki et. al. 1994), BRCA2 (Wooster et. al. 1994, Wooster et. al. 1995), P53 (Malkin et. al. 1990, Sidransky et. al. 1992), and PTEN (Liaw et. al. 1997) has been linked to hereditary breast tumors. Amplification of oncogenes, including ERBB2 (Slamon et. al. 1987), C-MYC (Escot et. al. 1986), and CYCLINDI (Buckley et. al. 1993), accounts for the second most common genetic alteration in breast carcinoma (Baselga and Norton 2002). In addition, the activation of certain receptors such as the estrogen receptors and the epidermal growth factor receptors is also implicated in the pathogenesis of breast malignancies (section 1.1.2). While 5-10% of all breast cancers in women are hereditary, most breast tumors are sporadic (Mincey 2003). 1.1.2 Classification of Breast Cancer Distinct subtypes of sporadic breast cancer have been identified using gene-expression microarray analysis. There are two main groups of breast carcinoma, namely the estrogen receptor-positive (ER(+)) and the estrogen receptor-negative (ER(-)) breast cancers (Perou et. al. 2000, Sorlie et. al. 2001, 2003). The ER(+) tumors are further classified into luminal subtype A and B, depending on their expression levels of cytokeratin-8 and cytokeratin-18. For ER(-) breast carcinoma, three biologically distinct subgroups are identified: 1) normal breast-like, 2) ERBB2+, and 3) basal-like. The normal breast-like tumors are characterized by high levels of many gene products expressed by the adipose tissue and other non-epithelial cell types. The ERBB2+ 3 subgroup over-expresses the ERBB2 protein (also known as Her-2), a member of the epidermal growth factor receptor subfamily, while the basal-like tumors express high levels of cytokeratin-5 and cytokeratin-7. Survival analyses on the subtypes have been conducted in two separate patient cohorts. In one cohort patients were treated with both pre-surgical chemotherapy and post-surgical endocrine therapy (Sorlie et. al. 2001) whereas patients from another group (van't Veer et. al. 2002) did not receive any systemic adjuvant treatment. Studies on both data sets revealed similar results in which patients with ER(+) tumors have a relatively favorable prognosis (Sorlie et. al. 2003). In contrast, the ER(-) ERBB2+ and basal-like subtype breast cancers are invasive and more likely to metastasize, and patients with these types of tumors have the shortest survival times (Sorlie et. al. 2003). 1.1.3 Standard Treatment of Breast Cancer For breast carcinomas that have not metastasized, the first line treatments include surgery and radiation therapies (Baselga and Norton 2002). Most breast cancer patients also receive adjuvant chemotherapies to eradicate residual cancer cells that may have spread to secondary sites (Baselga and Norton 2002, Houssami et. al. 2006). The most frequently used chemotherapeutic drugs are alkylating agents (cyclophosphamide), anthracyclins (doxorubicin), antimetabolites (5-fluorouracil), and antimicrotubule agents (paclitaxel and docetaxel) (Baselga and Norton 2002). These agents inhibit cancer growth by interfering with DNA synthesis or mitosis. For example, doxorubicin affects the activity of topoisomerase II, while paclitaxel interferes with microtubule functioning (Baselga and Norton 2002). Given that DNA and cell replication also occur in normal cells, traditional chemotherapies to cancers are often hindered by toxicity and lack of efficacy. Consequently, there is growing interest in developing treatments that more specifically target tumor cells. Such 4 treatment, termed targeted therapy, exploits the reliance of malignant cells on continued stimulation by hormones or growth factors as well as constitutive activation of downstream signaling pathways. The essential role of estrogen signaling in breast tumorigenesis led to the development of hormonal therapy. It was known that upon estrogen binding, ER becomes activated and associates with transcription factors such as AP-1 and Spl to promote gene expression and cell growth (Sommer et. al. 2001, Webb et. al. 1999, Porter et. al. 1997). One well known example of hormonal therapy is the selective estrogen receptor modulator (SERM) that competes with estrogen to bind to ER, thereby blocking the ligand-dependent activation function 2 (AF-2) domain of ER without inhibiting the activity of the ligand independent activation function 1 (AF-1) domain (Shao and Brown 2004). Currently, tamoxifen is the SERM frequently prescribed for the treatment of all stages of breast cancer (Shao and Brown 2004). Other SERMs, namely toremifen and raloxifene, have also been approved by the Food and Drug Association (FDA) for clinical use in the USA (Shao and Brown 2004). Faslodex, or fulvestrant, is a recently introduced novel "pure ER antagonist" that provides further option in hormonal therapy. Unlike tamoxifen, fulvestrant inhibits both AF-1 and AF-2 domains of ER and has no estrogen agonist activity (Fallowfield 2004, Howell 2005). Lastly, aromatase inhibitors (AIs) are hormonal drugs that act by inhibiting estrogen production in peripheral tissues in postmenopausal women. AIs that have started to be administered to postmenopausal breast cancer patients include anastrozole, letrozole, and exemstane (Fallowfield 2004). The efficacy of hormonal drugs such as tamoxifen in many breast cancer patients is limited by a few factors. First of all, although tamoxifen was shown to be beneficial to patients with ER(+) breast tumors (EBCT 1998a), approximately 30-40% of the breast tumors are ER(-) (Shao and Brown 2004). Herceptin, a humanized monoclonal antibody targeting ERBB2, was approved for treating ER(-) ERBB2-overexpressing metastatic breast cancers (section 1.2.4). However other ER(-) breast tumors, such as the aggressive basal-like breast cancer, are still difficult to manage by current therapies. Secondly, almost all patients with advanced breast cancer who initially responded to tamoxifen therapy developed resistance (Shao and Brown 2004). Reasons for tamoxifen resistance include the rare loss and mutation of ER, altered levels of ER co-regulators, and importantly, aberrant activation of additional growth factor signaling pathways (Shao and Brown 2004, Kurokawa and Arteaga 2003). Therefore, there is a need to gain more knowledge on other growth pathways that are deregulated in breast cancer, especially those that are in advanced stages and/or do not express ER, so that novel therapeutic strategies can be developed. 1.2 The PBK/Akt Signaling Pathway Of the growth factor signaling pathways, the phosphotidylinositol-3 kinase (PI3K)/Akt cascade (Figure 1) has crucial roles in regulating divergent cellular processes including apoptosis, proliferation, differentiation, and metabolism. In recent years, the PI3K/Akt signaling has attracted much attention as components of this pathway are frequently deregulated in a wide spectrum of cancers. 1.2.1 The Signaling Cascade Phosphatidylinositol-3 kinases (PI3Ks) constitute a lipid kinase family characterized by their ability to phosphorylate the 3'-OH group of inositol ring in inositol phospholipids (Fruman et. al. 1998). The PI3K family is divided into three classes (I, II, III) based on structure, substrate preference, and function (Vanhaesebroeck and Waterfield 1999, Cantley et. al. 2002, Okkenhaug and Vanhaesebroeck 2003, Wymann et. al. 2003, Deane and Fruman 2004). The primary PBK proteins that respond to growth factors belong to class la. The class la PBKs are 6 Figure 1 The PBK/Akt Signaling Pathway Upon growth factor binding, receptor tyrosine kinases dimerize leading to autophosphorylation and recruitment of PBK via the p85 subunit to the membrane. The pi 10 subunit of PBK then generates PIP3 from PIP2. The tumor suppressor PTEN can convert PIP3 to PIP2 thus inactivate the signaling. PIP3 acts as a second messenger to activate PDK1, which phosphorylates Akt at Thr308. For full activation, Akt is further phosphorylated at Ser473 by ILK, Akt itself, DNA-PK, rictor-mTOR complex, or an unidentified PDK2. The phosphorylated Akt then phosphorylates a wide range of proteins, leading to inhibition of apoptosis, drug resistance, cell cycle progression, translation and cell growth. Growth Factors 00 RTK Cell Membrane ^E> p11( p85 eg, ILK Akt DNA-PK Rictor-mTOR mTOR p27 p21 GSK3p MDM2 NF-KB F K H R B A D YB-1 i M / i i p53 • 7 1 Translation Cell Growth Apoptosis EGFR Expression? Cell Cycle Drug Resistance 7 heterodimers consisting of a p85 regulatory subunit and a pi 10 catalytic subunit (Fruman et. al. 1998). Upon binding by growth factors, tyrosine residues of transmembrane receptor tyrosine kinases (RTKs), such as the insulin-like growth factor-1 receptor (IGF-1R) and the epidermal growth factor receptors including ERBB-1 and ERBB-2, become phosphorylated. The phosphorylated RTKs then bind to the SRC-homology-2 domains (SH2 domains) of the p85 subunit of PI3K (Okkenhaug and Vanhaesebroeck 2001). The interaction between RTKs and p85 leads to the activation of pi 10, which then generates phosphatidylinositol 3,4,5-trisphosphate (PIP3) from phosphatidylinositol 4,5-bisphosphate (PIP2)- PIP3 serves as a second messenger to recruit protein kinase B (PKB), also known as Akt, to the membrane by binding to the pleckstrin-homology (PH) domain of Akt (Corvera et. al. 1998). The level of PIP3 is tightly regulated by phosphatase and tensin homologue (PTEN), which is a tumor suppressor capable of converting PIP3 back to PIP2 (Maehama and Dixon 1998). Once in the membrane, Akt is phosphorylated first at threonine 308 in the kinase domain by a serine/threonine kinase called 3-phosphoinositide-dependent protein kinase-1 (PDK1; Alessi et. al. 1997). Full activation of Akt requires the phosphorylation at a second site, serine 473, in the C-terminal hydrophobic tail. The protein responsible for phosphorylating Akt at serine 473 remains controversial, but possible candidates include integrin-linked kinase (ILK; Delcommenne et. al. 1998), Akt itself (Toker and Newton 2000), DNA-dependent protein kinase (DNA-PK; Feng et. al. 2004), the rictor-mTOR complex (Sarbassov et. al. 2005), and an yet to be identified "PDK2" (Alessi et. al. 1997, Toker and Newton 2000). Once Akt is fully activated, it acts as a master switch that turns on a spectrum of processes necessary to promote cell survival and growth. 1.2.2 The Functions of Akt Akt, originally identified as human homologues of the viral oncogene v-akt, has three 8 isoforms: Aktl, Akt2, and Akt3 (Staal et. al. 1988, Cheng et. al. 1992, Masure et. al. 1999, Nakatani et. al. 1999). Akt isoforms, encoded by distinct but highly related genes, share more than 80% identity at the amino acid level (Nicholson and Anderson 2002). Aktl and Akt2 are widely expressed whereas Akt3 is predominantly expressed in the brain, heart, and placenta (Masure et. al. 1999, Nakatani et. al. 1999). All Akt isoforms are activated by the same mechanism but may exert different functions. It has been shown that Aktl knockout mice exhibit increased apoptosis and growth retardation (Cho et. al. 2001, Chen et. al. 2001). On the other hand, Akt2 knockout mice were normal in size but displayed a diabetic phenotype (Cho et. al. 2001). Studies on Akt3 knockout mice suggested that Akt3 may be involved in brain development because these mice had reduced brain size but grew normally and exhibited functional glucose metabolism (Tschopp et. al. 2005). Mice lacking both Aktl and Akt2 were viable but died shortly after birth. The mice were dwarfs with abnormal skin and bone development (Peng et. al. 2003). Although Akt isoforms may exert specific functions, it is suggested that Akt isoforms share many substrates in the cellular survival and growth pathways as all Akt isoforms are able to induce tumor formation, which will be discussed in section 1.2.3. Akt is involved in many cellular pathways including cell apoptosis, proliferation, growth and angiogenesis (Figure 1). Akt inhibits apoptosis by directly phosphorylating and inactivating the pro-apoptotic factor BAD (BCL2-antagonist of cell death) and a pro-death protease caspase-9 (Batta et. al. 1997, Cardone et. al. 1998). Akt also indirectly inhibits apoptosis by regulating components of the cell-death system. For example, Akt phosphorylates the Forkhead family of transcription factors, FOXOl/FKHR (forkhead in rhabdomyosarcoma) and FOX04/AFX (ALL1 fused gene from chromosome X), and prevents their nuclear translocation, thus inhibiting the transcription of FKHR gene targets including several pro-apoptotic proteins such as BIM and FAS ligand (Brunet et. al. 1999). Moreover, Akt 9 activates MDM2, which targets the pro-apoptotic tumor suppressor p53 for degradation (Mayo and Dormer 2001, Zhou et. al. 2001b). Furthermore, by phosphorylating the I K B kinase (IKK), which induces the degradation of the N F - K B inhibitor ( IKB ) , Akt indirectly activates the nuclear factor kappa B ( N F - K B ) . N F - K B is then able to promote the expression of pro-survival genes (Nicholson and Anderson 2002, Romashkova and Makarov 1999, Kane et. al. 1999). Akt exerts a positive effect on cell viability not only by inhibiting apoptosis, but also by enhancing proliferation through the cell-cycle machinery. For example, Akt phosphorylates glycogen synthase kinase-3|3 (GSK3P), thereby blocking the ability of GSK3p to target cyclin D1 for degradation. The level of cyclin D1 is retained, and cell cycle progression occurs (Diehl et. al. 1998). In addition, Akt negatively regulates the expression of p21 and p27, which are cyclin-dependent kinase (CDK) inhibitors (Zhou et. al. 2001a, Vi ghetto et. al. 2002, Liang et. al. 2002, Shin et. al. 2002). Akt can also promote cell growth by up-regulating components involved in protein translation. The mammalian target of rapamycin, mTOR, which is a serine/threonine kinase that serves as a modulator sensor to regulate protein synthesis based on nutrient availability (Vivanco and Sawyer 2002), is phosphorylated by Akt. Upon phosphorylation, mTOR activates the p70 S6 kinase and the S6 ribosomal protein (Nave et. al. 1999), leading to protein translation (Dennis et. al. 1996). Activated mTOR also inhibits the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which is a translation repressor (Gingras et. al. 1999). The ability of Akt to regulate both apoptosis and cell growth implies that deregulation of Akt may lead to transforming events. In fact, many studies have demonstrated the oncogenic property of Akt (section 1.2.3). It is interesting to note that Akt can also affect angiogenesis by stabilizing the transcription factor hypoxia inducible factor-1 (HIF-la) via mTOR, resulting in increased expression of vascular endothelial growth factor (VEGF; Zhong et. al. 2000, Zundel et. al. 2000, Laughner et. al. 2001). As the fast-growing tumors depend heavily on blood vessels to 10 acquire oxygen, it is possible that deregulated Akt promotes cancer pathogenesis by "improving" the surrounding environment that is more suited for tumor growth. 1.2.3 The Oncogenic Property of Akt Immunohistochemical staining on tumor tissues revealed that the level of activated or phosphorylated Akt (pAkt) is associated with cancers of the colon (Itoh et. al. 2002), lung (Lee et. al. 2002), kidney (Horiguchi et. al. 2003), prostate (Malik et. al. 2002), and the breast ((Perea-Tenorio et. al. 2002, Schmitz et. al. 2004). Preclinical study using small animals demonstrated that activated Aktl transformed NIH3T3 cells, allowing malignant NIH3T3 cells to develop tumor in mice (Sun et. al. 2001). Mende et. al. further created Akt2 and Akt3 mutants by engineering a membrane-targeting myristylation signal of the c-Src kinase to the amino termini of Akt2 and Akt3. The myristylated Akt2 and Akt3, which exhibited kinase activities, were able to induce tumors in animals (Mende et. al. 2001). The role of pAkt in clinical breast cancer is more controversial. It has been shown by immunohistochemistry that pAkt is not associated with patient survival (Panigrahi et. al. 2004, Kucab et. al. 2005). In other studies, however, high level of pAkt correlated with relapse and distant metastasis (Perea-Tenorio et. al. 2002, Schmitz et. al. 2004). It was also demonstrated that pAkt was elevated in approximately 40% of breast cancers, most of which are in stage III/IV (Sun et. al. 2001). Moreover, AKT2 was found to be amplified in 3% of breast cancers and was associated with poor prognosis (Bellacosa et. al. 1995). Data from both in vivo and in vitro work support the idea that activated Akt contributes to breast cancer pathogenesis. First of all, activated Akt is able to transform breast epithelial cells into malignant phenotypes (Zhao et. al. 2003, Zhang et. al. 2003). In nude mice, co-expression of constitutively active Akt 1 and mutant polyomavirus T antigen resulted in mammary tumor formation in nude mice (Hutchinson et. al. 11 2001). In addition to tumorigenesis, Akt is also involved in tumor progression and metastasis. For example, exogenous over-expression of Aktl enhanced breast cancer cell growth both in monolayer and soft agar assays (Ahmad et. al. 1999). Akt was also shown to promote the motility and invasion of breast cancer cells by increasing the osteopontin mRNA and protein levels (Zhang et. al. 2003) as well as the metalloproteinase, MMP-9, protein level (Kim et. al. 2001). Besides, Akt2 also mediates invasion and metastasis in breast cancer cells by up-regulating P1 integrins protein expression (Arboleda et. al. 2003). Furthermore, activated Akt promotes resistance to radiotherapy and chemotherapeutic drugs including doxorubicin, trastuzumab, and tamoxifen by suppressing apoptosis (Liang et. al. 2003, Clark et. al. 2002). Together, these studies suggest an important role of activated or phosphorylated Akt in breast cancer. les 1.2.4 Akt as a Molecular Therapeutic Target for Breast Cancer The importance of pAkt in breast cancer has prompted researchers to develop therapi that block the activity and downstream effects of Akt. There are three approaches 1) targeting Akt itself, 2) blocking Akt upstream signaling, or 3) inhibiting Akt downstream substrates. Despite the effort in developing drugs that inhibit Akt or interfere with Akt:PIP3 interaction (Lin et. al. 2004, Castillo et. al. 2004), there are currently no Akt small molecule inhibitors on the market. However, adenoviral-mediated expression of a kinase-dead mutant of Akt, in which both Thr308 and Ser473 were changed to alanine, is able to induce apoptosis selectively in tumor cells and to reduce tumor growth in mice (Jetzt et. al. 2003). This study provided proof of principle that inhibiting Akt can suppress cancer progression. Targeting RTKs has been a popular therapeutic strategy for breast cancer. ERBB2, for example, is over-expressed in about 30% ER(-) aggressive breast carcinoma and is associated with poor prognosis and increased risk of relapse (Slamon et. al. 1987). Trastuzumab (Herceptin) is a humanized monoclonal antibody developed to target ERBB2. By preventing ligand-ERBB2 interaction, this antibody has shown an excellent clinical anti-tumor profile and was approved by the US Food and Drug Administration in 1998 for treating ER(-) ERBB2-overexpressing metastatic breast cancer (Baselga 2001, Slamon et. al. 2001). However, ERBB2- overexpressing breast cancer patients whose tumors are PTEN-negative often develop resistance to trastuzumab (Nagata et. al. 2004). In addition to ERBB2, the epidermal growth factor receptor (EGFR or ERBB1), is also commonly over-expressed in breast carcinoma (Salomon et. al. 1995, Osaki et. al. 1992, Klijn et. al. 1992, Bucci et. al. 1997). The expression of EGFR has been shown to be associated with tumor aggressiveness and poor prognosis in breast cancer patients (section 1.4.4). Therefore, there has been great interest in developing drugs to target EGFR to treat breast cancer. The small molecule inhibitor, gefitinib (also known as Iressa™; AstraZeneca, London, UK), which competes with ATP and inhibits the kinase domain of EGFR, is already approved for the treatment of patients with advanced non-small-cell lung cancer (NSCLC; Cohen et. al. 2004). Gefmitib therefore serves as an attractive candidate for breast cancer therapy. Unfortunately, the efficacy of gefitinib in treating metastatic breast tumors was found to be low in phase II studies (Albain et. al. 2002, von Minckwitz et. al. 2005). To date no clinical trials of gefitinib focusing on ER(-)/EGFR-overexpressing breast tumors have been reported. However, another phase II clinical trial of gefitinib revealed that it was largely ER(+) breast cancer patients who were resistant to tamoxifen and express relatively modest EGFR obtained clinical benefit from gefitinib (Robertson et. al. 2003, Gutteridge 2004). Patients with ER(-)/EGFR(+) breast tumors, however, did not respond very well to the drug (Robertson et. al. 2003, Gutteridge 2004). In fact, the efficacy of gefitinib in NSCLC seems to be independent of EGFR expression. It was recently discovered that gefitinib is only effective in NSCLC patients who harbor a specific mutation in 13 EGFR (Lynch et. al. 2004). Such a mutation has not been reported in breast tumors. In addition to the ERBB family, IGF-1R is implicated in breast cancer invasion and metastasis (Kucab and Dunn 2003), which led to the development of antibodies or small molecule inhibitors to target IGF-1R (Burtrum et. al. 2003, Garcia-Echeverria et. al. 2004, Mitsiades et. al. 2004). However, the efficacy of drugs targeting IGF-1R still requires further evaluation. In addition to drugs that target RTKs, small molecules including wortmannin and Ly294002 that inhibit PI3K activity were developed. However, these drugs are not suitable for treating cancers as wortmannin is not stable in an aqueous environment while LY294002 is too toxic. Recently, the cyclooxygenase-2 (COX-2) inhibitor, celecoxib, was discovered to inhibit PDK-1. Celecoxib derivatives, termed OSU-03012 and OSU-03013, were then developed to specifically target PDK-1 and not COX-2. These orally available analogs showed improved PDK1 inhibitory ability and were able to induce apoptosis by blocking Akt (Zhu et. al. 2004). Our lab previously demonstrated the ability of OSU-03012 and OSU-03013 to induce apoptosis in breast cancer cells (Kucab et. al. 2005). We also showed in breast cancer cells that OSU-03012 specifically inhibits PDK-1 without affecting other signaling pathways (Kucab et. al. 2005). Our lab felt that OSU-03012 may be a good drug candidate for treating breast tumors, and one of the objectives of this study is to further evaluate the potential of OSU-03012 in breast cancer therapy. Given the role of Akt2 in glucose metabolism, targeting Akt downstream substrates may be a preferable approach to treat breast cancer. Rapamycin, which is an immuno-suppressant, inhibits mTOR and is expected to have anti-proliferative effect on cancers (Vara et. al. 2004). Poor solubility and chemical instability of rapamycin led to the development of analogs with more favorable pharmaceutical characteristics (Carraway 2004). The rapamycin analog CCI-779 has shown good tolerability and response rates in clinical trials (Chan et. al. 2005). The ongoing phase III study will help define the role of this therapy in breast cancer (Morgensztern and McLeod 2005). Two other rapamycin analogs, RAD001 and AP23573, are in the early phase of development (Morgensztern and McLeod 2005). For the past few years, our lab has been focusing on identifying novel Akt downstream substrates specifically in the context of breast tumors in hope of developing alternative therapies for treating breast cancer. We screened tumor tissue microarrays (TMAs) representing 500 breast cancer patients and found that, among approximately 30 proteins examined, pAkt is co-expressed with a transcription/translation factor called the Y-box binding protein-1 (YB-1). Subsequent in vitro kinase assays indicated that Akt forms a complex with YB-1 and phosphorylates it on Seri 02, which is located in the cold-shock domain (CSD). Since YB-1 is known to be over-expressed in breast cancer (Bargou et. al. 1997, Rubinstein et. al. 2002, Janz et. al. 2002), our finding was significant as it suggested that PI3K7Akt may regulate YB-1 and that the action of these two proteins may contribute to breast cancer development or progression. Therefore, for the first part of the project we will elucidate whether YB-1 is phosphorylated at Seri 02 through PI3K/Akt signaling and how Seri 02 contributes to YB-1 functioning. 1.3 The Y-box Binding Protein-1 (YB-1) The YB-1 gene is located on chromosome lp34 and comprises of eight exons (Toh et. al. 1998). The mRNA is approximately 1.5 kb long and encodes a protein with 324 amino acids (Didier et. al. 1988). YB-1 was first isolated as a transcription factor that bound to the promoter of the major histocompatibility complex class II (MHC Class II). YB-1 was named as it recognized the Y-box in the MHC Class II promoter. The Y-box identified in the MHC Class II promoter is a 14-nucleotide conserved sequence that contains the inverted CCAAT box (Okada et. al. 1985), and the inverted CCAAT box is required for YB-1 binding (Didier et. al. 1988). At the 15 same time, another group identified a DNA binding protein that interacted with the EGFR enhancer (Sakura et. al. 1988). This protein, referred to DNA binding protein B (DbpB), was later shown to be identical to YB-1. Soon after, YB-1/DbpB was suggested to be the same as the nuclease-sensitive element protein-1 (NSEP-1), which was independently cloned based on its ability to bind to CT-rich elements in the C-MYC promoter (Kolluri and Kinniburgh 1991). More recently, YB-1 has also been called the human homolog of p50, which was originally found to be one of the major proteins in messenger ribonucleoprotein particles (mRNPs) in rabbit reticulocytes (Evdokimova et. al. 1995). 1.3.1 Gene Family and Expression YB-1 is a member of the Y box protein family. These proteins were identified in a number of eukaryotic and prokaryotic organisms, including human YB-1, DNA-binding protein A and C (DbpA or C; Tekur et. al. 1999); rat EF1A (Ozer et. al. 1990); frog FRGY1 and FRGY2 (Tafuri and Wolffe 1990); mouse MUSY1 and MUSY2 (Tafuri et. al. 1993, Yan and Tamm 1991); chicken chkYB-1 (Grant and Deeley 1993); rabbit p50 (Evdokimova et. al. 1995); and bacterial cspA and cspB (Wolffe 1993). In humans, while YB-1 has been studied extensively, little is known about DbpA and DbpC. Since DbpA is expressed in the heart and muscle whereas DbpC is detected in germ cells (Tekur et. al. 1999), perhaps their functions are tissue-specific. On the other hand, the expression of YB-1 varies among tissues and developmental stages. For example, YB-1 is widely expressed in fetal cerebrum, heart, muscle, liver, bone marrow, and kidney (Spitkovsky et. al. 1992). In addition, YB-1 is also produced in cell lines including fibroblasts, T-cells, B-cells, and monocytes (Spitkovsky et. al. 1992). However, YB-1 mRNA is not detected or is expressed at very low level in many adult tissues including breast tissues (Spitkovsky et. al. 1992), suggesting the expression of YB-1 must be tightly regulated. Multiple E-boxes and GF-boxes on the YB-1 gene promoter have been shown to be bound by c-Myc, which interacts with p73 to regulate YB-1 expression (Uramoto et. al. 2002). Another transcription factor, GATA-1, also binds to the 5'-UTR and activates the YB-1 promoter (Yokoyama et. al. 2003). Notwithstanding, mechanisms that control YB-1 expression remain largely unknown. 1.3.2 Protein Structure and Function YB-1, like all other vertebrate Y-box binding proteins, consists of three domains (Figure 2): a non-conserved variable N-terminal domain, a highly conserved cold-shock domain (CSD), and a C-terminal domain (CTD; Kohno et. al. 2003). The N-terminal domain, which is rich in alanine and proline residues, is involved in transactivation (Kohno et. al. 2003). The CSD, which contains two conserved RNP motifs (Graumann and Marahuel 1996), is homologous to bacterial cold-shock proteins (Wolffe et. al. 1992). In bacteria, cold-shock proteins respond to low-temperature by sustaining the expression of genes necessary for cell growth (Jones and Inouye 1994). The role of eukaryotic YB-1 in cold shock response is less defined, with only one report showing that at 33°C cells with the YB-1 locus disrupted do not replicate while the wild type cells grow slowly (Matsumoto et. al. 2005). It therefore seems that the CSD of eukaryotic YB-1 may not be involved in the cold shock response. In fact, the main function of CSD is to bind to oligonucleotides including RNA, double stranded DNA, and single strand DNA (Bouvet et. al. 1995, Didier, et. al. 1988, Hasagawa et. al. 1991, Kolluri et. al. 1992, MacDonald et. al. 1995). The CTD of YB-1 contains alternating regions of basic and acidic residues, termed charged zipper or the B/A repeats, and is suggested to mediate protein-protein interactions (Tafuri and Wolffe 1992, Swamynathan et. al. 1998, Nambiar et. al. 1998, Bouvet et. al. 1995). Proteins shown to interact with YB-1 include PCNA, MSH2, and DNA polymerase 5 (Ise et. al. 17 Figure 2 The Structure and Functions of YB-1 YB-1 contains an N-terminal variable domain which is rich in alanine and proline residues, a cold shock domain in which Seri02 is located, and a C-terminal domain which contains acidic and basic repeats. The nuclear localization signal and the cytoplasmic retention signal are also located within the B/A repeats in the C-terminal domain. YB-1 is multi-functional and is involved in translation, transcription, cell growth, DNA repair, and drug resistance. Y-box Binding Protein-1 Ser102 N C S D C T D NLS CRS B/A I B/A B/A 51 129 Trans- DNA/RNA activation binding 324 Protein-protein Interaction (eg, p53, PCNA, MSH2, DNA polymerase 6. P65/RelA. AP-2. CTCF. YY1, Smad3) Translation/ Transcription Cell growth DNA Repair RNA Chaperon (DNA replication, Cell cycle progression) Variable N-terminal domain (alanine proline-rich) CSD = Cold shock domain CTD = C-terminal domain; B/A = basic and acidic repeats; NLS = nuclear localization signal 1 CRS = cytoplasmic retention site Drug Resistance 18 1999, Gaudreault et. al. 2004). These alternating blocks of basic and acidic residues are also a common feature of proteins that bind to ribonucleoprotein complexes to shuttle between the nucleus and cytoplasm (Ranjan et. al. 1993). Noncanonical nuclear localization signals (NLS) and a cytoplasmic retention site (CRS) were also identified in CTD to control YB-1 cellular localization (Bader et. al. 2005). The mechanism for YB-1 trafficking remains unclear. It has been shown that the thrombin protease induced YB-1 nuclear translocation (Stenina et. al. 2001). However, whether the NLS and the CRS are involved in this process needs further investigation. 1.3.3 The Pleiotropic Functions of YB-1 YB-1 is a multi-functional protein involved in a multitude of biological processes. YB-1 shuttles between the cytoplasm and the nucleus. While in the cytoplasm, YB-1 acts as a translation factor. Once in the nucleus, YB-1 regulates transcription. Moreover, YB-1 is also involved in cell growth, DNA repair, and drug resistance (Figure 2). YB-1 in Translation YB-1 was identified as a major component of mRNPs and is essential for initiating translation (Evdokimova et. al. 1995, 1998). While the CSD binds to the sequence "AACAUC", the CTD interacts with RNA non-specifically (Bouvet et. al. 1995). Both the CSD and the CTD of YB-1 are required for incorporating mRNP into mRNA (Matsumoto et. al. 1996). Interestingly, whether YB-1 activates or represses translation depends on its concentration in a cell. It was shown in several in vitro studies that at low level, YB-1 binds to mRNA as monomers and "opens" up the transcript for translation, either by exposing the mRNA structure to translation factors or by facilitating 5'-UTR scanning. On the other hand, high concentration of YB-1 leads to homo-multimerization on mRNA to form the "beads-on-string" structure, preventing the 19 access of translation factors to the transcript (Evdokimova et. al. 1998, Skabkin et. al. 2004). YB-1 was recently shown to interfere with the translation initiation factor, eIF4G, to bind to mRNA, resulting in inhibition of translation (Evdokimova et. al. 2001, Nekrasov et. al. 2003). Moreover, the "beads-on-string" type of structure also suggests a role of YB-1 in packaging and storing cytoplasmic mRNA (Evdokimova et. al. 1995, Skabkin et. al. 2004). This hypothesis was further supported by the discovery that YB-1 binds to the cap (m7GpppG or GpppG) of mRNA thereby stabilizing the transcript by preventing degradation (Evdokimova et. al. 2001, Chen et. al. 2000). In addition to translation initiation and mRNA stabilization, YB-1 is also involved in recognizing alternative splice sites and stimulating pre-mRNA splicing (Chansky et. al. 2001, Stickeleref. al. 2001, Raffetseder et. al. 2003). While YB-1 binds mRNA and affects translation, the association between YB-1 and mRNA seems to in turn stabilize YB-1 in the cytoplasm. It has been shown by Sorokin et. al. that dissociation from mRNA leads to proteasomal cleavage of YB-1 (Sorokin et. al. 2005). Whether the cleaved YB-1 is eventually degraded or translocates into the nucleus, like the thrombin-cleaved YB-1 (section 1.3.2), was not determined in the study. YB-1 in Transcription As a transcription factor, YB-1 binds to the inverted CCAAT element in the Y-box (Didier et. al. 1988, Goldsmith et. al. 1993, Wilfred et. al. 1997, Norman et. al. 2001, Jurchott et. al. 2003). YB-1 is also able to recognize Y-box without the consensus inverted CCAAT box (Mertens et. al. 1997, Higashi et. al. 2003, Lasham et. al. 2003), and sequences flanking the Y-box have been suggested to contribute to YB-1 :DNA interactions as well (Didier et. al. 1988, Norman et. al. 2001). YB-1 is implicated in a wide range of cellular processes by functioning as either a transcription activator or repressor. First of all, YB-1 is involved in immune regulation as it blocks the expression of the class II major histocompatibility complex (Class II MHC) induced by interferon y ( IFN-y; Didier et. al. 1988, Ting et. al. 1994). The expression of FAS (Apo-l/CD95), which activates T cells and stimulates activated-induced cell death, is also suppressed by YB-1 (Lasham et. al. 2000). Considering the repressing role of YB-1 in the immune system, it appears to be a clever choice by pathogenic viruses to use YB-1 to escape from immuno-surveillance and to activate their own transcription. Indeed, YB-1 has been shown to regulate gene expression of many viruses, such as the Rous sarcoma virus d (RSV-d; Kandala and Guntaka 1994)), the human neurotropic papovavirus JC virus (JCV; Kerr et. al. 1994, Raj et. al. 1996, Safak et. al. 1999, 2002), the human T-cell lymphotropic virus type I (HTLV-I; Kashanchi et. al. 1994), the human immunodeficiency virus type 1 (HIV-1; Sawaya et. al. 1998), and adenovirus (Holm et. al. 2002). In the case of JCV, YB-1 was shown to regulate transcription by interacting with the JCV large T antigen (Safak et. al. 1999), agnoprotein (Safak et. al. 2002), and the NF-KB/rel family protein, p65/relA (Raj et. al. 1996). It is interesting to note that among these viruses, only adenoviral and HIV-1 promoters contain the inverted CCAAT box (Holm et. al. 2002), suggesting the existence of other regulatory sequences that can also be recognized by YB-1. Secondly, YB-1 is involved in cell growth. YB-1 up-regulates the transcription of CYCLIN A (Jurchotter. al. 2003), CYCLIN Bl (Jurchotte .^ al. 2003), TOPOISOMERASEII a (Shibao et. al. 1999), and DNA POLYMERASE a (En-Nia et. al. 2005), implying that YB-1 enhances cell growth possibly by promoting both cell cycle progression and DNA replication. It was further demonstrated by Swamynathan et. al. that cells with targeted disruption of one allele of YB-1 displayed defects in cell cycle and reduction in cell growth (Swamynathan et. al. 2002). A role of YB-1 in DNA replication, on the other hand, is not as clear. Although it has been suggested that YB-1 interacts with the proliferating cell nuclear antigen (PCNA; Ise et. al. 1999) and DNA polymerase 8 (Gaudreault et. al. 2004), whether YB-1 is part of the DNA replication 21 holoenzyme needs further investigation. Due to its role in cell growth, YB-1 has been implicated in cancer pathogenesis (section 1.3.4). In addition to inducing the growth-promoting genes described above, YB-1 was shown to control the expression of phosphatase PTP1B (Fukuda and Tonks 2003), matrix metalloproteinase-2 (MMP-2; Mertens et. al. 1997, 2002), matrix metalloproteinase-12 (MMP-12; Samuel et. al. 2005), collagen a 1(1) (Norman et. al. 2001), and collagen a2(I) (Higashi et. al. 2003), all of which are involved in cell adhesion, motility, and thus metastasis. The transcriptional activity of YB-1 on MMP-2 was demonstrated to be dependent on the interaction with two other transcription factors, namely the activating protein-2 (AP-2) and p53 (Mertens et. al. 2002). Furthermore, YB-1 promotes the expression of several players in drug resistance, including p-glycoprotein encoded by the multi-drug resistance gene {MDRI; Goldsmith al. 1993, Stein er. al. 2001), multi-drug resistance-related protein-1 (MRP 1; Stein et. al. 2001), as well as the major vault protein (MVP; Stein et. al. 2005). The ability of regulating genes related to metastasis and drug resistance implies that YB-1 may be a marker for tumor aggressiveness and may predict poor response to chemotherapies. YB-1 in DNA Repair YB-1 is suggested to be part of the cellular DNA repair system. It was shown by Lasham et. al. that YB-1 decreased p53 expression and thus p53 activity, suggesting YB-1 as a negative regulator of DNA repair (Lasham et. al. 2003). However, this is contradictory to another study demonstrating that YB-1 enhanced the activity of p53 (Okamoto et. al. 2000). The exact role of YB-1 in DNA repair requires further investigation, but there have been other data supporting the idea that YB-1 promotes DNA repair. For example, YB-1 was found to form complexes with the proliferating cell nuclear antigen (PCNA; Ise et. al. 1999), the MutS 22 homologue 2 (MSH2; Gaudreault et. al. 2004), and DNA polymerase 8 (Gaudreault et. al. 2004), all of which are involved in mismatch repair. Besides, YB-1 is able to bind to the Ku antigen, which is a protein implicated in base excision repair (Gaudreault et. al. 2004). The Werner syndrome (WRN) protein involved in both non-homologous end-joining and long patch base excision repair also interacts with YB-1 (Gaudreault et. al. 2004). Furthermore, YB-1 possesses several characteristics important for DNA repair. First of all, YB-1 is able to bind to single stranded DNA (Spitkovsky et. al. 1992, Gaudreault et. al. 2004), depurinated DNA (Hasegawa et. al. 1991), cisplatin-modified DNA (Ise et. al. 1999, Gaudreault et. al. 2004), as well as mismatched DNA pairs (Gaudreault et. al. 2004). Secondly, YB-1 acts as both 3'-> 5' exonuclease (Izumi et. al. 2001) and endonuclease (Gaudreault et. al. 2004) that may excise damaged DNA. Although these studies introduced a novel enzymatic function of YB-1, no further studies have been reported. Finally, it was demonstrated that YB-1 induces single-stranded conformation in DNA (MacDonald et. al. 1995). On the contrary, it was shown by another group that YB-1 also promotes DNA annealing (Skabkin et. al. 2001). The reason for the contradiction is unknown, but it was proposed that the ability of YB-1 to affect both DNA strand separation and renaturation may be associated with DNA repair (Skabkin et. al. 2001). YB-1 and Drug Resistance As described in section, YB-1 induces the expression of mdrl and mrpl, which encode the drug efflux pumps, ABC transporter P-glycoprotein and MRP 1, respectively (Goldsmith et. al. 1993, Stein et. al. 2001). YB-1 also facilitates the expression of MVP, which is another important player of drug resistance (Stein et. al. 2005). Over-expression of YB-1 has been shown to cause cells to become resistant to doxorubicin and etoposide (Bargou et. al. 1997), whereas knock down of YB-1 using antisense approaches sensitized cells to cisplatin and 23 mitomycin C (Ohga et. al. 1996, 1998). The role of YB-1 in drug resistance is further demonstrated by the report illustrating that mouse embryonic stem cells with targeted disruption of one allele of YB-1 (YB-l+/~) exhibited increased sensitivity to cisplatin and mitomycin C (Shibahara et. al. 2004). Moreover, upon exposure to UV radiation and anticancer drug such as doxorubicin and 5-fluorouracil, (Koike et. al. 1997, Ohga et. al. 1998, Bargou et. al. 1997, Stein et. al. 2005), YB-1 shuttles immediately into the nucleus followed by up-regulation of the drug resistance genes. This data is consistent with the finding that high level of YB-1 is detected in the nucleus of resistant cells (Ohga et. al. 1996). Immunohistochemical analyses performed in clinical studies have demonstrated the association between nuclear YB-1 and p-glycoprotein expression in cancers of the prostate (Gimenez-Bonafe et. al. 2004), bone (Oda et. al. 1998), ovarian (Kamura et. al. 1999), muscle (Oda et. al. 2003), and the breast (Bargou et. al. 1997, Saji et. al. 2003), suggesting YB-1 as a useful marker in predicting the effectiveness of chemotherapies. 1.3.4 The Oncogenic Property of YB-1 While many studies have focused on the role of YB-1 in drug resistance in cancers, there is growing evidence supporting the oncogenic property of YB-1. For example, the expression of YB-1 is controlled by the proto-oncogene C-MYC (Uramoto et. al. 2002). In turn, YB-1 may down-regulate the tumor suppressor gene P53 (Lasham et. al. 2003) and up-regulate several growth-related genes including TOPOISOMERASEII a, CYCLIN A, CYCLIN Bl, and DNA POLYMERASE a as described previously (Shibao et. al. 1999, Jurchott et. al. 2003, En-Nia et. al. 2005). Moreover, YB-1 represses the expression of the apoptosis-associated gene FAS (Lasham et. al. 2000). A recent study also demonstrated a physical interaction between YB-1 and Smad3 and proposed YB-1 as a component of the IFN-y-Janus kinase (Jak)-STAT pathway to 24 interfere with transforming growth factor-P (TGF-P) signaling (Higashi et. al. 2003). All these studies demonstrate a role of YB-1 in cell growth or cell survival processes, and it is not surprising that deregulation of YB-1 is implicated in the pathogenesis of cancer. Indeed, high level of YB-1 has been shown to be associated with many malignancies including colorectal carcinomas (Shibao et. al. 1999), prostate cancer (Gimenez-Bonafe et. al. 2004), osteosarcoma (Oda et. al. 1998), ovarian serous adenocarcinoma (Kamura et. al. 1999, Yahata et. al. 2002), lung cancer (Shibahara et. al. 2001, Gu et. al. 2001), synovial sarcoma (Oda et. al. 2003), and breast cancer (Bargou et. al. 1997, Janz et. al. 2002, Rubinstein et. al. 2002, Huang et. al. 2005, Wu et. al. 2006). 1.3.5 YB-1 and Breast Cancer In breast cancer, YB-1 first became recognized because of its ability to induce drug resistance. It has been confirmed that nuclear expression of YB-1 correlates with p-glycoprotein, the product of the MDR-1 gene, in breast tumor tissues (Bargou et. al. 1997). As YB-1 is known to promote cell growth and survival, the possibility that YB-1 can promote breast tumorigenesis and progression cannot be ruled out. In fact, it was discovered by several groups that YB-1 is over-expressed in breast carcinoma and that YB-1 is associated with tumor aggressiveness, relapse and poor survival (Bargou et. al. 1997, Rubinstein et. al. 2002, Janz et. al. 2002). It remains unclear whether up-regulation of transcription or gene amplification is the main cause of YB-1 over-expression in breast cancer. Furthermore, it has been shown in a transgenic mouse model that YB-1 induces mammary tumor formation with 100% genetic penetrance. Close examination of the mouse mammary tumors revealed a high content of binucleate cells, most of which are tetraploid. It was speculated that YB-1 may promote breast tumor formation and/or growth by accelerating cell cycle progression and escaping DNA damage check points (Bergmann et. al. 2005). Recently, our lab demonstrated that YB-1, but not the mutant YB-1 in which Ser 102 is changed to alanine (YB-1 A l 02), enhances the growth of breast cancer cells both in monolayer and anchorage-independent conditions (Sutherland et. al. 2005). Nonetheless, the mechanism by which YB-1 promotes breast tumor cell growth is unknown. 1.4 Epidermal Growth Factor Receptor (EGFR) As mentioned previously, YB-1 was originally identified as a DNA binding protein that interacts with the regulatory elements of EGFR (Sakura et. al. 1988). Interestingly, no follow-up studies have been reported. Since EGFR is commonly over-expressed in breast cancer, we questioned whether there is a functional link between YB-1 and EGFR in breast cancer. 1.4.1 Gene Family and Structure EGFR, a 170 kDa protein, is a receptor tyrosine kinase (RTK). It belongs to the epidermal growth factor (EGF) receptor superfamily that comprises of four closely related members: EGFR (also termed Her-1 or ERBB1), ERBB2/Her-2, ERBB3/Her-3, and ERBB4/Her-4. The promoter of the EGFR gene lacks a characteristic TATA box and CAAT box. It has high GC content and contains multiple transcription initiation sites (Ishii et. al. 1985). Both cis-and /ra«.y-acting elements have been suggested to control EGFR transcription. For example, multiple Sp-1 sites and an upstream enhancer (between -1429 to -1109) were found in the promoter region (Johnson et. al. 1988a, 1988b, Maekawa et. al. 1989). A downstream enhancer located in the first intron was also detected (Maekawa et. al. 1989). In epidermoid carcinoma cell lines, Sp-1 and the EGFR-specific transcription factor (ETF) were identified to bind and to regulate EGFR transcription (Johnson 1988a, Kageyama et. al. 1988a, 1988b). In normal 2 6 keratinocytes, an EGF-responsive DNA-binding protein-1 (ERDBP-1) was shown to interact with the EGFR promoter upon EGF stimulation; however, no functional studies were performed to illustrate the ability of ERDBP-1 to transactivate the EGFR promoter (Chen et. al. 1993). More recently, AP-1 was shown to bind to several sites in the EGFR promoter region, and c-Jun, which is a subunit of AP-1, was able to induce EGFR transcription in breast cancer cells and fibroblasts (Johnson et. al. 2000, Zenz et. al. 2003). Mice lacking c-jun in keratinocytes also exhibited reduced levels of EGFR mRNA and protein (Zenz et. al. 2003). In addition to transcription activators, two repressors namely the GC factor (GCF) and EGFR transcriptional repressor (ETR) were capable of repressing EGFR transcription by approximately two-fold (Kageyama et. al. 1989a, Kageyama 1989b, Hou et. al. 1994a). Finally, environmental signals including EGF, phorbol 12-myristate 13-acetate (PMA), cAMP (Hudson et. al. 1990), TGF-p (Hou et. al. 1994b), and hypoxia (Nishi et. al. 2002) can also induce EGFR transcription. In the case of PMA, it was demonstrated in epidermoid and HeLa cells that EGFR transcription stimulated by PMA is partly, if not all, through the transcription factor AP-2 (Johnson 1996). In a study performed in osteosarcoma cell lines, the early growth response factor-1 (Egf-1) was shown to play a role in hypoxia induced EGFR transcription (Nishi et. al. 2002). However, transcription factors that mediate other environmental stimuli to transactivate the EGFR promoter have yet to be identified. 1.4.2 Protein Structure and Function Like other receptors of the family, EGFR has an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular cytoplasmic tyrosine kinase domain (Hynes and Lane 2005). To date, at least ten growth factors were found to bind to the EGF receptors, and each receptor shows different affinity towards the ligand that it interacts with. For EGFR, there 27 are currently six EGFR-specific ligands, including EGF, transforming growth factor-a (TGF-a), amphiregulin, betacellulin, heparin-binding EGF (HB-EGF), and epiregulin (Yarden et. al. 2001). Upon ligand binding, the extracellular domain of EGFR undergoes conformational change, allowing the formation of homo- or heterodimers with other members of the EGF receptor family. Her-2, for which no specific direct ligand has been identified yet, is the preferred dimerization partner for EGFR (Graus-Porta et. al. 1997). Dimerization of the receptors in turn activates the kinase domain resulting in autophosphorylation of the specific tyrosine residues within the cytoplasmic tail. The phosphorylated tyrosine residues then serve as docking sites for adaptor proteins and downstream effectors, which leads to the activation of many cell signaling pathways. EGFR stimulates two major intracellular signaling cascades, the mitogen-activated protein kinase (MAPK) and the PI3K pathways, resulting in several cellular responses such as proliferation, survival, differentiation, and migration (Prenzel et. al. 2001, Slichenmyer and Fry 2001, Yarden et. al. 2001). After ligand binding and tyrosine kinase activation, the receptor-ligand complex is internalized for degradation or recycling (Yarden et. al. 2001). 1.4 .3 The Oncogenic Property of EGFR Signaling through EGFR not only regulates cell proliferation, but also controls a range of processes essential for tumor progression including cell survival, motility, adhesion, angiogenesis, and tumor invasion (Mendelsohn et. al. 2000). Direct evidence showing the oncogenic property of EGFR originated from a study demonstrating that over-expression of human EGFR by a retroviral vector induced a transforming phenotype in NIH 3T3 cells (Velu et. al. 1987, Di-Fiore et. al. 1987). Transgenic mice that over-expressed EGFR also developed tumors in the bladder and the breast (Cheng et. al. 2002, Brandt et. al. 2000). Enhanced or constitutive EGFR signaling is indeed observed in many tumors, such as lung, colon, ovarian, 28 and breast cancers (Laskin and Sandler 2004). Elevated EGFR signaling is thought to be caused by increased expression of the receptor and/or the ligands. 1.4.4 EGFR and Breast Cancer EGFR is commonly over-expressed in breast carcinoma (Salomon et. al. 1995, Osaki et. al. 1992, Klijn et. al. 1992, Bucci et. al. 1997). The expression of EGFR was found to be inversely correlated with ER (Tsutsui et. al. 2002, Lebeau et. al. 2003, Witton et. al. 2003, Quintela et. al. 2005, Wu et. al. 2006). More specifically, Nielson et. al. found that EGFR was highly expressed in the ER(-) basal-like subtype breast cancer (Nielson et. al. 2004). This finding corroborated with the fact that over-expression of EGFR is associated with tumor aggressiveness, metastasis, and poor patient prognosis (Klijin et. al. 1992, Tsutsui 2 et. al. 002, Nielson 2 et. al. 004, Wu et. al. 2006). Unlike other malignancies such as glioblastoma in which the EGFR gene is amplified, over-expression of EGFR in breast cancer is generally due to transcriptional up-regulation (Kersting et. al. 2004). Thus, it is possible that transcription factors that control EGFR expression may be de-regulated and contribute to tumor formation and/or progression. However, the association between EGFR and most of its transcription factors has not been indicated in the context of breast cancer. Recently, our lab showed that EGFR is co-expressed with YB-1 in breast tumor tissues (Wu et. al. 2006). In addition, it was demonstrated that normal epithelial cells expressing YB-1 cDNA exhibited elevated level of EGFR (Berquin et. al. 2005). These findings, together with the fact that YB-1 binds to the enhancer of EGFR, prompted us to further explore the possibility that YB-1 regulates EGFR expression in breast tumors and that targeting YB-1 may be a therapeutic intervention for treating breast cancers, especially those that are aggressive and over-express EGFR. 29 1.5 Thesis Hypothesis and Objectives Hypothesis YB-1 is a downstream component of the PBK/Akt pathway and promotes breast cancer cell growth by up-regulating EGFR expression. Objective 1: To examine whether YB-1 is regulated by PBK/Akt signaling ex vivo Aim 1: To determine whether YB-1 is phosphorylated via the PBK/Akt pathway ex vivo Aim 2: To assess whether YB-1 phosphorylation affects its cytoplasmic/nuclear trafficking Significance: It was previously shown that Akt phosphorylates YB-1 at Seri 02 using in vitro kinase assays. However, whether Akt phosphorylates YB-1 in the context of a living breast cancer cell has not been demonstrated. In addition, how Seri02 contributes to YB-1 functioning is unknown. It is anticipated that by accomplishing the two aims, the role of YB-1 in PBK/Akt pathway as well as the importance of Seri 02 in YB-1 activity will be determined. Objective 2: To verify whether YB-1 regulates EGFR expression Aim 1: To determine whether YB-1 promotes EGFR expression Aim 2: To examine whether YB-1 interacts with the promoter of EGFR Aim 3: To investigate whether YB-1 activates the promoter of EGFR Significance: It was found that YB-1 and EGFR are co-expressed in primary breast tumors, but evidence demonstrating functional link between the two proteins is lacking. This study will allow us to examine the possibility that YB-1 directly regulates EGFR expression in breast tumors. As well, by mutating Seri02 to alanine, we will be able to assess whether binding to the promoter and thus transcription of EGFR by YB-1 is dependent on the phosphorylation of Seri 02. 30 Objective 3: To evaluate whether OSU-03012 interferes with the function of YB-1 Aim 1: To assess whether OSU-03012 inhibits YB-1 nuclear translocation Aim 2: To examine whether OSU-03012 blocks the ability of YB-1 to bind to the EGFR promoter Significance: If the activity of YB-1 is regulated through the PBK/Akt pathway, small molecule inhibitors that block Serl02 phosphorylation should potentially inhibit the function of YB-1. By using the PDK1 inhibitor OSU-03012, the idea that Akt, YB-1, and EGFR all participate in the same pathway to promote breast cancer growth can be further verified. 31 CHAPTER 2 MATERIAL AND METHODS 2.1 Plasmids YB-1 or YB-1A102 was cloned into the 3x Flag-CMV-10 vector (Sigma, Oakville, ON, Canada) at the HindHI and Xbal restriction sites within the multiple cloning sites. These plasmids (Flag:YB-1 and Flag:YB-1 A102), along with the empty vector (Flag:EV), were transfected into MCF-7 cells to create stable clones as previously described (Sutherland, 2005), or transiently transfected in human embryonic kidney 293 cells (HEK293T) and MDA-MB-468 breast cancer cells for luciferase reporter assays (section 2.11). The pERl plasmid was obtained from Dr. Alfred Johnson of the National Cancer Institute, MD, U.S. The pERl construct was originally created by cloning approximately the first lkb of the EGFR promoter (-1109 to -16) into the HindHI restriction site of pGL3-Basic vector containing the firefly luciferase reporter gene (Promega, Madison, WI, USA). The pRL-TK plasmid (Promega, Madison, WI, USA) contains the Renilla luciferase gene, and was used as an internal control to measure transfection efficiency in the luciferase reporter assays. 2.2 Cell Culture MCF-7 (ATCC®# HTB-22; Manassas, VA) and MDA-MB-453 (ATCC®# HTB-131; Manassas, VA, USA) breast cancer cells were cultured in RPMI-1640 without phenol red (Invitrogen, Burlington, ON, Canada), supplemented with 5% fetal bovine serum (FBS) and 100 units/ml of penicillin and streptomycin (Invitrogen, Burlington, ON, Canada). MDA-MB-468 (ATCC®# HTB-132; Manassas, VA, USA) breast cancer cells and HEK293T (ATCC®# CRL-1573, Manassas, VA, USA) were grown in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Burlington, ON, Canada) containing 10% FBS and 100 units/ml of penicillin and streptomycin. MCF-7 stably transfected with Flag:EV, Flag: YB-1, or Flag: YB-1 A102 constructs were maintained in RPMI-1640 without phenol red, containing 5% FBS, 100 units/ml of penicillin and streptomycin, and 400ug/ml G418 (Invitrogen, Burlington, ON, Canada). The expressions of Flag: YB-1 and Flag: YB-1 A102 in the stable clones were constantly checked by western blotting (section 2.7). The levels of the transgene products and the endogenous YB-1 can be easily distinguished as the 3xFlag increased the recombinant protein from ~50 to ~55 kDa. All cells were maintained at 37°C with 5% C O 2 . Growth medium was changed every three days. At 90% confluency, cells were trypsinized in 0.25% trypsin with EDTA 4Na (Invitrogen, Burlington, ON, Canada), and one-tenth of the cells were subcultured. 2.3 Growth Factor or Drug Treatment After trypsinizing and re-suspending in growth medium, cells were diluted lOx in trypan blue (Invitrogen, Burlington, ON, Canada) and counted using a hemocytometer. In order to maintain the same confluency between cell lines in experiments, cells were seeded based on the size of the cells. In general MCF-7 and HEK293T cells, which are larger, were plated at lower cell number than MDA-MB-453 or MDA-MB-468. 2.3.1 Activation of PI3K/Akt pathway by IGF-1 in MCF-7 Cells To activate the PBK/Akt pathway, 8xl0 6 MCF-7 cells (80% confluent) were plated in 150mm dishes and serum-starved overnight. The next day the cells were treated with insulin-like growth factor-1 (IGF-1; GroPep, Thebarton, SA, Australia) in serum-free medium without antibiotics for lhour and harvested by scraping. To block the activation of the PBK/Akt 33 pathway by IGF-1, cells were treated with 30uM Ly294002 (Sigma, Oakville, ON, Canada) in serum-free medium for 30 minutes before adding IGF-1. 2.3.2 Inhibition of PI3K/Akt pathway by OSU03012 in MDA-MB-453 and MDA-MB-468 Cells To block the PBK/Akt signaling in MDA-MB-453 and MDA-MB-468, l x l 0 7 cells were plated in 150cm2 dishes. Since it takes longer for MDA-MB-453 cells to adhere to the dish, the cells were allowed to settle for 2 days after seeding. The cells were then treated with 10 uM OSU-03012, which is dissolved in dimethyl sulphoxide (DMSO), in serum-containing medium for 6 hours and harvested by scraping. OSU-03012 was obtained from Dr. Ching-Shih Chen from the Ohio State University, Ohio, USA. 2.4 Cell Lysis and Protein Extraction 2.4.1 Whole Cell Lysis Harvested cells were washed with ice-cold PBS and pelleted by centrifuging at 5,000 rpm for 5 minutes. The pellets were resuspended in five-packed-cell volumes of modified Radio-Immunoprecipitation Assay (RIPA) buffer (Appendix) supplemented with protease inhibitor cocktail (PIC; Appendix). To extract EGFR from MCF-7 cells, egg lysis buffer (ELB; Appendix) was used instead. The cells were incubated in the lysis buffer for 30 minutes on ice with vortexing every 10 minutes. The cells were then passed through a 22-gauge needle 10 times to release the cellular contents, and the lysates were purified by centrifuging at 13,000 rpm for 10 minutes at 4°C. The protein-containing supernatants were saved and quantified using Bradford assay (section 2.4.3). 34 2.4.2 Cytoplasmic and Nuclear Fractionation Harvested cells were rinsed with ice-cold PBS and pelleted by centrifuging at 5,000 rpm for 5 minutes. Cell pellets were resuspended in five-packed-cell volumes of Buffer A (Appendix) supplemented with PIC (Appendix) and incubated for 8 minutes on ice. The cells were then passed 10 times through a 21-gauge needle. The cytoplasmic fraction was separated from the nuclei by centrifuging at 10,000 rpm for 2 minutes at 4°C. The supernatant that contains the cytoplasmic proteins were transferred to a fresh tube, and the nuclei were washed in five-packed-cell volumes of the Borgatti Buffer B (Appendix) supplemented with PIC (Appendix). The nuclei were again pelleted by centrifuging at 10,000 rpm for 2 minutes at 4°C and the supernatant was removed. The nuclei were then resuspended in one-packed-cell volume of the Buffer C (Appendix) supplemented with PIC (Appendix) and incubated for 30 minutes on ice with vortexing every 10 minutes. The nuclear membrane was ruptured by passing through a 26-gauge needle 10 times, or by sonication (amplitude^ 40 watts; cycle= 2-3 seconds; 5 cycles) if the total sample volume is less than lOOul. The nuclear proteins were then purified by centrifuging at 13,000 rpm for 20 minutes at 4°C. The supernatant, which is the nuclear protein extract, was transferred to a new tube. Both the cytoplasmic and nuclear extracts were quantified by Bradford assay, which is describe in the subsequent section. 2.4.3 Protein Quantification Proteins were quantified by Bradford assay. To construct standard curves, increasing amounts of bovine serum albumin (1 ug, 2 ug, 5 ug, 10 ug, and 20 ug) were incubated in 1 ml of the Bio-Rad Protein Assay dye reagent (Bio-Rad, Hercules, CA, USA) for 15 minutes at room temperature for color development. The absorbance of each standard was measured at 595 nm using the GeneQuant Pro spectrophotometer (Biochrom, Cambridge, UK). A graph of 35 absorbance versus protein concentration was plotted, and the linear regression and the equation of the standard curve were determined. To quantify protein samples, 1 ul of protein extract was incubated in 1 ml of the dye reagent for 15 minutes at room temperature. The absorbance at 595nm was measured and used to calculate the protein concentration from the equation of the standard curve. 2.5 Immunoprecipitation To immunoprecipitate YB-1, 500 fig of whole cell protein extract (section 2.5.1) was diluted in RIPA buffer (Appendix) to give a final volume of 500 ul. The protein extract was pre-cleared with 50 pi of Preciphen goat anti-chicken IgY-agarose beads (Aves Labs, Tigard, Oregon, USA) for 2 hours at 4°C with rotation. The Preciphen beads were equilibrated in RIPA buffer beforehand such that the ratio of beads to RIPA buffer is 1 to 3. After 2 hours of pre-clearing, the beads were removed by centrifuging at 6,000 rpm for 5 minutes at 4°C, and the pre-cleared protein extract was incubated with 2 jag of chicken YB-lc-term antibody or pre-immune chicken IgY as a negative control (Aves Labs, Tigard, Oregon, USA) overnight at 4°C with rotation. The YB-lc-Term antibody, which recognizes the C-terminus of YB-1, was obtained from Dr. Isabelle Berquin of the Wake Forest University, NC, USA. The antibody:protein complexes were incubated with 60 ul of Preciphen for 3 hours at 4°C with rotation. The beads were then washed in Aves wash buffer A, Aves wash buffer B, and Aves wash buffer C (Appendix), two times each. To elute proteins, the beads were resuspended in 50 ul of RIPA buffer and 10 (al of 6x sample loading buffer (Appendix), vortexed briefly, and boiled for 5 minutes. By centrifuging at 6,000 rpm for 5 minutes at 4°C, the supernatant containing the purified proteins was saved and analyzed by western blotting (section 2.6). 36 2.6 Western Blotting 2.6.1 Electrophoresis The whole cell, cytoplasmic, or nuclear extract (10-50 ug) was mixed with 5x sample loading buffer (Appendix) and boiled for 5 minutes. The denatured proteins and 5ul of pre-stained broad range standards (Bio-Rad, Hercules, CA, USA) were loaded onto a SDS-polyacrylamide gel composed of a top 3% stacking gel and a bottom 12% or 6% separating gel (Appendix). For immunoprecipitation experiments, one-third of the total immunoprecipitated samples was loaded. The proteins were first compressed in the stacking gel at 82 Volts and then separated at 170 Volts for 1.5 hours. During electrophoresis, the whole system is submerged in running buffer (Bio-Rad, Hercules, CA, USA). 2.6.2 Transfer of Separated Proteins to Nitrocellulose Membrane The gel containing the separated proteins and a 0.45 (am pore-size nitrocellulose membrane were first soaked in transfer buffer (Appendix) for 20 minutes. The proteins were transferred to the membrane in chilled transfer buffer for 1 hour at 110 Volts using the Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). The membrane or the blot was then stained with Ponceau-S (0.1% in 5% acetic acid; Sigma, Oakville, ON, Canada) for 1 minute and rinsed with distilled water to ensure complete protein separation and proper transfer. 2.6.3 Primary Antibody Incubation and Detection To minimize non-specific binding, the blot was blocked with 5% non-fat milk in PBS with 0.1% Tween-20 (Fischer Scientific, Ottawa, ON, Canada) for 1 hour at room temperature. The blot was then incubated with primary antibody overnight on a rocking platform at 4°C. 37 Antibodies against the following proteins were used in western blotting: actin (Cell Signaling, Danvers, MA, USA), total Akt (Cell Signaling, Danvers, MA, USA), phospho-Aktm (Cell Signaling, Danvers, MA, USA), creb (Cell Signaling, Danvers, MA, USA), EGFR (Stressgen, Victoria, BC, Canada), total S6 ribosomal protein (Cell Signaling, Danvers, MA, USA), phospho-S6 ribosomal protein (Cell Signaling, Danvers, MA, USA), and vinculin (Upstate, Charlottesville, VA, USA). The YB-1 N-Term antibody, which recognizes the N-terminus of YB-1, was obtained from Dr. Colleen Nelson of the Prostate Centre, Jack Bell Research Centre, BC, Canada. All the antibodies were diluted 1:1000 in 5% BSA in PBS with 0.1 % Tween-20. To detect EGFR in the MCF-7 cells on western blots, a dilution of 1:275 of EGFR antibody was used. To detect protein-antibody complexes, the blot was incubated with an anti-mouse or anti rabbit IgG horseradish peroxide-linked antibody (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) in 5% non-fat milk in PBS with 0.1% Tween-20 for 1 hour on a rocking platform at room temperature. The blot was then washed three times, 5 minutes each, in PBS with 0.1 % Tween-20 and subjected to the enhanced chemiluminescence (ECL) detection system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) and film exposure (Kodak, Burnaby, BC, Canada). 2.7 Immunofluorescence MCF-7 cells expressing either Flag:EV, Flag:YB-l, or Flag:YB-1 A102 were plated on coverslips at 50-60% confluency in RPMI-1640 without phenol-red containing 5% FBS and allowed to attach overnight. After 2 days, cells were fixed with 2% paraformaldehyde in PBS for 20 minutes, washed with PBS, and permeabilized with 0.1% Triton X-100 (Sigma, Oakville, ON, Canada) in PBS for 10 minutes at room temperature. The anti-Flag M2 monoclonal antibody (Sigma, Oakville, ON, Canada) was used at a concentration of 20 ug/ml and visualized with Texas red fluorescence-conjugated anti-mouse secondary antibody (1:500 dilution). The nuclei 38 were stained with a DAPI-containing mounting medium, Vectashield (Vector Laboratories, Burlingame, CA, USA). Cellular images were captured on an Axioplan 2 Zeiss fluorescence microscope (Carl Zeiss Microimaging, Thornwood, NY, USA) at 400x magnification. 2.8 Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) RNA was isolated from log-growing MCF-7 (4xl05 cells in 35 mm dish) expressing either Flag:EV, Flag:YB-l, or the Flag: YB-1 A102 using the RNeasy mini kit (Qiagen, Mississauga, ON, Canada). The isolated RNA was quantified using the GeneQuant Pro spectrophotometer (Biochrom, Cambridge, UK). The purity of the sample was determined by the ratio of absorbance (260 nm / 280 nm), of which all samples must be greater than 1.5. Isolated RNA (1 ug) was reverse-transcribed using the Superscript™III First-Strand Synthesis System (Invitrogen, Burlington, ON, Canada). The cDNA (1 ul) was then amplified using EGFR specific primers and probes (Applied Biosystem, Foster City, CA, USA) in TaqMan® Universal PCR Master Mix (Applied Biosystem, Foster City, CA, USA). Raw fluorescence data was processed to produce threshold cycle (CT) by the ABI PRISM® 7000 Sequence Detection System. The mRNA of the house keeping gene, TATA box binding protein (TBP; Applied Biosystem, Foster City, CA, USA), was included as loading control to assess differences in sample input. Each sample was analyzed in replicates of four on two separate occasions. 2.9 Chromatin Immunoprecipitation (ChIP) 2.9.1 ChIP in MCF-7 Expressing Flag:EV, Flag:YB-1, or FIag:YB-lA102 ChIP assays in MCF-7 expressing Flag:EV, Flag:YB-l, or Flag:YB-lA102 were performed using the ChlP-IT™ kit (Active Motif, Carlsbad, CA, USA). The protocol can be downloaded from the Active Motif website (www, Buffers and reagents were 39 provided by the ChlP-IT kit unless otherwise indicated. In brief, 8xl0 6 MCF-7 expressing Flag:EV, Flag:YB-1 or Flag:YB-1 A102 cells were seeded in 150 mm plates in normal medium and fixed for 10 minutes with 1 % formaldehyde the next day. After quenching the reaction with glycine for 5 minutes, cells were lysed in Lysis Buffer supplemented with protease inhibitor cocktail (PIC) with 10 strokes using a dounce homogenizer. The nuclei was isolated and sonicated in Shearing Buffer containing PIC using a Cole Parmer Ultrasonic Processor (Cole Parmer Canada Inc., Anjou, QU, Canada) at 25% power with 10 pulses of 20 seconds each and a 30 second rest on ice between each pulse to give chromatin fragments between 200 bp and lkb. The nuclear extract was then pre-cleared with salmon sperm DNA/protein G agarose and incubated with 3 pg of anti-Flag M2 monoclonal antibody (Sigma, Oakville, ON, Canada) overnight at 4°C. The extract incubated with 3 ug mouse IgG was used as a negative control. Salmon sperm DNA/protein G agarose was added to the DNA:protein:antibody complexes and incubated for 1.5 hours at 4°C. The beads were washed, and the DNA:protein:antibody complexes were eluted from the beads. In order to reduce non-specific binding, the washing steps were modified from the ChlP-IT protocol so that the beads were washed twice with ChIP IP Buffer containing PIC, 5 times with Wash Buffer 1, twice with Wash Buffer 2, and 3 times with Wash Buffer 3. To reverse crosslink and to remove RNA, the immunoprecipitated material was incubated in 200 mM NaCl and 10 ug RNase A overnight at 65°C. The proteins were then removed by proteinase K treatment, and DNA was purified using the DNA purification mini-columns provided with the kit. Eluted DNA (5 ul) was amplified by polymerase chain reaction (PCR) and analyzed on an agarose gel (section 2.9.3). 2.9.2 C h I P in M D A - M B - 4 6 8 The protocol for immunoprecipitating endogenous YB-1 .-chromatin complexes in 40 MDA-MB-468 cells was originally adapted from the Upstate Cell Signaling Solutions Chromatin Immunoprecipitation Assay Kit manual. The protocol was modified so that the conditions were optimized for the use of chicken anti-YB-1 antibody and the Preciphen beads (Aves Labs, Tigard, Oregon, USA). MDA-MB-468 cells (9x106 cells) were plated in 150 mm dishes in regular growth medium. After OSU-03012 treatment, the cells were fixed with 1% formaldehyde in growth medium for 10 minutes, washed and harvested in cold PBS supplemented with protease inhibitors phenylmethylsulfonyl fluoride (PMSF; Sigma, Oakville, ON, Canada), aprotinin (Sigma, Oakville, ON, Canada), and pepstatin (Sigma, Oakville, ON, Canada). The cell pellet was resuspended in 300 ul of SDS lysis buffer (Appendix) with protease inhibitors and sonicated as described previously (section 2.9.1). The sonicated lysate was then diluted 5-fold in ChIP dilution buffer (Appendix) and pre-cleared with 100 ul of Preciphen beads. One-tenth of the diluted lysate was saved as an input control. The Preciphen beads were equilibrated in SDS lysis buffer beforehand such that the ratio of beads to SDS lysis buffer was 1 to 3. After pre-clearing for 2 hours, the lysate was incubated with chicken anti-YB-1 or pre-immune chicken IgY as a negative control overnight with rotation at 4°C, followed by incubation with 100 ul Preciphen for 2 hours with rotation at 4°C. Immunoprecipitates were then washed once with 1 ml of low salt buffer (Appendix), once with 1 ml of high salt buffer (Appendix), once with 1 ml LiCl buffer (Appendix), and finally twice with 1 ml of TE buffer (Appendix). Protein:DNA complexes were eluted twice by incubating with 250 ul of elution buffer (Appendix) for 15 minutes with rotation at room temperature. Eluted samples and input control were then treated with 5 M NaCl at 65°C overnight followed by proteinase K (lOug; Invitrogen, Burlington, ON, Canada) treatment at 42°C for 1 hour. DNA was purified by phenol/chloroform extraction and alcohol precipitation. The DNA pellet was resuspended in 40 pi distilled water. PCR was then performed, and products were analyzed on an agarose gel (section 2.9.3). 41 2.9.3 Polymerase Chain Reaction (PCR) DNA obtained from ChIP experiments were amplified with 1 unit/ul of Platinum Taq polymerse (Invitrogen, Burlington, ON, Canada) by PCR with the following primers: EGFR lb 5'-TCGCCGCCAACGCCACAAC-3' (forward) 5'-ACACGCCCTTACCTTTCTTTTCCTCCAG-3' (reverse) EGFR2a 5 '-CCGCGACTTTCCCTCGCATTTCT-3' (forward) 5'-CCTTCCCCCTTTCCCTTCTTTTGTTTTAC-3' (reverse) EGFR2b 5 '-TCCCATTTGCCTTTCTCTAGTTTTGTTTTC-3' (forward) 5'-GTCCACCCCATCCCCACTGTTCCTTCTC-3' (reverse) EGFR3 5' -TTCAGCAAACCCATTCTTCT-3' (forward) 5'-GCTTCCTGCACACCTGGGCTGAG-3' (reverse) GAPDH 5 '-TACTAGCGGTTTTACGGGCG-3' (forward) 5 '-TCGAACAGGAGGAGCAGCGAGCGA-3' (reverse) The PCR program was set with an initial melting step at 94°C for 3 minutes followed by 35-40 cycles of 94°C for 20 seconds, 59°C for 30 seconds, and 72°C for 30 seconds. The PCR products and 100 bp DNA markers (New England Biolabs, Ipswich, MA, USA) were diluted in 6x DNA loading buffer (Appendix) and then analyzed on a 2% agarose gel by electrophoresis in Tris-Acetate-EDTA buffer (TAE buffer; Appendix). The DNA bands were stained with ethidium bromide and viewed with the Chemi Genius2 Bio Imaging Systems (Perkim Elmer, Woodbridge, ON, Canada). The intensities of the DNA bands were quantified using the SynGene Gene Tools and Gene Snap softwares (Perkim Elmer, Woodbridge, ON, Canada). 42 2.10 Transfection 2.10.1 Transfection in HEK293T Cells The HEK293T cells were counted by trypan blue exclusion (section 2.3) and seeded in 6-well plates (4xl05 cells per well). The following day, the cells were transfected in triplicate wells using Lipofectamine™ (Invitrogen, Burlington, ON, Canada) according to manufacturer's instructions. In brief, 0.5 ugofpERl, 0.1 ug of pRL-TK, and 0.1-1.0 ug of the expression vector (Flag:EV, Flag: YB-1, or Flag:YB-lA102) were diluted in 50 ul of serum-free DMEM without antibiotics. The co-transfected pRL-TK reporter gene was used as an internal control. In a separate tube, 5 ul of Lipofectamine™ was added in 45 pi serum free medium without antibiotics. The diluted DNA and Lipofectamine™ were then mixed together. The cells were rinsed with warm phosphate buffered saline (PBS; Appendix) and 400 pi of serum free medium without antibiotics was added to each well. The DNA:Lipofectamine complexes were then added drop-wise to the cells. After 5-hour incubation, the medium containing the DNA:Lipofectamine complexes was removed and replaced with regular growth medium without antibiotics. The cells were incubated for another 18-19 hours, and luciferase reporter assay was then performed (section 2.11). 2.10.2 Transfection in MDA-MB-468 Cells MDA-MB-468 cells were seeded in 6-well plates (5xl0 5 per well) and transfected in triplicate wells using Lipofectamine™ 2000 (Invitrogen, Burlington, ON, Canada). DNA (0.5 ug of pERl, 0.1 ug of pRL-TK, and 1.0 pg of either Flag:EV, Flag: YB-1, or Flag:YB-lA102) was diluted in 250 ul serum-free Opti-Mem (Invitrogen, Burlington, ON, Canada). In another tube, 10 pi of Lipofectamine™ 2000 was added to 240 pi of serum-free Opti-Mem and incubated at room temperature for 5 minutes. The diluted DNA and Lipofectamine™ 2000 were mixed 43 together and incubated at room temperature for 20 minutes. The cells were rinsed with warm PBS, and 2 ml of regular growth medium without antibiotics was added to each well. The DNA:Lipofectamine™ 2000 complexes were then added drop-wise to the cells. The cells were transfected for 5 hours, and the medium containing the transfection complexes was replaced with regular growth medium without antibiotics. The cells were incubated for 18-19 hours, followed by luciferase reporter assay (section 2.11). 2.11 Luciferase Reporter Assay The luciferase reporter assay was performed using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA), and the protocol can be downloaded from the website ( Al l buffers and reagents were supplied with the kit. In brief, the transfected cells (section 2.4) were washed with cold PBS and incubated with Passive Lysis Buffer (PLB) for 15 minutes. The Luciferase Assay Reagent II (LARII) was then mixed with each lysate, and the firefly luciferase activity was measured using a Lumat LB 9507 luminometer (Perkin Elmer, Fremont, CA, USA). Quenching of firefly luciferase luminescence and simultaneous activation of Renilla luciferase was performed by adding the Stop and Glow® Reagent to each sample. The Renilla activity was again measured by the same luminometer. The luminometer was programmed to perform a 2-second pre-measurement delay, followed by a 20-second measurement period for each reporter assay. To account for the differences in transfection efficiencies among samples, the activity of the firefly luciferase was normalized to that of the Renilla luciferase. 44 CHAPTER 3 RESULTS 3.1 YB-1 is a Downstream Component of the PI3K/Akt Cascade 3.1.1 YB-1 is Phosphorylated at Serl02 via PBK/Akt Signaling ex vivo It was previously found that Akt forms a complex with YB-1 and phosphorylates YB-1 specifically at Ser 102 in the cold shock domain (CSD; Sutherland et. al. 2005) by in vitro kinase assays. Thus, we would like to examine phosphorylation of YB-1 by Akt in the context of breast cancer cells ex vivo. MCF-7 breast cancer cells, which have low level of phosphorylated or activated Akt (pAkt; Figure 3, lane 3), provided a suitable system in which the PBK/Akt pathway can be stimulated by growth factors such as the insulin-like growth factor-1 (IGF-1). To activate Akt, MCF-7 cells were serum-starved overnight and treated with IGF-1 for 1 hour. To measure YB-1 phosphorylation at Ser 102, the most direct approach is to use a phospho-Ser 102-YB-1 specific antibody. Unfortunately, we do not have a suitable antibody for this purpose. In fact, we have attempted to make a phospho-Serl02-YB-l antibody by two different companies, but the antibodies generated were proven to be non-specific. Alternatively, total YB-1 was immunoprecipitated and phospho-Ser-YB-1 (pSer-YB-1) was detected using a phospho-serine specific antibody by western blotting. We were able to detect a single band, which was considered to represent pSer-YB-1. The molecular weight of pSer-YB-1 appeared to be higher than YB-1 (58 kDa and 50kDa, respectively; Figure 4A, panel 2) in a SDS-PAGE gel. As shown in Figure 4A, basal level of pSer-YB-1 was observed in serum starved cells (Figure 4A, lane 1). Upon treatment of IGF-1, the PBK/Akt pathway was activated as indicated by the increased level of pAkt (Figure 4A, panel 3). Activation of the pathway also induced serine 45 Figure 3 Characterization of the Cell Lines Used in the Study The protein levels of YB-1, phospho-Akt, total Akt, phospho-S6 ribosomal protein, total S6 ribosomal protein, and EGFR were examined by western blotting in cell lines used in the study. Vinculin was used as a loading control. The cell lines used include the human embryonic kidney HEK293T cells as well as two breast cancer cell lines: MCF-7 and MDA-MB-468. The immortalized normal epithelial breast cell line, 184hTERT, was included as a reference. / Total YB-1 Phospho-AktSer473 Total Akt Phospho-S6 Total S6 EGFR Vinculin 46 Figure 4 YB - 1 was Serine Phosphorylated via the PI3K/Akt Signaling Cascade (A) YB-1 was immunoprecipitated from MCF-7 cells that were serum starved (S.S.), IGF-1 stimulated (IGF-1) for 1 hour, or pre-treated with Ly294002 for 30 minutes before IGF-1 stimulation (Ly294002+IGF-1). YB-1 phosphorylation at Ser 102 was then detected with a phospho-serine specific antibody on a western blot. Actin was used as a loading control. ( B ) The experiment was repeated three times. The band intensities of phospho-Ser-YB-1, total-YB-1, and IgY were measured using densitometry, which were then used to calculate the amount of induction or suppression of YB-1 phosphorylation by IGF or Ly294002 with the formula: (phospho-Ser-YB-1 - IgY)/total-YB-l. The basal level of phospho-Ser-YB-1 in serum starved cells was set at "1" . A student t-test was performed to calculate the p-value. A. Immuno-Precipitated sample Total cell lysate B. MCF-7 kDa 93 50 Phospho-Ser-YB-1 a.Ph0spho-ser Phospho-Ser- YB-1a.YB-i Total YB-1 a . y B . . , P h o s p h o - A k t S e r 4 7 3 Total Akt Actin 1 2 3 4 Phosphorylation of YB-1 via PI3K/Akt Pathway S.S. IGF-1 Treatment Ly294002+IGF-1 47 phosphorylation of YB-1 (Figure 4A, lane 2). By measuring the intensities of the bands using densitometry, it was found that IGF-1 stimulation caused almost 1.9 fold increase in YB-1 serine phosphorylation (p=0.018; Figure 4B). Ly294002, on the other hand, blocked PBK/Akt signaling as well as YB-1 serine phosphorylation (Figure 4A, lane 3). It was interesting to note that the level of pSer-YB-1 in Ly294002 treated cells was even lower than that in the untreated cells (Figure 4A, lane 3). Indeed, Ly294002 suppressed YB-1 serine phosphorylation approximately 5 fold (p=0.05; Figure 4B), suggesting the basal serine phosphorylation of YB-1 was partly, if not all, induced by the low level of PBK/Akt signaling in MCF-7 cells. Together these results indicated that the Seri 02 residue of YB-1 was phosphorylated through the PBK/Akt pathway ex vivo. 3.1.2 Disruption of Serl02 Suppresses YB-1 Nuclear Translocation Akt is known to directly or indirectly affect the cytoplasmic-nuclear shuttling of transcription factors by phosphorylation. It has been suggested that the CSD, in which Seri02 is located, cooperates with the C-terminal domain to control the cellular localization of YB-1 (Jurchott etal. 2003). Therefore, we questioned whether Serl02, the site of Akt phosphorylation, plays a role in YB-1 cellular trafficking. To investigate this possibility, the cytoplasmic and nuclear fractions of log-phase proliferating MCF-7 cells expressing either Flag: YB-1 or the mutant YB-1, in which Serl02 is changed to alanine (Flag:YB-l A102), were first analyzed on a western blot. The recombinant proteins were detected using either Flag (Figure 5A, first panel) or YB-1 antibody (Figure 5A, second panel). Flag: YB-1 was detected both in the cytoplasm and the nucleus, (Figure 5A, lanes 3 and 4), whereas Flag: YB-1 A102 was retained in the cytoplasm (Figure 5A, lanes 5 and 6). The densities of Flag: YB-1 and Flag:YB-lA102 bands were quantified using densitometry to determine the relative amounts of the recombinant proteins in 48 Figure 5 1 Loss of Serl02 Suppressed Nuclear Translocation of YB-1 (A) Cytoplasmic and nuclear proteins of MCF-7 cells were fractionated and examined by western blotting. Cytoplasmic and nuclear Flag:YB-l and Flag:YB-l A102 were detected using either the Flag or the YB-1 antibodies. The levels of endogenous YB-1 in the cytoplasm and nucleus were also monitored. Vinculin and creb were used as loading controls for the cytoplasmic and nuclear fractions, respectively. (B) The cellular localization of Flag: YB-1 and Flag:YB-1 Al02 (red signal) was investigated using immunofluorescence. DAPI (blue signal) was used to detect the nucleus. This was performed in collaboration with Min Chen (PhD candidate, Dr. Catherine Pallen's Lab) and presented here with permission. MCF-7:Flag EV YB-1 YB-1A102 cyto nucl cyto nucl cyto nucl B. Flag:YB-1 a . F / a g Flag:YB-1 a. y B.., EndogenousYB-1 a . y B . 7 Vinculin Creb 1 2 3 4 5 6 Merge Flag DAPI MCF-7 Flag: EV MCF-7 Flag:YB-1 MCF-7 Flag:YB-1 A102 'Published in Sutherland et. al. Oncogene. 2005. 49 the cytoplasmic and nuclear fractions. The nuclear/cytoplasmic ratios for Flag:YB-1 and Flag:YB-lA102 were 1.23 and 0.41, respectively, when proteins were detected by YB-1 antibody. Similarly, when measured by Flag antibody, the nuclear/cytoplasmic ratios for Flag: YB-1 and Flag: YB-1 A102 were 0.99 and 0.63, respectively. It was noted that the level of endogenous YB-1 in the nucleus of cells expressing Flag: YB-1 A102 was lower than that in the nucleus of cells expressing either Flag:EV or Flag:YB-l, implying that the Flag:YB-1 A102 may have a dominant negative effect (Figure 5A, third panel). To examine Flag:YB-1 and Flag:YB-1 A102 localization at the cellular level, immunofluorescence was performed in collaboration with Min Chen (PhD candidate, Dr. Catherine Pallen's Lab). Again, under log-growing conditions Flag:YB-1 was detected both in the cytoplasm and the nucleus (Figure 5B, middle panel). Nonetheless, Flag: YB-1 A102 was predominantly found in the cytoplasm (Figure 5B, bottom panel). The staining from Flag antibody appeared to be specific since only a small amount of non-specific signal on the plasma membrane, but not throughout the cytoplasm or the nucleus, was observed in MCF-7 cells expressing the Flag empty vector (Flag:EV; Figure 5B, top panel). Interestingly, while the results from western blotting demonstrated an equal distribution of Flag: YB-1 in the cytoplasm and the nucleus (Figure 5A, second panel), the data obtained from immunofluorescence showed that there were more Flag:YB-1 in the cytoplasm (Figure 5B, middle panel). The results given by western blotting should be analyzed with caution. For western blotting, 50ug of total cytoplasmic or nucleic proteins were loaded. However, the amounts of proteins in the cytoplasm and the nucleus in a living cell are not necessarily the same. The levels of the cytoplasmic and nuclear Flag: YB-1 detected by western blotting may not reflect the actual protein distribution in a single cell. In conclusion, data obtained by both techniques demonstrated that mutation of Ser 102 affected the cytoplasmic-nuclear shuttling of YB-1 under log growing conditions. 50 3.2 Activated YB-1 Induces EGFR Expression 3.2.1 YB-1 Promotes EGFR mRNA and Protein Expression Having shown that YB-1 is regulated via the PI3K7Akt signaling, we next investigated the downstream effector of activated YB-1. Our lab has recently demonstrated that YB-1, but not YB-1 Al02, enhanced breast cancer cell growth both in monolayer and anchorage-independent conditions (Sutherland et. al. 2005). Since YB-1 was originally isolated as a DNA binding protein that interacts with the regulatory elements of EGFR (Sakura et. al. 1989), we revisited the possibility that YB-1 promotes breast cancer cell growth by inducing the expression of EGFR. MCF-7 cells were again chosen as a model system because they express low level of EGFR (Figure 3, lane 3), which enabled us to compare the effect of exogenous YB-1 and YB-1A102 on EGFR expression. In comparison with Flag:EV, the levels of EGFR mRNA and protein were increased by the expression of Flag: YB-1 in cells (Figure 6 A and Figure 6B, lanes 1 and 2). However, inductions of the EGFR mRNA and protein were not observed when the mutant Flag: YB-1 A102 was present (Figure 6A). Interestingly, the level of EGFR proteins was even less in MCF-7 Flag:YB-1 A102 compared to Flag:EV (Figure 6B, lanes 1 and 3), which was in agreement with previous observation that the mutant YB-1 A l 02 may have a dominant negative effect on the endogenous YB-1 (section 3.1.2). The data shown in Figure 5A and 5B were obtained in courtesy of Dr. Sandra Dunn and Daniel Yokom (summer student, Dr. Sandra Dunn's lab), respectively. 3.2.2 YB-1 Interacts with the Promoter of EGFR The data from section 3.2.1 suggested that YB-1 regulates EGFR expression in part, if not all, at the transcriptional level. We have identified several putative YB-1 responsive elements (YREs) that contain the inverted CCAAT boxes (ATTGG or ATTG; Figure 7), by retrieving the 51 Figure 62 YB-1, but not YB-1A102, Induced EGFR Expression (A) The level of EGFR mRNA transcript was measured by qRT-PCR in MCF-7 cells expressing Flag:EV, Flag: YB-1, or Flag:YB-lA102. The amount of EGFR mRNA in MCF-Flag:EV was considered as the basal-level and was set as " 1 " . qRT-PCR was performed by Dr. Sandra Dunn and presented here with permission. (B) The amount of EGFR protein in MCF-7 expressing Flag:EV, Flag:YB-l, or Flag:YB-l A102 was measured by western blotting. Vinculin was used as loading control. This western blotting was performed by Daniel Yokom (summer student, Dr. Sandra Dunn's lab) and presented here with permission. 2Published in Wu et. al. Cancer Res. 2006. 52 -2 kb EGFR promoter sequence from the UCSD genome browser ( In addition, YB-1 has been shown to interact with the antisense strand of the DNA (McDonald et. al. 1995). Therefore we also scanned the -2 kb of the reverse strand of the EGFR promoter for inverted CCAAT boxes. In total, nine putative YREs were found in the -2 kb of the EGFR promoter. While the occurrence of these YREs may be random, it is possible that these YREs are functional in regulating EGFR transcription. Chromatin-immunoprecipitation (ChIP) was therefore performed to first investigate whether there is an interaction between YB-1 and the EGFR promoter. Since the CSD of YB-1 is known to bind to DNA, we also speculated whether disruption of Ser 102 would effect YB-1 :DNA binding and thus EGFR gene expression. Central to addressing this question, YB-1A102 was subjected to the ChIP assay as well. Four sets of primers were designed to analyze different regions containing the YREs in the -2 kb of EGFR promoter (Figure 7). The first 1 kb region was examined by primers lb and 2a, while the region between -1 to -2 kb was investigated by primers 2b and 3. A primer set that amplified the gapdh gene was used as a negative control. Prior to ChIP, the efficiencies of the primers were assessed by amplifying genomic DNA from MCF-7 cells. Al l primer sets were able to amplify DNA templates producing PCR products of the expected sizes (Figure 8A). Next, chromatin from MCF-7 Flag:YB-1 or Flag:YB-1 Al02 was sheared into fragments between 200 bp and 1 kb (Figure 8B) by sonication, and protein:DNA complexes were isolated using the Flag antibody. To ensure that same amounts of input samples were used and that PCR was in the linear range, input DNAs from MCF-7 Flag:YB-1 and Flag: YB-1 A102 were amplified and titrated with the 2a primers (Figure 8C). As shown in Figure 9A, Flag: YB-1 was able to interact with the first 2 kb of the EGFR promoter (Figure 9A, lanes 1-4). Intriguingly, while the interactions with the 2b and 3 region were retained (Figure 9A, lanes 8 and 9), binding to the lb region by Flag:YB-lA102 was abolished (Figure 9A, lane 6). Also, Flag:YB-lA102 did not bind to the 2a region as strongly as 53 Figure 7 3 The First 2 kb of the EGFR Promoter The first 2 kb of the egfr promoter was retrieved from the UCSD Genome Browser ( Nine putative YREs were found, including one YRE in the lb region, two YREs in the 2a region, one YRE in the 2b region, and 5 YREs in the 3 region. Four sets of primers (lb, 2a, 2b, and 3) were designed, and each primer set covers one or more YREs in the egfr promoter. EGFR p r o m o t e r -2kb -fkb +1 ATG 2b 2a 1b i p r o m o t e r • •• i n t r o n > • f o r w a r d p r i m e r | A T T G | C A A T • e x o n S 5 ' - U T R < r e v e r s e p r i m e r | A T T G G 3Published in Wu et. al. Cancer Res. 2006. 54 Figure 84 Validation of the Primers and Conditions for Chromatin-immunoprecipitation (ChIP) (A) Genomic DNA from MCF-7 cells was amplified using the EGFR primers (lb, 2a, 2b, and 3) as well as the GAPDH primers by PCR. The GAPDH primers were used as a negative control in the ChIP assay. The PCR products were analyzed on a 2% agarose gel. (B) Genomic DNA from MCF-7 expressing Flag:YB-l or Flag:YB-l A102 was sheared by sonication. The sheared DNA was analyzed on a 2% agarose gel to confirm that the size of DNA fragments were between 200 bp and 1 kb. (C) Increasing amounts of input DNA from each sample was examined by PCR using the EGFR 2a primer set. A. M C F - 7 g e n o m i c D N A n o t e m p l a t e 5 0 0 b p 2 0 0 b p B . S h e a r e d Input M C F - 7 F l a g : CM O 1 k b 0.2 k b 4 5 6 7 8 9 10 c. Input : M C F - 7 F l a g : Y B - 1 Y B - 1 A 1 0 2 3 3 3 co m 3 3 3 t - co in 5 0 0 b p 1 1 2 3 4 5 6 1 2 Published in Wu et. al. Cancer Res. 2006. 5 5 Figure 9 s YB-1, but not YB-1A102 Interacted with the -1 kb of the EGFR Promoter (A) ChIP was performed in MCF-7 cells expressing Flag:YB-1 or Flag:YB-lA102. The DNA obtained from the ChIP assay was amplified using the four EGFR primer sets (lb, 2a, 2b, and 3) as well as the GAPDH primers and analyzed on a 2% agarose gel. IgG was used as a negative control for the ChIP Assay. (B) The assay was repeated three times, and the intensities of DNA bands were quantified using densitometry. The degree of Flag: YB-1 or Flag: YB-1 A102 binding to each EGFR promoter region was calculated using the formula: (binding by YB-l/input)-(binding by IgG/input). The level of DNA binding by Flag:YB-l was set as "1" . • * 1 Ii 1 II * EGFR promoter -1kb I * I'll +1 ATO ' 1M 1 II < I < 'I! < 2b 1b 3 2a MCF-7 Flag:YB-1 MCF-7 Flag:YB-1A102 IgG 500 bp 200 bp J3 ro T - CM n CM co GAPDH I n ro CM CM CO GAPDH I n T — n CM n CM co GAPDH | mmm — mm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Inhibition of YB-1 :EGFR Promoter Interaction by Mutation at Ser102 Promoter Region of EGFR Published in Wu et. al. Cancer Res. 2006. Flag: YB-1 did (Figure 9A, lane 7). Quantification of the intensities of the DNA bands revealed that while binding to the 2b and 3 regions by Flag:YB-lA102 was retained, disruption of Ser 102 completely prevented YB-1 from interacting with the first 1 kb of the EGFR promoter (Figure 9B). The inability of Flag: YB-1 A l 02 to bind to the -1 kb region may be the reason why Flag:YB-lA102 was not capable of inducing EGFR gene expression (section 3.2.1). Furthermore, although a small amount of non-specific binding by IgG was observed (Figure 9A, lanes 11 to 15), there was no amplification by the GAPDH primers (Figure 9A, lanes 5 and 10). Thus, the binding of Flag: YB-1 or Flag: YB-1 A102 to the EGFR promoter was considered specific. Taken together, we concluded that Ser 102 is involved in DNA binding. Specifically, YB-1 interacts with the EGFR promoter, and Ser 102 is required for optimal binding to the -1 kb region. 3.2.3 YB-1 Activates the Promoter of EGFR The ability of Flag: YB-1, but not Flag: YB-1 A102, to bind to the -1 kb of the EGFR promoter suggested that YB-1 may regulate EGFR transcription via this region. To address this possibility, reporter assays were performed using a luciferase construct (pERl) harboring the -16 to -1109 sequence of the EGFR promoter, which was a generous gift from Dr. Alfred Johnson from Maryland, U.S. The construct was first transfected in MCF-7 expressing Flag:EV, Flag:YB-l, or Flag: YB-1 A102. Unfortunately, we could hardly detect luciferase activity in any of these cell lines (data not shown). One reason could be that since MCF-7 cells are estrogen receptor positive (ER(+)) and express very little EGFR (Figure 3, lane 3), there could be strong repressors acting on the EGFR promoter in this cell line. Besides, phosphorylation of YB-1 may be minimal as Akt is not activated in MCF-7 cells. We also learned that luciferase activity is relatively hard to measure to produce significant and consistent data in MCF-7 possibly due to the nature and the low transfection efficiency of the cell line (personal communication with Dr. 57 Figure 10 Disruption of Seri 02 Inhibited YB-1 to Activate EGFR Transcription Luciferase Assays were performed by co-transfecting pERl with either (A) 1 pg of the expression vector in HEK293T, (B) increasing amounts (0.1, 0.5, and 1 pg) of the expression vector in HEK293T cells, or (C) lug of the expression vector in MDA-MB-468 cells. Each assay was performed in triplicates and repeated three times. The luciferase activity measured from cells containing 1 pg of the Flag YB-1 A l 02 expression vector was set as "100%". A student t-test was performed to calculate the p-value. A. o (0 TO i t > o on 300 250 200 150 100 50 0 Luciferase Assay (HEK293T; 24h) 0.003 r f « F l a g : E V • F l a g : YB - 1 B F I a g : Y B - 1 A 1 0 2 Expression vector (1.0ug) co-transfected with pER1 o w 3 & > o l < 5 a: Luciferase Assay (MDA-MB-468; 24h) p=0.0005 \— « F l a g : E V • F l a g :YB -1 H F I a g : Y B - 1 A 1 0 2 Expression vector (1.0ug) co-transfected with pER1 58 Catherine Pallen). As a result, we changed the model system to the human embryonic kidney 293 (HEK293T) cells, as they are easier to transfect. After transient co-transfection of pERl with the Flag: YB-1 expression vector or the empty vector, no induction or very small induction of the luciferase activity was observed (Figure 10A). However, luciferase activity was significantly lower in cells co-transfected with the Flag: YB-1 A102 mutant (p=0.003; Figure 10A). The decrease in luciferase activity by Flag:YB-1 A102 was also dose-dependent (Figure 10B). To examine whether the result was reproducible in a breast cancer cell line, luciferase assay was performed in MDA-MB-468 breast cancer cells. This cell line was chosen because it expresses high levels of EGFR and activated Akt (Figure 3, lane 4). Consistently, we did not observe a significant increase in luciferase activity in the presence of the Flag: YB-1 expression vector (Figure 10C). However, Flag:YB-1 A102 was again able to significantly suppress the reporter activity (p=0.0005; Figure 10C). The inability of Flag: YB-1 to further induce EGFR promoter activity in neither HEK293T nor MDA-MB-468 cells may be due to intrinsically high levels of YB-1 and pAkt in these cell lines (Figure 3, lanes 2 and 4). Together, these data demonstrated that when Seri 02 was mutated, YB-1 lost its ability to activate the -1 kb of the EGFR promoter. 3.3 OSU-03012 Inhibits the Functions of YB-1 3.3.1 OSU-03012 Blocks YB-1 Nuclear Translocation For the past few years, our lab has been interested in identifying small molecular inhibitors for treating breast cancer. Recently, we have demonstrated that the celecoxib derivative, OSU-03012, a stable and orally available potent PDK1 specific inhibitor, decreased the growth of breast cancer cells with high level of pAkt (Kucab et. al. 2005). Having demonstrated YB-1 as a downstream component of PBK/Akt pathway, it is therefore possible to inhibit the activity of YB-1 using OSU-03012. First, we examined whether OSU-03012 prevented YB-1 from 5 59 translocating into the nucleus. The MDA-MB-453 breast cancer cells in which Akt was highly activated (Noh et. al. 2004) were treated with OSU-03012 for 6 hour, and YB-1 nuclear trafficking was inspected by cytoplasmic-nuclear fractionation and western blotting. Under normal log growing conditions, YB-1 was detected both in the cytoplasm and the nucleus (Figure 11 A, lanes 1 and 3). However, when log-phase proliferating cells were treated with OSU-03012, the level of nuclear and cytoplasmic YB-1 decreased and increased, respectively (Figure 11 A, lanes 2 and 4). In fact, the ratio of nuclear to cytoplasmic YB-1 decreased almost 2 fold in OSU-03012 treated cells (p=0.05; Figure 1 IB). The efficacy of the drug to inhibit PDK1 signaling was assessed by a decrease in the phosphorylation of Akt and the S6 ribosomal protein (Figure 11 A, panel 2 and 4). It was noted that the level of total S6 ribosomal protein changed after drug treatment. In fact, we and others have observed inconsistent levels of total S6 ribosomal protein after drug treatment or growth factor stimulation. Nonetheless, the phosphorylation of S6 ribosomal protein is still widely used by researchers as a downstream marker of the PBK/Akt signaling. The levels of vinculin and creb were not affected by OSU-03012 (Figure 11A, panels 6 and 7), indicating that decrease in YB-1 protein level by OSU-03012 was specific. 3.3.2 OSU-03012 Prevented YB-1 from Binding to the EGFR Promoter Since it was determined that disruption of Seri 02 prevented YB-1 from binding to the -1 kb of the EGFR promoter as detected by ChIP using the lb and 2a primer sets, we then asked if the interaction between YB-1 and the -1 kb of EGFR promoter can be abolished by OSU03012. MDA-MB-468 cells provided a good model system for this study because they express high levels of pAkt and EGFR (Figure 3, lane 4). MDA-MB-468 cells were treated with OSU03012 for 6 hours, and the effect of the drug was indicated by decreases in the levels of pAkt and pS6 60 Figure 11 OSU-03012 Inhibited YB-1 Nuclear Translocation (A) MDA-MB-453 cells were treated with OSU-03012, and cytoplasmic and nuclear protein fractions were extracted. The cellular localization of YB-1 was examined by western blotting. Vinculin and creb were used as loading controls for cytoplasmic and nuclear fractions, respectively. (B) The experiment was repeated three times, and the band intensities of cytoplasmic and nuclear YB-1 were measured by densitometry. The difference in YB-1 cellular localization upon OSU-03012 treatment was assessed by the ratio of (nuclear YB-1/cytoplasmic YB-1). A student t-test was performed to calculate the p-value. MDA-MB-453 Cyto Nucl OSU-03012 YB-1 Phospho-AktSer473 Total-Akt Phospho-S6 Total-S6 Vinculin Creb B. DO > O E tn ro Q . o O o *-» >. ro O or T— CQ >-i _ ro <D o 3 Z Inhibition of YB-1 Nuclear Localization by OSU03012 in MDA-MB-453 DMSO OSU03012 Treatment 61 ribosomal protein (Figure 12A, panels 2 and 4). ChIP was performed and there was a clear loss of binding of YB-1 to the first 1 kb of the EGFR promoter after treatment with OSU-03012 (Figure 12B, top and middle panels). The reduced binding of YB-1 to the EGFR promoter in OSU03012-treated cells was not due to unequal inputs (Figure 12B, lanes 5 to 7). Furthermore, the specificity of the ChIP assay was confirmed as there was no non-specific binding by IgY (Figure 12B, lane 3) and DNA was not amplified by the GAPDH primers (Figure 12B, bottom panel). The ability of OSU-03012 to inhibit the binding of YB-1 to the lb and 2a regions was quantified using densitometry. Interestingly, while the binding of YB-1 to the lb region was prevented more than 2 fold (p=0.036; Figure 12C), the interaction between YB-1 and the 2a region was almost completely abrogated (p=0.024; Figure 12D). Whether the interaction between YB-1 and 2b and 3 is also affected by OSU-03012 is currently under investigation. In summary, OSU-03012 was able to block YB-1 to shuttle into the nucleus and to bind to the EGFR promoter. Moreover, the data generated here further verified the relationship between Akt, YB-1, and EGFR. 62 Figure 12 OSU-03012 Prevented YB-1 from Binding to the -1 kb of the EGFR Promoter (A) MDA-MB-468 cells were treated with OSU-03012. The cell lysates were examined by western blot to ensure that OSU-03012 inhibited the PBK/Akt signaling. Vinculin was used as a loading control. (B) DNA of untreated or OSU-03012 treated cells obtained from ChIP assays, which were repeated three times, were amplified using the EGFR lb or 2a primers. The PCR products were analyzed on a 2% agarose gel. IgY was used as a negative control for the ChIP assays. The intensities of DNA bands were quantified using densitometry. The degree of YB-1 binding to the (C) EGFR lb region or the (D) EGFR 2a region were calculated using the formula: (OSU-03012Chip/OSU-03012input)- (IgYcwp/IgYinput). The level of DNA binding by YB-1 in cells treated with DMSO was set as "1" . A student t-test was performed to calculate the p-value. MDA-MB-468 OSU-03012 YB-1 Phospho-Akt S e r 4 7 3 Total Akt Phospho-S6 Total S6 Vinculin B. EGFR promoter -1kb 2a MDA-MB-468 +1; ATG —> < 1b O CO ChIP o c o o I co o >-O) Q o o CO Input CM o c o c p CO o >-63 o .S — TJ d) C S m 11 •2 Q -*-» re t -1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Inhibition of YB-1 :egfr Promoter (1 b) Interaction by OSU-03012 p=0.036 r '1 DMSO OSU0301 2 Treatment o •= — TJ © C 5 m .2 Q +* .. re ,-r2* Inhibition of YB-1 :egfr Promoter (2a) Interaction by OSU-03012 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 p=0.024 f~ , r DMSO OSU03012 Treatment 64 CHAPTER4 DISCUSSION The importances of Akt, YB-1, and EGFR have been separately implicated in breast cancer. In this study, we have demonstrated a functional link that connects all three proteins in the same pathway. We first showed that YB-1 is phosphorylated at serine residues through PBK/Akt signaling in the context of breast cancer cells by immunoprecipitation and western blotting (section 3.1.1). Since the CSD has been suggested to contribute to YB-1 cellular localization, we then assessed whether Serl02 may be involved in YB-1 nuclear translocation. We found that disruption of Ser 102 prevented YB-1 from moving into the nucleus by western blotting and immunofluorescence (section 3.1.2). We next investigated the possibility that YB-1 up-regulates the expression of EGFR. It was discovered that YB-1 but not YB-1 A102 increased both the EGFR mRNA and protein levels (section 3.2.1). This result suggested that YB-1 may regulate EGFR transcription. To assess whether this was the case, we determined if YB-1 bound the EGFR promoter. We first identified nine putative YREs in the -2 kb of the EGFR promoter. Results from chromatin immunoprecipitation revealed that while YB-1 interacted with the -2 kb of the EGFR promoter, YB-1 A l 02 could only bind to the -1 kb region (section 3.2.2). By using luciferase reporter assays, we discovered that transfection of the Flag: YB-1 expression vector could only activate the EGFR promoter to a small extent. However, Flag: YB-1 A102 suppressed EGFR transcription significantly (section 3.2.3). Together, these data support the rationale that PBK/Akt activates YB-1, which in turn stimulates EGFR expression resulting in enhanced cancer cell growth. Finally, we used OSU-03012 to target PDK1 and thus Akt to further validate the association among Akt, YB-1, and EGFR. It was found that OSU-03012 inhibited YB-1 65 nuclear localization and binding to the EGFR promoter (section 3.3). In light of the work presented here, YB-1 may be considered as a novel therapeutic target to treat breast cancer. 4.1 Phosphorylation of YB-1 by PBK/Akt In this study, we first demonstrated that YB-1 is a component of the PI3K/Akt pathway. Upon Akt activation by IGF-1 stimulation, YB-1 is phosphorylated at serine residues ex vivo. It is interesting to note that the location of Ser 102 in the CSD is in a flexible loop region of YB-1, which is accessible to the crevice formed by Aktl in its active conformation (Sutherland et. al. 2005). In this experiment we observed that pYB-1 migrated slower than YB-1 on a SDS-PAGE gel. Phosphorylation is known to affect protein mobility on a gel. Taking FKHRL1 as an example, when it is phosphorylated by Akt, a band shift was observed (Biggs et. al. 1999 or Brunet et. al. 1999). The mechanism for band shifting is not clear but is thought to involve the negative charge of the phosphate group, which interferes with SDS to bind to the protein. It is possible that the single band detected by the phospho-serine antibody did not represent pYB-1 as other proteins might be co-immunoprecipitated with YB-1. To further confirm that the up-shifting band is indeed pYB-1, phosphatase treatment, which should cause the up-shifting band to disappear, can be performed. Alternatively, the protein can be extracted from the gel and sequenced by mass spectrophotometry. The advantage of the second approach is that other phosphorylation sites on YB-1 can be discovered. Indeed, evidence suggests that Akt is not the only protein to phosphorylate YB-1, which is discussed below. 4.1.1 Potential Proteins that Phosphorylate YB-1 via P B K Signaling This year Evdokimova et. al. similarly demonstrated that IGF-1 stimulation leads to YB-1 phosphorylation. In their study, YB-1 was immunoprecipitated with a custom-made YB-1 66 antibody, and phosphorylation of YB-1 was detected by radio-labeled orthophosphate (Evdokimova et. al. 2006). Wortmannin, which is another PI3K inhibitor, fully blocked phosphorylation of YB-1. However, mutating Seri 02 to alanine did not completely prevent YB-1 phosphorylation, suggesting that other components of PI3K/Akt cascade may be involved (Evdokimova et. al. 2006). To explore this possibility, we used the motif scanner prediction tool ( to identify putative YB-1 phosphorylation sites (Figure 13). Interestingly, protein kinase C (PKC)-e, a PKC isoform characterized as a calcium-independent and phorbol ester/diacylglycerol-sensitive serine/threonine kinase, could potentially phosphorylate YB-1 at Seri 02. It has been shown that PI3K converts PIP2 to PIP3, which activates PKC-e, resulting in cell proliferation and survival (Moriya et. al. 1996, Graness et. al. 1998). PKC-e has been implicated in tumorigenesis, invasion, and metastasis (Mischak et. al. 1993, Perletti et. al. 1998, Wu et. al. 2002, Jansen et. al. 2001, Berrier et. al. 2000). The role of PKC-e in breast cancer is less defined, and whether phosphorylation of YB-1 at Seri 02 by PKC-e contributes to the pathogenesis needs further investigation. Moreover, the glycogen synthase kinase 3 (GSK3), which is negatively regulated by Akt (Diehl et. al. 1998; section 1.2.2), could also potentially phosphorylate YB-1 at Ser21 in the N-terminal domain and affect the transcriptional activity of YB-1 (Figure 13). GSK3 has been implicated in cancers as it is involved in the degradation of p-catenin, a marker of the epithelial-mesenchymal-transition (EMT) and metastasis (Aberle et. al. 1997). It is therefore motivating to examine whether GSK3 phosphorylates YB-1 and whether this event has any significance in breast cancer progression. Furthermore, Since Evdokomova et. al. demonstrated YB-1 phosphorylation via the PI3K/Akt signaling in cells by using radioactive ATP (Evdokimova et. al. 2006), phosphorylation of YB-1 detected by this approach could also be due to phosphorylation at residues other than serines. Indeed, results from the motif scanning prediction tool suggested that the p85 subunit of 67 Figure 13 Putative Proteins that Phosphorylate YB-1 via the PI3K or the MAPK Signaling Pathway To examine possible kinases that could also phosphorylate YB-1 through the PI3K or the MAPK signaling pathway, the motif scanning prediction tool ( was used. It was found that in addition to Akt, P K C - E may also phosphorylate YB-1 at Seri 02. Since RSK recognizes the same sequence as Akt in its binding partners, RSK is another potential kinase to phosphorylate YB-1 at Seri 02. GSK3, ERK1, and the p85 subunit of PI3K were also suggested by the motif scanning prediction tool to phosphorylate YB-1 at Ser21, Ser36, and Tyrl97, respectively. Y-box Binding Protein-1 ERK1 PKC-e Akt RSK p85 I S21S36 S102 Y197 N- CTD B/A I B/A B/A 51 129 NLS CRS 324 Trans- DNA/RNA activation binding Protein-protein Interaction (eg, p53, PCNA, MSH2, DNA polymerase 6, P65/RelA, AP-2, CTCF, YY1, Smad3) Variable N-terminal domain (alanine proline-rich) CSD = Cold shock domain CTD = C-terminal domain; B/A = basic and acidic repeats; NLS = nuclear localization signal | CRS = cytoplasmic retention site ^ Signaling through the PI3K pathway ""*" Signaling through the MAPK pathway 68 PBK may phosphorylate YB-1 at Tyr 197 in the C-terminal domain (Figure 13). Curiously, Tyrl97 is located within the NLS (residue 183-202; Bader et. al. 2005) of YB-1, which raises the question whether YB-1 subcellular localization is affected by phosphorylation at Try 197 by PBK. If YB-1 is phosphorylated by one or more proteins discussed above, inhibiting the PBK signaling by Ly294002 or wortmannin should block YB-1 phosphorylation via PBK signaling. Mutation of Ser 102, however, will not completely inhibit YB-1 phosphorylation, as observed by Evdokimova and coworkers (Evdokimova et. al. 2006). 4.1.2 Potential Proteins that Phosphorylate YB-1 via MAPK Signaling The possibility that YB-1 may be phosphorylated by other signaling pathways cannot be ruled out. Our lab has recently discovered that neither Ly294002 nor OSU-03012 decreased YB-1 serine phosphorylation in aggressive breast cancer cell lines such as MDA-MB-453 and MDA-MB-468 (data not shown). In these cell lines, both PBK/Akt and MAPK pathways are highly activated. Thus, it is possible that the MAPK pathway also contributes to YB-1 serine phosphorylation. In support of this hypothesis, data obtained from the motif scanning prediction tool suggested a few players of the MAPK/ERK pathways as putative proteins to phosphorylate YB-1 at serine residues (Figure 13). For example, the Ser36 residue located in the transactivating N-terminal domain of YB-1 may be a target for the extracellular-signal-regulated kinase 1 (ERK1). ERK is activated by MAPK in response to growth factor stimulation, resulting in cell proliferation and survival (Chang and Karin 2001, Sebolt-Leopold and Herrera 2004). Elevated MAPK/ERK signaling also leads to malignancies such as breast cancer (Sivaraman et. al. 1997, El-Ashry et. al. 1997, Coutts and Murphy 1998, Donovan et. al. 2001). It would be interesting to pursue whether YB-1 and the MAPK/ERK pathway work cooperatively in breast cancer pathogenesis. Small molecules such as the MEK inhibitor, PD98059, would be valuable in 69 determining the role of YB-1 in the MAPK/ERK pathway in breast cancer cells. Finally, Akt binds to and phosphorylates YB-1 by recognizing the RxRxxS/T motif, in which S is the SI02 residue (Alessi et. al. 1996). It was noted that the p90 ribosomal S6 kinase (RSK), a downstream component of MAPK/ERK signaling (Frodin and Gammeltoft 1999), also recognize the same sequence in its binding partner (Alessi et. al. 1996). Therefore, RSK could be another candidate protein that activates YB-1 (Figure 13). 4.2 The Role of Serl02 in the Regulation of YB-1 by Akt We have examined the importance of Serl02 in the regulation of YB-1 by Akt by using the YB-1 A102 mutant. It was revealed that Ser 102 is involved in YB-1 nuclear localization and DNA binding, which ultimately influence the ability of YB-1 to activate transcription. 4.2.1 Serl02 and Nuclear Translocation The disruption of Serl02 retained Flag:YB-lA102 in the cytoplasm (section 3.1.2). Besides, treatment of OSU-03012 prevented endogenous YB-1 to traffic into the nucleus, resulting in the accumulation of YB-1 in the cytoplasm (section 3.3.2). Therefore, nuclear translocation of YB-1 can be controlled by Ser 102 phosphorylation via the PBK/Akt pathway. To further confirm that activation of PBK/Akt pathway leads to YB-1 nuclear localization, we have previously stimulated the cells with IGF-1 for 1 hour and examined YB-1 cellular localization by western blotting. Treatment of IGF-1 for 1 hour did not induce YB-1 nuclear translocation (data not shown). In a study conducted by Higashi et. al., the ability of interferon-y (IFN-y) to promote YB-1 nuclear translocation was demonstrated (Higashi et. al. 2003). Interestingly, IFN-y induced nuclear translocation of YB-1 was observed 6 hours after stimulation (Higashi et. al. 2003). Thus, it is possible that stimulation of IGF-1 for 1 hour may 70 not be long enough to induce YB-1 nuclear translocation. Akt is known to directly or indirectly control the cytoplasmic-nuclear localization of proteins, or transcription factors, through different mechanisms. First of all, Akt can phosphorylate the nuclear localization signal (NLS) or the nuclear export signal (NES) of its substrates, such as the FOXO forkhead transcription factors and p27. In the case of FOXO, hyperphosphorylation of FOXO proteins by Akt at NLS and NES facilitates binding with 14-3-3, which promotes the nuclear export of FOXO proteins through the exportin CRM1 (Brunet et. al. 2002, Rena et. al. 2001, Biggs et. al. 1999, Brownawell et. al. 2001). On the other hand, Akt phosphorylation at the NLS of p27 inhibits p27 nuclear import resulting in cytoplasmic sequestration (Liang et. al. 2002, Shin et. al. 2002). Recently, YB-1 was shown to have a noncanonical NLS as well as a cytoplasmic retention site (CRS) that prevails over the NLS (Bader et. al. 2005). However, both of NLS and CRS are located in the C-terrninal domain of YB-1 whereas Ser 102 is resided in the CSD. Therefore, it is unlikely that Akt regulates YB-1 cellular localization directly through NLS or CRS phosphorylation. Nonetheless, it is possible that Ser 102 cooperates with NLS and/or CRS in YB-1 nuclear trafficking. In fact, it has been suggested by Jurchott et. al. that both the C-terminus and the CSD are involved in YB-1 nuclear shuttling (Jurchott et. al. 2003). Another approach utilized by Akt to affect protein cellular localization involves the phosphorylation of an inhibitor that is bound to the NLS of the protein. For instance, under basal condition the transcription factor N F - K B forms a complex with the inhibitor I K B , which masks the NLS on N F - K B and prevents translocation of N F - K B to the nucleus. Upon growth factor stimulation, Akt becomes phosphorylated and activates the I K B kinase (IKK). The activated IKK in turn phosphorylates I K B and targets it for degradation by the 26S proteasome. Consequently, the NLS is revealed and N F - K B nuclear import occurs (Henkel et. al. 1992, Beg et. al. 1992). To date, no proteins that bind the NLS or CRS of YB-1 have been found. Although it has been reported that functional p53 is required for YB-1 nuclear translocation, physical interaction between the two proteins is not involved (Zhang et. al. 2003). It was suggested that p53 causes nuclear translocation of YB-1 by activating a downstream target gene, the product of which mediates this translocation process (Zhang et. al. 2003). In normal cells, YB-1 mainly locates in the cytoplasm, where it is bound to mRNA. As illustrated by Stenina et. al, the release of YB-1 from mRNA induced by P D G F and the thrombin protease results in YB-1 nuclear localization (Stenina et. al. 2001). It was further demonstrated that phosphorylation of YB-1 at Serl02 by Akt reduced the interaction of YB-1 with the capped 5' terminus of mRNA (Evdokimova et. al. 2006). Therefore, the binding of YB-1 to mRNA may be analogous to the interaction of N F - K B with IKB in the cytoplasm, the effect of both is the prevention of transcription factors to translocate into the nucleus. Taken together, we propose a model that may explain how YB-1 cellular trafficking occurs. In unstimulated cells, YB-1 mainly resides in the cytoplasm where it is bound to mRNA. The association between YB-1 and mRNA may prevent YB-1 from degradation. In support of this view, it has been demonstrated that proteasome cleavage of YB-1 is protected by mRNA binding (Sorokin et. al. 2005). Upon stimulation by growth factors, PBK/Akt signaling phosphorylates YB-1 at Ser 102 located in CSD, resulting in the dissociation of YB-1 from mRNA in the cytoplasm. Furthermore, phosphorylation of Ser 102 may also induce a conformational change in YB-1, which may uncover NLS and/or mask CRS. Consequently, YB-1 can be transported into the nucleus through nuclear pores via the conventional NLS-importin a/p pathway. Once in the nucleus, YB-1 then binds to DNA and functions as a transcription factor, which will be discussed in subsequent sections. Since the NLS of YB-1 is noncanonical, whether this NLS interacts with importins needs to be examined. Experiments 72 inspecting the association between the noncanonical NLS of YB-1 and importins will not only validate the idea proposed above but also identify another type of NLS sequence recognized by importin a. 4.2.2 Ser 102 and DNA Binding Since CSD of YB-1 is known to bind to nucleic acids, we questioned whether Ser 102 contributes to the ability of YB-1 to interact with the promoter of EGFR. We found that while Flag: YB-1 bound to the first 2 kb of the EGFR promoter, Flag: YB-1 A l 02 was not able to interact with the first 1 kb promoter region. In addition, OSU-03012, which blocked YB-1 phosphorylation at Ser 102 by Akt, prevented the interaction between YB-1 and the -1 kb of EGFR promoter. These data suggest that the binding of YB-1 to the first 1 kb of the EGFR promoter is dependent on the phosphorylation of Ser 102 by Akt. One may argue that disruption of Ser 102 blocked nuclear translocation of YB-1 thus prevented the binding of YB-1 to the EGFR promoter. While this may be correct, we believe that Ser 102 plays a role in YB-1 :DNA interaction due to the following reasons. First, upon careful inspection, neither mutation of Ser 102 to alanine nor treatment of OSU-03012 completely blocked nuclear shuttling ofYB-1. In fact, there was still approximately 50% of YB-1 detected in the nucleus after disruption of Serl02 or OSU-03012 treatment. Second, abolishment of Serl02 almost fully prevented Flag: YB-1 A102 to bind to the -1 kb of EGFR promoter. Similarly, the binding of YB-1 to the lb region decreased to less than 50% upon OSU-03012 treatment. More intriguingly, OSU-03012 fully prevented the interaction between YB-1 and the 2a region of the EGFR promoter. Therefore, we suggest that in addition to nuclear translocation, Ser 102 is also important for YB-1 to bind to DNA. Furthermore, the observation that YB-1 but not YB-1A102 bound to the -1 kb of EGFR promoter implies that this region may contribute to the regulation of EGFR transcription by YB-1, which is discussed in section 4.3 below. The presence of YB-1 A102 in the nucleus explains the observation that YB-1A102 interacted with the 2b and 3 regions of the EGFR promoter. The ability of YB-1 A l 02 to bind to these sites equally strongly as the wild type YB-1 indicates that the binding of YB-1 to the 2b and 3 regions is Seri02 independent and is not regulated via PBK/Akt signaling. It is possible that YB-1 binds to this region solely due to the ability of YB-1 to recognize YREs containing the inverted CCAAT boxes without exerting any regulatory effect on EGFR expression. Alternatively, YB-1 may indirectly bind to this part of the promoter through another transcription factor, which then activates or repress EGFR transcription. Since the CSD of YB-1 is not involved in protein-protein interaction, phosphorylation at Seri 02 by Akt may not be required for this complex formation and DNA binding. If this is true, then pathway(s), besides PBK/Akt signaling, that control the regulation of EGFR by YB-1 may exist. 4.3 Regulation of EGFR Gene Expression by YB-1 By using luciferase reporter assays we further examined whether YB-1 activates the -1 kb of EGFR promoter. Transfection of the YB-1 expression vector hardly induced the luciferase activity. In this experiment, two cell lines were used. The HEK293T cells were originally derived from embryonic kidney. It was shown that YB-1 is highly expressed in fetal tissues (Spitkovsky et. al. 1992), and indeed we found that HEK293T cells expressed high levels of YB-1. The level of pAkt in HEK293T was also observed to be elevated (Figure 3). Another cell line used in the luciferase reporter assays was the MDA-MB-468 cells, which also exhibit constitutively activated Akt signaling and express high level of EGFR (Figure 3). Therefore, it is possible that the EGFR promoter cannot be further activated by exogenous YB-1 in cell lines chosen for this study. Alternatively, since the region between -1 kb and -2 kb of the EGFR promoter contains 74 many YREs and was bound by YB-1, it is possible that these YREs may be required for optimal transcriptional activity of YB-1. The pER construct excluded all sites in the 2b and 3 regions of the EGFR promoter, and thus transcriptional activity could not be increased in cells co-transfected with YB-1. Despite the inability to enhance EGFR transcription by exogenous YB-1, transfection of YB-1 A l 02 expression vector significantly suppressed EGFR promoter activity, suggesting YB-1 A102 may exert a dominant negative effect to interfere with the function of endogenous YB-1 in regulating EGFR transcription. This notion can be supported by the observation that YB-1 A l 02 also interfered the nuclear translocation of the endogenous YB-1 (section 3.1.2). Although exogenous YB-1 could not activate EGFR transcription in HEK293T and MDA-MB-468 cells, we were able to show that expression of Flag: YB-1, but not Flag: YB-1 A l 02, increased the level of EGFR protein and mRNA in MCF-7 cells. While the amount of mRNA in MCF-7 Flag:EV and MCF-7 Flag: YB-1 A102 were comparable, the level of EGFR protein was further reduced in MCF-7 Flag:YB-1 A102, suggesting that YB-1 Al02 may also have a dominant negative effect on protein translation. Collectively, these data indicated that YB-1 contributes to the regulation of EGFR gene expression. In a similar study, Berquin et. al. recently illustrated in normal epithelial cells that transfection of YB-1 cDNA also increased EGFR mRNA and protein levels. However, promoter-reporter assays demonstrating a direct role of YB-1 in EGFR transcription were not performed in their study (Berquin et. al. 2005). There are several ways in which YB-1 may regulate EGFR transcription. The most straightforward approach is to bind to the promoter of EGFR directly. In this study we demonstrated the interaction between YB-1 and EGFR promoter using ChIP; however, this method does not reveal whether the interaction is direct or is mediated by other co-activators. YB-1 is known to form complexes with other transcription factors including p53, Rel A (p65), 75 activating protein-2 (AP-2), the multivalent zinc finger factor (CTCF), and YY1 (Okamoto et. al. 2000, Raj et. al. 1996, Mertens et. al. 2002, Chernukhin et. al. 2000, Li et. al. 1997). None of these YB-1 binding partners has been shown experimentally to regulate EGFR transcription in breast cancer cells. In addition, by examining the -2 kb of the EGFR promoter using CONSITE (, we did not find any of the above YB-1 binding partners as potential transcription factors that regulate the first 2 kb of the EGFR promoter (Figure 14). YB-1 and other proteins that regulate EGFR transcription (section 1.4.1) were not identified as possible regulators for the EGFR promoter by CONSITE because these transcription factors were not included by CONSITE as candidate transcription factors. Interestingly, N F - K B and p50, both of which are members of the NF-KB/rel protein family, were discovered to be possible regulators for the first 1 kb of the EGFR promoter by CONSITE (Figure 14). One member of the NF-KB/rel protein is p65/RelA (Pikarsky and Ben-Neriah 2006). The p65/RelA protein is able to form heterodimers with p50 and is a subunit of the N F - K B complex (Pikarsky and Ben-Neriah 2006). The N F - K B complexes are regulated by Akt (Nicholson and Anderson 2002, Romashkova et. al. 1999, Kane et. al. 1999) and have been implicated in cancer including breast carcinoma (Bours et. al. 2000, Garg et. al. 2003, Zhou et. al. 2005, Wu and Krai 2005). Since YB-1 has been shown to interact with p65 to regulate the transcription of the JC virus (Raj et. al. 1996), it is possible that YB-1 induces EGFR transcription by interacting with the p65 subunit of the N F - K B complex. If it is true, then the N F - K B complex may be the protein that binds to the DNA while YB-1 is the co-activator to enhance transcription. Alternatively, YB-1 may be the protein that interacts with the inverted CCAAT boxes of EGFR whereas N F - K B acts as the co-activator. Recently, our lab was able to demonstrate by EMS A that YB-1 binds directly to the 2 YREs in the 2a region of the first 1 kb of the EGFR promoter. Mutations of the inverted CCAAT boxes in one or both YREs inhibited 76 Figure 14 Potential Transcription Factors that Regulate the -2 kb of the EGFR Promoter Predicted by CONSITE Several transcription factors, including N F - K B , p50, bZIP910, Myf, and COUP-TF, were predicted as potential regulators of the EGFR promoter by CONSITE ( The -2 kb of the egfr promoters of both human and mouse were retrieved from the USCS genome browser ( and analyzed for all the transcription factors available in CONSITE. A 75% conservation between the human and mouse EGFR promoter and an 80% of transcription factor threshold were the criteria used to predict potential regulators of the EGFR promoter. H I " -COUP-TF Myf bZIP910 P50 NF-kappaB T • 2 k b • -1 kb • +1 A T G n u c l e o t i d e p o s i t i o n 2246 mam -2 kb of the Human egfr Promoter — -2 kb of the Mouse egfr Promoter Putative YB-1 Binding Sites 77 YB-1 :DNA complex formation (unpublished data). Therefore, we argue that YB-1 binds directly to the -1 kb of the promoter to activate EGFR transcription. It would however be interesting to examine if N F - K B plays a role in YB-1 -mediated EGFR transcription activation. Finally, the C-terminal domain of YB-1 is known to facilitate protein-protein interaction or homo-multimerization (Tafuri and Wolffe 1992, Swamynathan et. al. 1998, Nambiar et. al. 1998, Bouvet et. al. 1995); thus, the possibility that the binding of YB-1 to EGFR promoter is enhanced by YB-1 homo- or hetero-multimerization cannot be ruled out. 4.4 The Role of Akt, YB-1, and EGFR in Breast Cancer The relationship between Akt and YB-1 in cancers has been demonstrated in several studies. It was recently shown that YB-1 but not YB-1 A102 enhanced breast cancer cell growth, suggesting that Akt and YB-1 together exert a stimulatory effect on tumor growth (Sutherland et. al. 2005). This is in contrast to the study in which YB-1 suppresses Akt-mediated oncogenic transformation of chicken embryo fibroblasts by inhibiting protein synthesis (Bader et. al. 2003, 2005). However, this work advocating YB-1 as a tumor suppressor was contradicted by another study, which showed that YB-1 activates mRNA translation upon phosphorylation by Akt (Evdokimova et. al. 2006). We suspect that the discrepancy may be due to the different model systems used in the studies. In the context of breast cancer, much of evidence supports the oncogenic function of YB-1. For example, YB-1 is over-expressed in breast tumors, and over-expression of YB-1 is associated with poor prognosis (Bargou et. al. 1997, Rubinstein et. al. 2002, Janz et. al. 2002). The fact that pAkt is co-expressed with YB-1 (Sutherland et. al. 2005) further validates the idea that Akt and YB-1 work together to promote breast cancer growth. Since we have shown that the expression of EGFR by YB-1 is dependent on Ser 102, it is considered that YB-1 stimulates breast cancer cell growth by up-regulating EGFR transcription. This proposition is verified by our recent work demonstrating that knocking-down YB-1 using siRNA suppressed EGFR expression and breast cancer cell growth (Wu et. al. 2006). It will be beneficial to examine whether inhibiting EGFR would block YB-1-mediated tumor cell growth. In fact, it has been demonstrated that while transfection of YB-1 cDNA leads to increased EGFR in the membrane, antibody targeting EGFR decreased the growth of cells that are transfected with YB-1 cDNA (Berquin et. al. 2005). Taken together, we propose that upon PBK/Akt activation, YB-1 promotes breast cancer cell growth in part via inducing EGFR expression. It is now well recognized that there exist subtypes of breast cancer, namely the ER(+) luminal subtype A and B, the ER(-) normal breast like, ER(-) ERBB2+, and ER(-) basal like breast carcinoma (Perou et. al. 2000, Sorlie et. al. 2001, 2003). At least two studies have shown that the expression of EGFR is associated with the basal-like breast cancer (Nielson et. al. 2004, Livasy et. al. 2006). The question raised here is whether YB-1 also correlates with the basal like breast tumors. In a study conducted by van't Veer et. al. using tumor tissue microarrays, YB-1 was clustered in the basal-like subtype in a cohort of women who possessed BRCA-1 mutation (van't Veer et. al. 2002, Sorlie et. al. 2003). In another study, YB-1 falls into a novel unknown cluster (Sorlie et. al. 2001). One possible explanation for this contradiction may be that the validated basal-like markers, such as cytokeratin 5, are highly expressed in both normal breast tissue and basal-like breast cancer. However, YB-1 is only detected in breast tumor tissues. Thus, in this study YB-1 failed to cluster with markers of the basal-like breast carcinoma. Interestingly, upon closer inspection of their data it is evident that YB-1 is most intensely stained in tumors that fall into the basal-like subtype. A connection between YB-1, EGFR, and basal-like subtype has been implicated in the SUM 149 cell line. The SUM 149 cells display the gene expression profile representing the basal-like breast cancer. It was observed that both EGFR and YB-1 are highly expressed in these cells (manuscript in preparation, Habibi et. al. 2006). Equally important, our lab also showed that YB-1 and EGFR are co-expressed in breast tumor tissues (Wu et. al. 2006). Given that YB-1 regulates EGFR expression (section 3.2), there is no reason why we should not assess the role of YB-1 in the basal-like breast carcinoma. Such investigation should definitely broaden our knowledge towards the basal-like disease that is difficult to manage with current treatments. 4.5 Targeting YB-1 in Breast Cancer As activated Akt is associated with tumor aggressiveness and drug resistance, the components of the Akt signaling pathway have become attractive targets for developing new therapeutic interventions for breast cancers. Drugs such as trastuzumab that target the RTKs has been shown to be effective in some aggressive breast tumors. However, resistance to the RTK inhibitors has been observed in patients whose tumors are PTEN-negative. Therefore, it is of interest to look for small molecule inhibitors that target proteins that are more downstream of the PBK/Akt pathway. Although many Akt downstream substrates have been found, YB-1 is an attractive therapeutic target because YB-1 is over-expressed in breast tumors but not in normal breast tissue. In addition, the importance of YB-1 in breast cancer has been established by our lab and others. Here, we have further demonstrated that activation of PBK/Akt pathway activates YB-1, which up-regulates EGFR expression and promotes breast tumor cell growth. In this study, we examined the ability of a PDK-1 inhibitor, OSU-03012, in inhibiting the functions of YB-1. Another advantage of OSU-03012 is that since OSU-03012 inhibits PDK1, which is downstream of PTEN, this drug may have the potential to treat breast tumors that are PTEN-negative. We found that OSU-03012 was able to block Akt activity both in PTEN-positive MDA-MB-453 (Noh et. al. 2004) and PTEN-negative MDA-MD-468 cells (Nicholson et. al. 2003). Furthermore, OSU-03012 also prevented YB-1 nuclear translocation and EGFR promoter binding. This data not only showed in principle that blocking YB-1 may be a novel approach to target EGFR over-expressing breast cancers, but also demonstrated the potential of OSU-03012 to be a therapeutic drug for PTEN-negative breast carcinoma. In addition to breast cancer, OSU-03012 has been studied in other cancers such as chronic lymphocytic leukemia (CLL) and pancreatic cancer (Johnson et. al. 2005, Li et. al. 2006). In both studies, OSU-03012 decreased CLL and pancreatic cancer cell growth. Previously, our lab illustrated that OSU-03012 decreased the level of EGFR in mouse xenograft (unpublished data). However, OSU-03012 failed to reduce EGFR protein level in MDA-MB-468 in cell culture (data not shown). MDA-MB-468 was chosen for this in vitro drug study because it expresses high level of EGFR. The extremely high expression of EGFR in this cell line is partly due to gene amplification (Filmus et. al. 1985), but the region(s) of gene amplification is unknown. It is also possible that the expression caused by gene amplification may be too high to be reduced by OSU-03012 under the conditions studied. Alternatively, the amplified EGFR may harbor different transcription regulatory mechanism; thus, OSU-03012 which target PBK/Akt and YB-1 may not be effective. Furthermore, as discussed previously OSU-03012 did not inhibit phosphorylation of YB-1 in MDA-MB-468 cells. Evidently, there is still much work to be performed to assess the efficacy of OSU-03012 in treating EGFR over-expressing breast cancer. Suppressing EGFR expression by OSU-03012 in other breast cancer cell lines is currently under investigation. This study will also provide insights into whether the mechanism causing EGFR over-expression is useful in selecting patients who may respond to OSU-03012 in future clinical trials. 81 4.6 Conclusion and Future Direction In conclusion, YB-1 is phosphorylated at Seri02 through the PBK/Akt signaling pathway. The phosphorylation of Seri 02 is important for YB-1 to translocate into the nucleus and to bind to the first 1 kb of the EGFR promoter. This leads to activation of EGFR transcription, resulting in increased level of mRNA and protein. The elevated expression of EGFR then can promote breast cancer cell growth (Figure 15). This study has provided novel insights into how YB-1 is regulated and how YB-1 promotes breast cancer cell growth. Undoubtedly, many experiments are still required to further verify the model proposed here. First of all, we assessed Seri 02 phosphorylation of YB-1 by immunoprecipitation and western blotting using a phospho-serine specific antibody. The disadvantage of this approach is that other phosphorylated serines are also detected. To specifically examine Serl02, our lab is currently developing a phospho-Serl02-YB-l antibody. This antibody will also allow easier screening for small molecule inhibitors such as OSU-03012 that inhibits YB-1 activity. Secondly, we have established the importance of Ser 102 in YB-1 functioning by comparing the behaviors of YB-1 and the YB-1 A l 02 mutant. To further validate the role of Seri02, one can generate an "activating" YB-1 mutant in which Ser 102 is changed to glutamate or aspartic acid to mimic the negative charge of the phosphate group. The activating YB-1 mutant is expected to locate primarily in the nucleus and to bind to the promoter of EGFR more efficiently. Consequently, breast cancer cells expressing the activating mutant are anticipated to grow much faster. For the past few years, our lab has focused on the capability of YB-1 to enhance breast tumor cell growth. It is now time to extend this knowledge into animal models. For instance, by using an inducible system, one can investigate whether YB-1 Al02 or the activating YB-1 mutant decreases or increases the size of the tumors that are pre-formed in mice. 82 Figure 15 YB-1 Up-regulates EGFR Expression and Enhances Breast Cancer Cell Growth via the PI3K/Akt Signaling Cascade Upon growth factor stimulation, the PBK/Akt pathway is activated. YB-1 is then phosphorylated at Serl02 by Akt and translocates into the nucleus. Once in the nucleus, YB-1 binds to the first 1 kb of the egfr promoter and activates transcription. The Expression of EGFR is therefore induced, resulting in enhanced breast cancer cell growth. Growth Factors, eg, IGF-1 0 0 83 Furthermore, since it has been shown that YB-1 promotes mammary tumor formation in nude mice, it would be interesting to examine whether YB-1 A l 02 or the activating mutant exert any effect on tumorigenesis in small animals. Thirdly, we have shown that YB-1 regulates the first 1 kb of the EGFR promoter. There are several YREs contained in this region; thus, we are interested in fine-mapping the YB-1 binding site(s). In fact, by using EMSA our lab has recently found that both YREs in the 2a region contribute to YB-1 binding. Luciferase assays using constructs with mutated YREs will further confirm the role of these YREs in EGFR transcription regulation by YB-1. Finally, the ability of OSU-03012 to block YB-1 nuclear trafficking and DNA binding was illustrated. We are in the process to examine if OSU-03012 may also inhibit the binding of YB-1 to the 2b and 3 regions of the EGFR promoter. Since the interaction between YB-1 and these sites seems to be Ser 102 independent, the inhibition of such interaction would further suggest the presence of other phosphorylation sites in YB-1 recognized by components of the PBK pathway. Furthermore, whether OSU-03012 inhibits YB-1 phosphorylation and suppresses EGFR expression require further investigation. Our lab has previously investigated the efficacy of OSU-03012 in mice. Initial results revealed that OSU-03012 decreased tumor size, inhibited YB-1 to bind to the EGFR promoter, and suppressed EGFR expression. Thus, it is motivating to expand the study to further evaluate the potential of OSU-03012 as a therapeutic drug. We have shown that YB-1 stimulates tumor cell growth by up-regulating EGFR expression. Since the expression of EGFR is associated with the basal-like breast carcinoma, it is of special value to assess whether YB-1 also plays a role in the basal-like subtype. It is envisioned that such a study will certainly advance the field of basal-like subtype, which we know very little about. More importantly, this investigation will allow us to further evaluate whether YB-1 serves as a potential target to treating the basal-like breast tumors. Currently more than 10 genes have been identified to be regulated by YB-1. However, the relationship between YB-1 and most of them has not been determined in cancers. Our lab is interested in discovering novel YB-1 target genes in the context of cancers by using the ChlP-on-chip technology. Moreover, since we are most interested in learning more about the basal-like breast cancer, it is valuable to inspect which genes are deregulated by YB-1 in this subtype of the disease. Finally, YB-1 is known to be over-expressed in a spectrum of malignancies including colon, lung, and ovarian cancers. However, how YB-1 contributes to these cancers is poorly understood. Since EGFR is also implicated in these tumors, the knowledge obtained from this study may be applied to other cancers and may be of value in treatment of other tumor types. 85 REFERENCES Aberle H., Bauer A., Stappert J., Kispert A., Kemler R. beta-Catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 1997;16(13):3797-804. Agrawal A., Gutteridge E., Gee J.M., Nicholson R.L, and Robertson J.F. 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APPENDIX BUFFERS AND REAGENTS Aves wash buffer A 10 mM Tris buffer (pH 8.0) 0.02% sodium azide 0.01% BSA 150 mM NaCl 0.1% Triton X-100 Aves wash buffer B 10 mM Tris buffer (pH 8.0) 0.02% sodium azide 150 mM NaCl Aves wash buffer C 50 mM Tris buffer (pH 6.8) Rnrp;atri Buffer B 10mMTris-HCL(pH7.2) 2 mM MgCh Buffer A 10 mM HEPES (pH 7.9) lOmMKCl 0.1%NP-40 1.5mMMgCl 2 1 mM EDTA Buffer C 0.42MNacl 1.5mMMgCl 2 20 mM HEPES (pH 7.9) 20% glycerol ThlP dilution buffer 0.01% SDS 2 mM EDTA 150 mM NaCl 1.1% Triton X-100 16.7mMTris-HCl (pH 8.1) ELB Buffer Av r>NAloafti"r,buffeg 25 mg bromophenol blue 5 ml glycerol 25 mg xylene cyanol 100 pi of I M Tris-HCl (pH8.0) j m i make up to 10 ml with distilled water Egg Lysis Buffer (ELB) 100 mM HEPES (pH 7.4) lOmM EDTA (pH 8.0) 1 mM DTT High salt buffer 0.1% SDS 2 mM EDTA 500 mM NaCl 500 mM NaCl 0.2% NP-40 1% Triton X-100 20 mM Tris-HCl (pH 8.1) LiCl buffer 0.25 mM LiCl 1% deoxycholate lOmM Tris-HCl (pH 8.0) Low salt buffer 0.1% SDS 2 mM EDTA 150mMNaCl RIPA buffer \50mM Nacl 0.5% sodium deoxycholate 50mM Tris (pH7.2) adjust pH to 8.0 Phosphate buffered saline (PBS) 8 g NaCl 1.44gNa2HP04 make up to 1 L with distilled water adjust pH to 7.4 1% IGEPAL 1 mM EDTA 1% Triton X-100 20 mM Tris-HCl (pH 8.1) 1% NP-40 0.1% SDS 0.2gKCl 0.24 g K H 2 P O 4 118 Protease Inhibitor Cocktail (PIC) 2.5mM sodium pyrophosphate ImM sodium vanadate ImM PMSF 1 ug/ml aprotinin ImM (3-glycerol phosphate 1 ug/ml leupeptin ImM NaF Sample loading buffer 0.5 M Tris-HCl (pH 6.8) 0.8 ml glycerol 10% SDS 1 % bromophenol blue 3.8 ml distilled water add 0.4 ml of 14.3 M 2-P-mercaptoethanol before use SDS lysis buffer 1%SDS 10 mM EDTA 50 mM Tris-HCl (pH 8.1) 3% Stacking gel for SDS-PAGE 4% acrylamide/bis 0.1% SDS 0.1% TEMED 0.125 M Tris (pH 6.8) 0.05% ammonium persulfate 6% Separating gel for SDS-PAGE 6% acylamide/bis 0.1% SDS 0.1% TEMED 0.375 M Tris (pH 8.8) 0.05% ammonium persulfate 12% Separating gel for SDS-PAGE 12% acylamide/bis 0.375 M Tris (pH 8.8) 0.1 % SDS 0.05% ammonium persulfate 0.1% TEMED lOx Transfer buffer 140 g glycine 30.3 g Tris-base To make lx transfer buffer containing 20% methanol, dilute 100 ml of lOx transfer buffer in 700 ml distilled water and 200 ml of methanol. Chill before use. T A E buffer 242 g Tris-base 100 ml of 0.5 M EDTA make up to 1 L with distilled water adjust pH to 8.5 57.1 ml acetic acid T E buffer ImM EDTA 10 mM Tris-HCl (pH 8.0) 


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