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UBC Theses and Dissertations

Inhibition of YB-1 alone or in combination with TMZ may improve GBM treatment Gao, Yuanyuan 2009

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Inhibition of YB-i alone or in combination with TMZ may improve GBM treatment by Yuanyuan Gao B.Sc., Peking University, China, 2007 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASThR OF SCIENCE in The Faculty of Graduate Studies (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April, 2009 © Yuanyuan Gao 2009 Abstract Glioblastoma is a rare, but devastating brain tumor. Recent studies showed that GBM is correlated with Y-box binding protein-i (YB-i) overexpression both in pediatric and adult patients. YB-i is a member of the cold shock domain protein superfamily. It performs a wide variety of cellular functions, including reg ulation of cell growth, apoptosis, transcription, translation and drug resistance. Our goal was to determine the potential therapeutic value of targeting YB-i in GBM. We carried out both transient and stable YB-i knock-down, using siRNA and shRNA in GBM ceLl lines. The loss of YB-i inhibited the cologenicity of SF188 in soft agar, and suppressed cell invasiveness in matrigel invasion assay. Furthermore, YB-i stable knock-down delayed the onset of tumor formation in mice and suppressed tumor growth by 30%. Beyond its role in tumor growth and invasion, YB-i is also strongly linked to drug resistance. We therefore addressed whether it plays roles in taxol (TXL) and temozolomide (TMZ) sensitivity. TXL is a commonly used anti-tumor drug and TMZ is the leading compound in GBM treatment. We evaluated YB i stable knock-down GBM cells sensitivity to these drugs both in monolayer and soft agar. Interestingly, we see a significant increase of apoptosis induced by TXL and TMZ in YB-i silenced cells. Also, an enhanced suppression of cologenicity by TXL in soft agar was examined in YB-i silenced cells. These results suggest that YB-i modulates cell invasive ability, cologenicity, tumorgenicity in vivo and response to chemotherapy. With improved understanding of YB-i roles in GBM, novel therapeutic approaches developed to target it are expected to provide a promising therapeutic benefit in treatment of GBM. 11 Table of Contents Abstract ii Table of Contents iii List of Figures vi List of Abbreviations vii Acknowledgements x Dedication xi Statement of Co-Authorship xii Thesis Hypothesis and Objectives xiii 1 Introduction 1 1.1 Giloblastoma Multiforme (GBM) 1 1.1.1 Definition 1 1.1.2 Origin and Frequency 1 1.1.3 Diagnosis 2 1.2 Treatment of GBM 2 1.2.1 Surgery 3 1.2.2 Radiation Therapy 4 111 Table of Contents 1.2.3 Chemotherapy 4 1.2.4 Targeted Therapies 7 1.3 The Y-Box Binding Protein-i (YB-i) 10 1.3.1 Gene Family and Protein Structure 10 1.3.2 Regulation of YB-i Expression and Activation 11 i.3.3 Functions of YB-i i2 i.3.4 Drug Resistance 13 i.3.5 YB-i and Cancers 14 References 16 2 Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozolomide 27 2.1 Introduction 27 2.2 Material and methods 28 2.3 Results and Discussion 33 Figures 37 References 47 3 Discussion 49 3.1 The Effect of YB-i Knock-down on Cell Growth and Cellular Functions 49 3.2 Inhibition of YB-i Suppressed Xenograft Growth in Nude Mice 51 3.3 Silencing YB-i Improved the Sensitivity of SFi 88 and U25 i Cells to Temozolomide and Paclitaxel Treatment 52 3.3.1 YB-i Modulates Drug-sensitivity via Regulation of MDR1JP-glycoprotein Ex pression 53 3.3.2 YB-i May Mediate DNA Repair as a Possible Explanation for It Associated TMZ Insensitivity 55 3.3.3 Interaction Between YB-i and Microtubules Might Perturb TXL-sensitivity . . . 56 3.4 Preliminary Study of YB-i Potential Role in GBM Cancer Stem Cells Properties 57 iv Table of Contents 3.5 Summary and Future Directions 60 Figures 62 References 68 Appendix 74 V List of Figures 2.1 Cellular invasion through Matrigel was inhibited by transient and stable YB-i knock-down 38 2.2 Knock-down of YB-i reduced cologenicity and tumor formation ability of SF188 cells . . 40 2.3 Induction of apoptosis and inhibition of colony formation in soft agar after Paclitaxel and Temozolomide treatment 42 2.4 Hierarchical cluster analysis revealed elevated YB-i expression association with aGBM. 44 2.5 Inhibition of YB-i in aGBM increases sensitivity to TMZ 46 3.1 Cell proliferation of transient and stable YB-i knock-down cells 63 3.2 CD i 33 expression and Neurosphere formation of SFi 88 shYB- i cells 65 3.3 YB-i mediated chemo-sensitivity mechanism model 67 vi List of Abbreviations aGBM adult glioblastoma BBB Blood-brain-barrier BER Base excision repair BSC Best supportive care CDK Cyclin dependent kinase CHIP Chromatin immunoprecipitation CSD Cold shock domain CSPs Cold shock proteins CNS Central Nervous System CTD C-terminal domain DbpA DNA binding protein A DbpB DNA binding protein B DbpC DNA binding protein C DMSO Dimethyl Sulfoxide EGF Epidermal growth factor EGFR Epidermal growth factor receptor ELB Egg lysis buffer ERK Extracellular regulated kinase GBM Glioblastoma GDP Guanine diphosphate GRPs Glycine-Rich Proteins GSK3 Glycogen Synthase Kinase 3 GTP Guanine triphosphate hNthl human endonuclease ifi vii List of Abbreviations LOH Loss of heterozygosity MAPK Mitogen activated protein kinase MEK Mitogen activated protein MDR- 1 Multidrug resistance 1 MG methylguanine MGMT 06-methylguanine methyltransferase MHC Major histocompatibility complex MMP-2 Matrix metalloproteinase-2 MMP-12 Matrix metalloproteinase-12 MMR Mismatch repair MRP 1 Multidrug resistance-related protein 1 mTOR mammalian target of rapamycin NLS Nuclear Localization Signal NTD N-terminal Domain pGBM Pediatric glioblastoma PCNA Proliferating cell nuclear antigen PDGF/R Platelet derived growth factor/receptor PDK1 3-phosphoinositide-dependent protein kinase-1 Pgp P-glycoprotein PH Pleckstrin homology PKC Protein kinase C P13K Phosphatidylinositol-3 kinase PTEN Phosphate and tensin homolog deleted on chromosome lOq RNP Ribonucleoprotein RTOG Radiation Therapy Oncology Group RT Radiotherapy RTK Receptor tyrosine kinase TMZ Temozolomide TP Tumor protein TXL Taxol viii List of Abbreviations uPA Urokinase-type Plasminogen Activator UV Ultraviolet VEGFR Vascular Endothelial Growth Factor Receptor WBRT Whole Brain Radiotherapy WHO World Health Organization YB-i Y-box binding protein-i ix Acknowledgements I would like to thank my supervisor Sandi who gives me great support and instructions on my studies. Also it is Sandi who helped me go through so many tough times in my life and gave spiritual encourage ment. Also, I would like to thank my dear lab mates Chathy, Michelle, Abbas, Kaiji, Anna, Karen, Jessie, Jennifer, Kristen, Arezoo and Alastair who generously offered me great help during my studies. Thank my parents’ support and Ian’s generous help. x To my parents and Ian xi Statement of Co-Authorship Chapter 2: I have conducted all the assays independently or cooperatively. Cooperative parts: • The immunostaining parts were accomplished with help from Cathy Lee and Dr. Abbas Fotovati. Please refer to Figure 2.1A and 2.5B. • The animal experiments were accomplished with help from Michelle Wang. Please refer to Figure 2.1 C&D, Figure 2.2 B, C&D. • Hoechst staining measurement was accomplished with help from Dr. Kaiji Hu. Please refer to Figure 2.3 E. • Adult GBM data was acquired by Dr. Gilbert Cote’s lab. Please refer to Figure 2.4. Chapter 3: I have conducted all the assays and figures independently. xii Thesis Hypothesis and Objectives Hypothesis YB-i may be a possible target for the novel treatment of GBM. Objectives Objective 1: To examine the changes in cellular function of transient and stable YB-i silenced models in GBM cell lines. Aim 1: To establish transient and stable YB-i knock-down models in pGBM cell line SF188. Aim 2: To evaluate cell invasive ability of YB-i knock-down cells by matrigel invasion assay. Aim 3: To assess the colony formation ability of knock-down cells in soft agar assay. Significance Studies have shown the over-expression of YB-i in pGBM and aGBM compared with normal brain. However, the role of YB-i in GBM cellular functions is largely unknown. In SF188 cells, a model of pGBM, we examine the cell invasiveness and colony formation of YB-i silenced cells. The results indi cated YB-i might play potential roles in the regulation of tumor formation and metastasis. Objective 2: To characterize tumorigenicity of YB-i silenced cells in vivo. Aim 1: To optimize the injection condition in mice and examine the tumor initiating ability of SF188 control cells. Aim 2: To study the tumorigenicity of YB-i knock-down cells. Significance Although YB-i has been shown to be over-expressed in GBM tumors, there has not been any study examining the effects of silencing YB-i in GBM development. Therefore, we evaluate the tumor formation ability of SF188 YB-i knock-down cells in vivo to understand the biological importance of this protein. xlii Thesis Hypothesis and Objectives Objective 3: To examine drug-sensitivity of YB-i knock-down cells. Aim 1: To evaluate apoptosis induced by paclitaxel and temozolomide in pGBM SF188 YB-i knock down cells in monolayer. Aim 2: To study apoptosis induced by paclitaxel and temozolomide in aGBM U25 i YB-i knock-down cells in monolayer. Aim 3: To preliminarily explore the mechanism by which silencing YB-i enhances drug-sensitivity. Significance Although TMZ shows impressive therapeutic benefits in clinical studies, its efficacy is still limited and GBM remains incurable. Since YB-i has been reported to confer drug-resistance in cancers, we proposed that a combination of chemotherapeutic agents and YB-i inhibition through siRNA may be more effective than single agents given alone. These preliminary studies provide us valuable information to develop combination therapy clinically in future. xiv Chapter 1 Introduction 1.1 Giloblastoma Multiforme (GBM) 1.1.1 Definition Glioma, the most common primary central nervous system tumor, develops from glial cell in brain. Glial cell is not neuronal cell but the surrounding cell that protects and supports neuronal cell; therefore glial cells are known as the glue of the nervous system. Among the various types of glial cells, astrocytes are the origin of approximately 77.5% of gliomas (Raizer et al 2005). Tumors rising from astrocytes are named astrocytomas. Based on morphologic evidence, the current World Health Organization (WHO) scheme (Louis et al. 2007) established a four-tiered grading (Grade I-TV) guideline for this disease. WHO grade IV astrocytoma, commonly known as glioblastoma multiforme or simply glioblastoma (GBM), is graded because of its high degree of cellularity, anaplasia, vascular proliferation and necrosis. 1.1.2 Origin and Frequency GBM may arise through 2 distinct pathways of neoplastic progression, which were defined by their distinct disease subtypes and the patients age. Secondary GBM with mutations in the tumor suppressor gene p53 that progress from lower-grade (II and ifi) astrocytoma typically affects younger patients and is preferentially located in the cerebral hemispheres. Primary GBM, which occurs more frequently, are present de novo after a short clinical history without evidence of a less malignant precursor lesion. This form of GBM are characterized by egfr amplification (36% of the cases) and pten mutations (25%) (Ohgaki et al 2007). The incidence of primary brain tumors has increased dramatically over the past several decades (Bran- 1 Chapter 1. Introduction des et al. 2003). Among them, GBM accounts for approximately 23.0% of brain cancers (Raizer et al. 2005) and is the most common primary brain tumor in adults (Grossman et al. 2004). In the U.S. popula tion, the annual incidence of GBM is around 2 per 100,000 (seer.cancer.org). Each year, more than half of the 18,000 patients with malignant primary are diagnosed with GBM (Grossman et al. 2004). Although being a relatively rare form of cancer, GBM is indeed one of the most aggressive and difficult tumors to treat. From the Surveillance Epidemiology and End Results (SEER) study, in the U.S., the relative survival for adults diagnosed with GBM was < 5% at three years and < 3% at five years. Despite the fact that GBM occurs in patients of all ages, the incidence is highest in the elderly. From Central Brain Tumor Registry of the United States (CBTRUS) 2005-2006 statistical report, the median age at diagnosis of this disease is 64, with the majority of the patients being over the age of 55. 1.1.3 Diagnosis The common symptoms of early-stage GBM include headache, seizure, speech problems, personality changes and visual loss. In addition, a history form provided by family or friends is helpful in confirming the disease as patients themselves may not recognize the onset of mental changes (Truong et al. 2006). Many patients were diagnosed several months after appearance of initial symptoms. To date, no primary prevention is recommended for brain tumors and no screening procedure is available (Brandes et al. 2008). The current standard procedure for diagnosis is magnetic resonance imaging (MM), which characterizes tumor locations, areas of contrast enhancement, areas of necrosis and mass effect (Truong et al. 2006). It has not been demonstrated whether an early diagnosis leads to greater survival, but it is assumed that patients with small tumors are more likely to benefit from surgery and/or to respond better to radiation and chemotherapies. 1.2 Treatment of GBM Definitive therapeutic options for GBM include various combinations of surgery, radiation therapy (RT) and chemotherapy. The primary aim of surgery is total tumor resection. However, it is often difficult and has never been completely achieved because of the special location of this disease, that is, the brain. Invasive GBM cells tend to infiltrate into the surrounding normal brain, further complicating complete surgical resection. Radiotherapy is currently the standard adjuvant treatment for GBM. It improves overall 2 Chapter 1. Introduction survival compared with surgery alone in randomized studies (Brandes et al. 2008). Chemotherapy is another method to treat GBM. However, efficacies of a majority of these chemotherapeutic agents are limited by the Blood-Brain-Barrier (BBB) due to molecular sizes of the drugs. The alkylating agent Temozolomide (TMZ) is an excellent oral drug that readily crosses the BBB with only mild adverse effects and has been shown to prolong overall survival of GBM patients. In addition, novel targeted molecular therapies against critical components of GBM signaling pathways such as EGFR, VEGFR and mTOR, appear promising in preliminary studies. Each of these therapies will be described in more detail below. 1.2.1 Surgery Surgery is often considered the first therapeutic modality for GBM. The ultimate goal of a surgery is complete resection. However, if tumor cells show early infiltrative ability into areas of a normal brain, the disease is believed to be surgically incurable. In this case, the achievable goal of a surgery is maximal resection of the tumor. Surgery is very effective in reducing tumor volume while remaining tumor would optimally be eliminated with other therapies. It also effectively removes the accessible tumor cores, which may be difficult to reach by chemotherapy and are resistant to radiation therapy. Surgery may, moreover, delay progression. No brain tumor should be treated with radiation or chemotherapy without a definitive pathological diagnosis and the valuable patient tumor tissues from surgical procedure thus provide an opportunity for tissue diagnosis. Even if a tumor is so extensive that an effective resection is impossible, stereotactic brain biopsy can still be performed to provide tissue for diagnosis. Surgical resection improves survival of most patients and provides time for additional therapies. Since the benefit of surgery to GBM patients is undeniable, to date, no clinical trial has been done to evaluate the result of surgery versus no surgery in patients. Yet, research has demonstrated a correlation between survival and extent of resection (Chang et al. 1983). In a review of three consecutive Radiation Therapy Oncology Group (RTOG) trials, the patients with complete surgical excision showed longer median sur vival (11.3 months) compared with the patients receiving biopsy alone (6.6 months) (Simpson et al. 1993). However, since the extent of resection is greatly influenced by patient conditions (age and performance status), size and site of tumors, the aforementioned review may be subjected to selection bias (Brandes et al. 2008). Also, brain surgery carries a number of risks which are more serious than most other opera tions. The major specific complications of brain surgery are damage to the brain at the time of surgery and bleeding within the head after the operation. Meningitis and epilepsy occasionally follow craniotomy. 3 Chapter 1. Introduction 1.2.2 Radiation Therapy In radiation therapy (RT), the photons and electrons present in radiation damage to cellular DNA. Normal cells possess functional DNA repair systems while cancer cells show diminished ability to repair DNA damage and are thus more susceptible to the stress induced by radiation. To date, external beam conventional radiation therapy remains the mainstay of disease management after surgical resection. This therapy results in a approximately doubling overall survival rate compared with surgery alone or surgery followed with chemotherapy in randomized studies (Brandes et al. 2008). Kristiansen et al. (1981) has shown that glioma patients receiving RT demonstrated significantly improved survival (from 5.2 months to 10.5 months) compared with patients receiving only best supportive care (BSC). Therefore, the post operative radiotherapy is currently the standard adjuvant treatment for GBM. Whole brain radiotherapy is not commonly employed in treatments. In the majority of cases, 60 Gy radiotherapy is delivered for a total of 6 weeks to a target volume defined as a 2-3 cm ring of the lesion tissue. RT doses higher than 60 Gy does not offer additional therapeutic benefit (Chang et al. 1983). The majority of GBM recurrence occurs within 2cm of the edge of the original tumor (Hochberg et al. 1980), while multifocal lesions are uncommon. Fractionated stereotactic radiation therapy is introduced to maximally localize therapy to the tumor bed without increasing harmful exposure to surrounding brain. It is a technique that allows delivering radiation through the use of a three-dimensional coordinate system to locate intracranial targets precisely (Tsao et al. 2005). It is likely that more GBM patients will benefit from this newly developed technique in the near future. Despite of the effectiveness, RT inevitably results in several short-term and long-term side-effects. The main disadvantage is the damage to normal cerebral tissue. This would lead to cerebral edema that poten tially increases intracranial pressure, leading to headaches, nausea, vomiting, and worsening of preexisting neurologic deficits (Chiro et al. 1988). Also, some late reactions can be permanent and progressive. The symptoms, varying from mild to severe, include decreased intellect, memory impairment, confusion and personality changes (irsa.org). 1.2.3 Chemotherapy In the late 1970s, several randomized clinical trials were conducted to examine the role of adjuvant chemotherapy in improving survival of brain tumor patients. Fine et al. (1993) reported that the combina 4 Chapter 1. Introduction tion of radiotherapy and adjuvant chemotherapy yielded increased survival by 10.1% at 1 year and 8.6% at 2 years compared with radiotherapy alone. A new standard of care emerged in 2005 with publication of a large scale study of patients receiving 60 Gy radiotherapy and temozolomide (TMZ) treatment. 1.2.3.1 Paclitaxel Paclitaxel is a renowned intravenous injected anti-cancer drug that is effective against ovarian car cinomas and advanced breast carcinomas. It was initially extracted from the bark of the Pacific yew in the 1960’s and has been recognized by Monroe Wall as a compound with outstanding anticancer activity (cfs.nrcan.gc.ca). Paclitaxel binds to microtubules, prevents their depolymerization into tubulin and thereby inhibiting the break-down of mitotic spindle during mitosis. Therefore, cells cannot replicate successfully. Cancer cells are characterized by their ability to divide rapidly and as a result, are more susceptible to paclitaxel treatment. In addition, a second mechanism by which paclitaxel works was reported by Cheng et al. (2001) demonstrating that paclitaxel could bind to anti-apoptosis protein Bcl-2 thus arresting its function. In GBM studies, paclitaxel has been shown to be effective in in vitro studies (Cahan et al.1994). There is also research suggesting that paclitaxel inhibits glioma xenograft growth in nude mice (Riondel et al. 1992). In human clinical trials, paclitaxel was applied in combination with radiotherapy (Lederman et al. 1998; Fountzilas et al. 1999; Langer et al. 2001) but was not shown to be a good radiation sensitizer with significant activity. Pharmacokinetic evidence from clinical trials (Rowinsky et al. 1989) and animal studies (Klecker et al. 1994) indicated that paclitaxel minimally penetrates the BBB. These results may explain why paclitaxel demonstrates outstanding anti-cancer activities in several cancer models except the brain. Ongoing investigations have been carried to resolve this problem. Laccabue et al. (2001) have developed new generations of paclitaxel family compounds (taxanes) with enhanced brain penetration. Liquid crystalline cubic phases (von Eckardstein et al. 2005) and Biodegradable Polymer Implant (Walter et al. 1994) have been employed to encapsulate paclitaxel in order to effectively deliver the drug into brain tumor targets. We believe there remains a large uninvestigated research area in the potential of paclitaxel in GBM treatment. 5 Chapter 1. Introduction 1.2.3.2 Temozolomide (TMZ) TMZ is the leading chemotherapeutic agent in GBM treatment. It has shown to improve overall median survival from 12.1 months to 14.6 months (Stupp Ret al 2005). The ability of this drug to cross the BBB is crucial to its activity in central nervous system (CNS) tumors. TMZ was synthesized at Aston University in the 1980s as part of a rational drug development initiative (Newlands et al. 1997). The drug can be efficiently absorbed by body, exhibits 100% bioavailability within 1-2 hours of oral administration, reaches 5—50tM in target brain area (Beier et al. 2008) and demon strates a schedule-dependent anti-tumor activity. Clinical studies reported that TMZ is well-tolerated and produces mild-to-moderate side-effects such as nausea and constipation when administered once daily for 5 days (100 -200 mg/m2/day) in a 4-week cycle (O’eilly et al. 1993). Severe or life-threatening toxicity includes thrombocytopenia, neutropenia, and opportunistic infections but are rare incidences. With all the given information, TMZ is believed to improve the quality of life of these patients. On the basis of the relatively safe toxicity and findings achieved in adult malignant gliomas, phase I and II clinical trials were set up to take advantage of this novel drug in pediatric GBM (Barone et al. 2006). Although pediatric GBM patients benefit more from several chemotherapeutic agents than adult GBM patients (Finlay et al. 1995), they are not more sensitive to single agent TMZ compared to the adults (Barone et al. 2006). Several investigators have studied the biologic effects of TMZ on adult GBM cell lines. TMZ treatment of lOOjiM for 96 hours has been shown to suppress cell growth and to cause cell cycle arrest in U251-MG and U87-MG cells (Fisher et al. 2007). In primary GBM cells, a dose- and time-dependent decline of the stem cell subpopulation was observed after TMZ treatment (Beier et al. 2008). 1.2.3.3 Mechanism of TMZ Action and MGMT Induced Resistance The principal mechanism responsible for cytotoxicity of TMZ in GBM cells is achieved by DNA methylation. At physiologic pH, TMZ spontaneously converts to MTIC, a reactive methylating agent. This conversion of TMZ is a chemically controlled reaction with little or no enzymatic component. MTIC transfers the methyl group to DNA and turns into the final degradation product, AIC, which is excreted via kidneys (Friedman et al. 2000). The most common methylation sites in DNA after TMZ treatment were N7 (70%) position of guanine and N3 (10%) position of adenine, followed by a few number of 06 positions of guanine. Formation of 06 methylguanine(06-MG), which accounts for 8% of the total adducts, is believed 6 Chapter 1. Introduction to initiate DNA mismatch leading to apoptosis (Tentori et al. 2002).6-methylguanine methyltransferase (MGMT) is the DNA repair enzyme that removes cytotoxic06-MG. The MGMT-mediated repair process is unique and differs from other DNA repair pathways in that it specifically removes the methyl group from the 06 position of guanine, thereby protecting the nucleotide in its native form without causing DNA strand breaks. Once the transfer of an alkyl group is complete, MGMT becomes irreversibly inactivated (Hegi et al. 2008). MGMT is believed to be an integral enzyme involved in TMZ resistance in both adult (Hegi et al. 2008) and pediatric GBM (Donson et al. 2007). 45% (92=206) of newly GBM diagnosed patients with MGMT promoter hypermethylation (MGMT silencing) benefit from TMZ, with an improvement in median survival (21.7 versus 12.7 months) compared with patients having high level of MGMT (unmethylated MGMT promotor) (Hegi et al. 2008). This result indicated that newly diagnosed glioblastoma patients can be stratified into two prognostic groups based upon MGMT promoter methylation status (Hegi et al 2005). Given the central role of MGMT in TMZ resistance, this protein is a promising potential target for biochemical modulation of drug resistance. Research has been done to explore methods that down-regulate MGMT expression and to investigate possible combination therapies to treat MGMT over-expressing GBM tumors. 1.2.4 Targeted Therapies Recently, the numerous genetic changes that occur in GBM progression have been documented. A stepwise progression of molecular genetic events involving over-expression of oncogenes and loss of tu mor suppressor genes underlies the development of GBM. More than half of the low-grade astrocytomas harbor mutations in tp53, a gene on chromosome l’7p, which encodes the tumor suppressor protein p53. In addition, over-expression of platelet-derived growth factor (PDGF) and PDGF receptors (PDGFR) is com monly observed. Further, primary GBM show multiple genetic alterations including epidermal growth factor receptor (EGFR) amplification and mutation, and deletion or mutation of phosphatase and tensin homologue (PTEN) (Brandes et al. 2008; Norden et al. 2006). The understanding of GBM pathogenesis has led to advances in improved use of agents for disease treatment and the development of new targeted therapies. 7 Chapter 1. Introduction 1.2.4.1 Inhibition of Growth Factor Signaling Pathway In GBM, several growth factor receptors (e.g. EGFR, VEGFR, PDGFR) are over-expressed, leading to stimulation of the downstream signaling pathways and activation of cell proliferation, survival, invasion and secretion of angiogenic factors. Kinase inhibitors and monoclonal antibodies of these ligands or receptors have been developed in clinical trials to inhibit cell signaling in GBM. Two kinase inhibitors, Erlotinib and Gefitinib, have been evaluated in tumors with egfr amplification to improve radiation response. In a phase II trial of gefitinib in GBM, tumor regression was not observed based on MRI (Rich et al. 2004) (Franceschi et al 2007). In addition to small-molecular inhibitors, mono clonal antibodies targeting EGFR, for example, cetuximab, has demonstrated anti-tumor activity as single agent or in combination with radiation in egfr-amplified GBM in vitro (Combs et al. 2007). However, due to the large molecular weight of monoclonal antibody, there is still concern about the efficiency of drug delivery through BBB and its clinical performance. PDGF signaling is important for growth and angiogenesis of gliomas. Imatinib, an inhibitor of PDGFR, has shown anti-glioma activity in preclinical studies (Kilic et al. 2000). In clinical trails, imatinib exhib ited anti-tumor ability when combined with hydroxyurea (Reardon et al. 2005) but failed to work as monotherapy in patients (Wen et al. 2006). The growth and survival of GBM are dependent on adequate blood supply and it is therefore not sur prising that malignant gliomas are highly vascularized (Reardon et al. 2006). As a result, anti-angiogenic agents became a promising therapeutic strategy in treatment of this disease. A VEGF-neutralizing anti body, bevacizumab provided preclinical benefits in a glioma xenograft model (Rubenstein et al. 2000). Other antiangiogenic drugs such as cediranib, is an oral inhibitor of VEGFR that benefited GBM patients in clinical trials (Batchelor et al 2007). 1.2.4.2 Inhibition of Intracellular Factors Following growth factor receptor activation, downstream signaling molecules such as RAS, P13K and PKC are recruited to the cell membrane. Sequential activation of other downstream proteins through phos phorylation subsequently regulates important cellular processes. Aberrant cell signaling can be detrimental to cells and can contribute to malignancy. Proteins such as RAF, mitogen-activated protein extracellular 8 Chapter 1. Introduction regulated kinase (MEK), extracellular regulated kinase (ERK, or MAPK), AKT and mammalian target of rapamycin (mTOR) are some of the most crucial intracellular signaling mediators. Various inhibitors of these molecules have been evaluated in preclinical and clinical studies of malignant gliomas. In the RAS-RAF-MEK-ERK (MAPK) pathway, RAS protein regulates many cellular functions such as proliferation, differentiation, and secretion of angiogenic factors (Sathornsumetee et al. 2008). Acti vation of ERK is associated with poor outcome in GBM patients (Pelloski et al. 2006); thus, targeting RAS pathway may be effective in treating this type of tumor. Since RAS activation involves prenylation (addition of farnesyl or geranyl groups), several farnesyltransferase inhibitors have been introduced as RAS inhibitors. Two famesyltransferase inhibitors, tipifanib and lonafarnib have been evaluated in ma lignant gliomas. Tipifarnib showed modest clinical activity in recurrent GBMs (Cloughesy et al. 2006). Although some side-effects were reported, positive clinical activity was observed in clinical studies of lonafarnib (Caponigro et al. 2003). The development of RAS-targeting inhibitors is currently underway (Sathornsumetee et al. 2008). Activation of the PI3K-AKT-mTOR pathway is also associated with poor prognosis in glioma (Chakravarti et al. 2004). Several malignant phenotypes including anti-apoptosis, enhanced cell growth, proliferation, and invasion, are regulated by the P13K pathway. In addition, loss of pten is a common genetic feature in GBM. mTOR is downstream target of the AKT and RAS pathways. Rapamycin and its analogues alone failed to provide therapeutic benefits in clinical trials of malignant gliomas (Chang et al. 2005). P1-103, a novel inhibitor of both P13K and mTOR, has shown activity in both in vitro and in vivo models of malig nant gliomas and may therefore be a potential agent for GBM treatment (Fan et al. 2006). Also, inhibition of P13K by LY294002 broadly sensitizes GBM cells to chemotherapy induced apoptosis in vitro (Oped et al. 2008). Protein kinase C (PKC) is a serine/threonine kinase that regulates cell proliferation, invasion, and angiogenesis. The PKC-3 inhibitor has shown anti-tumor activity as single agent or when combined with radiotherapy in xenograft models (Graff et al. 2005) (Tabatabai et al. 2007). 9 Chapter 1. Introduction 1.3 The Y-Box Binding Protein-i (YB-i) 1.3.1 Gene Family and Protein Structure The yb-i gene is located on chromosome 1p34 and comprises 8 exons (Toh et al. 1998). The mRNA is approximatedly 1.5 kb long and encodes a protein with 324 amino acids (Didier et al. 1988). YB-i was first isolated as a transcription factor that bound to the promoter of the major histocompatibility complex class II (MHC Class II). Since it recognized the Y-box in the MHC Class II promoter, this DNA-binding protein was consequently named Y-box binding protein 1 (YB-i). During the same period of time, Sakura et al. indentified DNA binding protein B (Dbp B) that interacted with the EGFR enhancer (Sakura et al. 1988). Soon after, a protein called nuclease-sensitive element protein-i (NSEP-i) was shown to bind to the CT-rich elements in the c-MYC promoter (Kolluri and Kinniburgh 1991). It was subsequently found that Dbp B and NSEP-1 both referred to the same protein, that is, YB-i. YB-i is a member of the cold-shock domain (CSD) superfamily. Members of this family have a conserved 65-70 amino acid long sequence, the cold-shock domain, which is highly homologous to cold- shock proteins (CSPs) and possess similar RNA-binding properties as CSPs (Matsumoto et al. 1998). In CSD family, the most important components are the Y-box proteins that are involved in regulation of several transcription and translation processes. Other examples of the CSD proteins are plant glycine-rich proteins (GRPs) (Graumann et al. 1998), which are associated with cell wall with an unknown function, and Lin 28 proteins that are essential for post-transcriptional regulation of stage-specific genes (Moss et al. 1997). In bacteria, cold-shock proteins respond to low-temperature by stimulating the expression of genes involved in transcription and translation, thereby up-regulating cell growth (Jones and Inouye et al. 1994). However, the CSD in YB-i may not be involved in cold shock response (Matsumoto et al. 2005). YB-i consists of three domains: a non-conserved variable N-terminal domain (NTD), a highly con served cold-shock domain (CSD), and a C-terminal domain (CTD) (Kohno et al. 2003). The aniline and proline-rich N-terminal domain is involved in trans-activation (Kohno et al. 2003). The conserved CSD of YB-i exhibits DNAIRNA binding but not cold shock response ability (Swamynathan et al. 1998). In addition, the CSD of YB-i contains two conserved Ribonucleoprotein motifs conferring transport and translational control of mRNA (Graumann and Marahuel 1996). The CTD is composed of alternating regions of basic or acidic amino acids, and is suggested to mediate protein-protein interactions (Bouvet et al. 1995). A non-canonical nuclear localization signal (NLS) and a cytoplasmic retention site (CRS) were 10 Chapter 1. Introduction identified in the CTD to regulate YB-i cellular localization (Bader et al. 2005). 1.3.2 Regulation of YB-i Expression and Activation The yb-i gene resides on chromosome ip34 however it is not clear whether it is commonly amplified in primary tumors. Based on a limited study of iO cases, YB-i does not appear to be commonly amplified in breast cancer however these would need to be confirmed on a larger cohort of samples (Wu et al. 2007). Thus, transcriptional activation may be the primary factor contributing to YB-i up-regulation in disease states. The structural analysis of human yb-i gene promoter revealed multiple E- and GC-boxes. c-MYC interacting with p73, binds to E-box and results in transactivation of yb-i gene (Uramoto et al. 2002). Recently, Shiota et al. (2008) has demonstrated that another protein, Twist, up-regulated YB-i expression by also binding to the E-boxes in the yb-i promoter. Putative YB-i regulatory sites in the yb-i promoter were predicted using CONSITE (Wu et al. 2007), which indicated that the members of MYC family and Snail could be potentially involved in YB-i regulation. Lutz et al. (2006) identified F-box protein 33 (FBX33) to be a down-regulator of YB-i by targeting the protein to proteosomal degradation. Recent studies suggested that retinoblastoma binding protein 6 (RBBP6) was able to induce proteosomal degradation and thus decrease YB-i level in vivo (Chibi et al. 2008). Phosphorylation is an important post-translational modification affecting protein functions. We have previously reported that activated AKT phosphorylated YB-i at SeriO2 site in the CSD to promote YB-i nuclear localization and transcription regulation (Sutherland et al. 2005). It was subsequently confirmed that a PI3K/AKT inhibitor (Wortmannin) completely inhibited the phosphorylation of YB-i (Evdokimova et al. 2006). Interestingly there have been contradictory studies by Bader et al. (2003, 2005) propos ing YB-i to be a tumor suppressor protein that suppressed AKT-mediated oncogenic transformation of chicken embryo fiborblasts by inhibiting protein synthesis. In a recent study of Bader et al., they discov ered that phosphorylation of YB-i by AKT encouraged YB-i nuclear localization and therefore abrogated the ability of YB-i to function in the cytoplasm as a “translation-repressor”. As a consequence, AKT phosphorylation promotes oncogenic activity of YB-i (Bader et al. 2008). Taken together, these data suggested the phosphorylation of YB-i by AKT abolishes the translation-suppressing activity of YB-i in cytoplasm, and further stimulates nuclear translocation and transcription regulation of this protein. As the importance of AKT phosphorylation at YB-i serine i02 site had already been demonstrated, our lab has recently identified p90 ribosomal S6 kinase (RSK) to be a novel activator of YB-i. Protein kinase C ii Chapter 1. Introduction (PKC) has also been evaluated as another potential activator due to its ability to recognize the consensus phosphorylation sequence (RxRxxSIT) on YB-1(Stratford et al. 2008). In addition to SeriO2 in CSD, other phosphorylation sites of YB-i have been investigated. Coles et al. (2005) have reported the phos phorylation of YB-i at Ser2i in the NTD by GSK3 and the phosphorylation at Ser36 in the NTD by ERK led to enhanced binding of YB-i to the vegf promoter. Analysis using Motif Scanner indicated Tyr197 in the NLS may be phosphorylated by the p85 subunit of P13K (Wu et al. 2006). Further studies are required to characterize the specific functions of these phosphorylation events. 1.3.3 Functions of YB-i As a transcription factor, YB-i binds to the sequence motif CTGATTGG, or Y-box, in the promoter regions of several growth-associated genes (Didier et al. 1988). It is implicated in a wide range of the cellular processes by functioning as either a transcription activator or repressor. The role of YB-i in promoting cell growth has been greatly investigated in the past 20 years. YB-i up- regulates the transcription of cyclin A (Jurchott et al. 2003), cyclin B (Jurchott et al. 2003), topoisomerase II a (Shibao et al. 1999), DNA polymerase a (En-Nia et al. 2005), proliferating cell nuclear antigen (PCNA) (Ise et al. 1999), and DNA polymerase ö (Gaudreault et al. 2004). Recently, we found that silenc ing YB-i decreased EGFR and HER-2 expression (Wu et al. 2006). Furthermore, YB-i has been shown to regulate the expression of protein tyrosine phosphatase-iB (PTPiB) (Fukada and Tonks. 2003), ma trix metalloproteinase-2 (MMP-2) (Mertens et al. 2002), matrix metalloproteinase-i2 (MMP-i2) (Samuel et al. 2005), matrix metalloproteinase-13 (MMP-i3) (Samuel et al. 2007), collagen a 1 (Norman et al. 2001), CXCR4 (Basaki et al. 2007), and collagen a 2 (Higashi et al. 2003) in cell adhesion, motility, invasion and thus metastasis. Also, our lab has previously found metastasis-related gene uPA is correlated with YB-i expression in breast cancer (in press). YB-i is also involved in immune regulation and drug resistance. It represses transcription of class II MHC (Didier et al. 1988), FAS (Lasham et al. 2000), and CC (adjacent N-terminal cysteines) chemokine ligand-5 (CCL5) (Krohn et al. 2007). The expression of multi-drug resistance gene (MDRi) (Goldsmith et al. 1993) (Stein et al. 2001), multi-drug resistance-related protein-i (MRP-1) (Stein et al. 200i) and the major vault protein (MVP) (Stein et al. 2005) is modulated by YB-i. The ability of YB-i to regulate genes related to cell growth, metastasis and drug-resistance indicates that it may be a potential molecular target for cancer treatment. 12 Chapter 1. Introduction Despite the profound and extensive impact of YB-i in transcriptional control, the majority of YB-i is found in the cytoplasm in complex with various cellular mRNAs. Cytoplasmic YB-i is responsible for translational silencing and storage of mRNAs crucial for cell growth or viability (Evdokimova et al. 2006). While low concentrations of YB-i facilitate the binding to mRNA, high concentrations of YB-i destabilize the interaction of the cap-binding protein and thus enhance mRNA stability. Most mRNAs in eukaryotes are translated in a cap-dependent manner. The mRNA 5’ cap-binding protein eIF4E is a key player in the recruitment of mRNAs to translation initiation complex (Evdokimova et al. 2006). YB-i is associated with a number of growth- and stress-related mRNAs, many of which are regulated by eIF4E in translation (Evdokimova et al. 2006). There is a model proposing that YB-i could compete with e]F4E-driven translation initiation complex for binding to the capped 5’ mRNA terminus. However AKT phosphorylation impairs the 5 ‘-mRNA cap binding capacity of YB-i. The function of YB-i in translation repression is therefore negatively regulated by the PI3KIAKT pathway. The RNA-binding site specificity of YB-i was not found to be directly related to its translational function (Dong et al. 2009). While YB-i binds mRNA and affects translation, this association seems to in turn stabilize YB-i in cytoplasm. YB-i dissociated from mRNA is subject to proteosomal degradation (Sorokin et al. 2005). 1.3.4 Drug Resistance In addition to transcription and translation, YB-i plays a role in drug resistance. YB-i is involved in drug resistance in cancers of the breast (Bargou et al. 1997; Saji et al. 2003), prostate (Gimenez-Bonafe et al. 2004), bone (Oda et al. i998), ovary (Kamura et al.i999), muscle (Oda et al. 2008), colon (Vaiman et al. 2007), and skin (Schittek et al. 2007). Elevated levels of nuclear YB-i is believed to be the key to drug resistance. Nuclear localization of YB-i is observed under stress induced by chemotherapy (eg. Doxorubicin and 5-fluorouracil), ultraviolet (UV), irradiation (Koike et al. i997, Ohga et al. 1996) and hyperthermia (Stein et al. 200i). A probable explanation for YB-i-mediated drug resistance may be that some anti-cancer therapeutics activate YB-i, resulting in its translocation to the nucleus where it up-regulates the expression of mdr 1, mrp-J and mvp genes (Stein et al. 200i, 2005). Indeed, clinical studies have shown an association between YB-i and pglycoprotein (Pgp), an ATP-binding transporter, involved in multi-drug resistance in various cancers (Huang et al. 2005; Gimenez-Bonafe et al. 2004; Oda et al. i998, 2003; Fujita et al. 2005). Increased nuclear YB-i and Pgp was observed in breast cancer cells treated with paclitaxel (Fujita i3 Chapter 1. Introduction et al. 2005). These results collectively suggest that YB-i may contribute to drug resistance by augmenting transcription of specific drug-resistant genes. Nuclear YB-i functions in base excision and mismatch repair pathways (Gaudreault et al. 2004). Although the exact role of YB-i in DNA repair is not fully understood, there is evidence suggesting YB-i promotes DNA repair. For example, it was reported that YB-i functioned as a recognition protein for cisplatin-damaged DNA (Ise et al. i999). Therefore YB-i may be essential in DNA repair or in directing cellular response to DNA damage. Moreover, the protein forms a complex with DNA polymerase 6 in mismatch repair and Ku antigen in base excision repair (Gaudreault et al. 2004). Radiotherapy and various chemotherapeutic agents (eg. Temozolomide and Doxorubicin) act by causing DNA damage to cancer cells. YB-i may confer resistance to these therapies by cooperating with the proteins in DNA repair pathways. Together, these results suggest that YB-i may be a useful marker in predicting the effectiveness of chemotherapies. It is also a promising molecular target in drug-resistant cancers. 1.3.5 YB-i and Cancers There is growing evidence suggesting YB-i may be oncogenic. Proto-oncogene c-MYC (Uramoto et al. 2002) and novel oncogene, Twist, (Shiota et al. 2008) have been reported to control YB-i transaction. YB-i as a transcription factor up-regulates several growth-promoting genes including cyclin A, cyclin B] (Jurchott et al. 2003), DNA polymerase a (En-Nia et al. 2005), topoisomerase II a (Shibao et al. i999), egfr (Stratford et al. 2007) and her-2 (Fujii et al. 2008). Tumor suppressor gene p53 (Lasham et al. 2000) and apoptosis-associated gene fas are transcriptionally suppressed by YB-i (Lasham et al. 2003). By modulating transcription of these genes, YB-i exerts its impact on a broad range of factors, influencing cell fate. Because these proteins are crucial in cell growth and survival, it is therefore not surprising that aberrant YB-i expression may contribute to cancer progression. In clinical studies, YB-i has been found to be over-expressed in various cancers including breast (Wu et al. 2006), prostate (Gimenez-Bonafe et al. 2004), lung (Shibahara et al. 200i), muscle (Oda et al. 2003), and brain (Cheung et al. 2008). Recently, our lab confirmed the significance of YB-i as a prognostic factor in breast cancer in a large-scale tumor tissue microarray study. YB-i was expressed in 4i % of breast cancers (n=i 644/4049) and was associated with poor survival (p< 7.3 x 10—26) regardless 14 Chapter 1. Introduction of tumor subtype (p< 6.7 x i0, HR= 1.45) (Habibi et al. 2008). These studies suggest the crucial role of YB-i in cancer pathogenesis. To date, there have only been a few studies on YB-i in glioblastoma (GBM). YB-i is detectable in most of primary pediatric GBM (pGBM) tumor tissues based on both qRT-PCR and immunohistochemistry (Faury et al. 2007; Haque et al. 2007). 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Yang CY, Dantzig AH, Pidgeon C. Intestinal peptide transport systems and oral drug availability. Pharm Res. 1999; 16(9):1331-1343. 26 Chapter 2 Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozolomide1 2.1 Introduction Glioblastoma multiforme (GBM, WHO grade IV astrocytomas) is the most common type of primary brain tumor. Despite advances in surgical and adjuvant therapies, their prognosis remains poor. Pediatric glioblastoma (pGBM; pediatric grade IV astrocytoma) is a rare and deadly brain tumor that accounts for 8- 12% of all pediatric brain tumors and is the third leading cause of death in children younger than 16 years of age (Gottardo et al. 2008). The 3-year survival of pGBM patients is less than 20% (Faury et al. 2007). Current treatment for pGBM is radical surgical resection followed by radiation therapy and chemotherapy. Like adult GBM (aGBM), chemotherapy combined with surgery and radiotherapy significantly increases survival of pGBM (Donson et al. 2007). The most potent anti-tumor agent against aGBM and pGBM thus far is temozolomide (TMZ), an orally administered DNAmethylating agent. Patients receiving TMZ treatment demonstrate enhanced response rates, for example those on TMZ treatment have a 10-12 month survival rate compared to 4 months without such treatment (Friedmann et al. 2000). Furthermore, a combination of TMZ and radiotherapy further increases survival from 12.1 to 14.6 months (Stupp et al. 2005). Perhaps there is the potential to enhance the therapeutic benefits of TMZ by a combinatory drug regime including molecular targeted therapy to ultimately improve cure rates. We recently reported that the Y-box binding protein-i (YB-i) an oncogenic transcription/translation factor is highly expressed in primary pGBM (Faury et al. 2007) yet its functional role in these tumors has not been described. YB-i controls the oncogenome by shuttling between cytoplasm and nucleus. In the cytoplasm YB-i functions as ‘A version of this chapter has been submitted for publication. Gao Y, Fotovati A, Lee C, Wang M, Cote G, Guns E, Toyota B, Faury D, Jabado N, Dunn SE. Inhibition of Y-Box binding protein-i (YB-i) slows the growth of Giioblastoma Multiforme and Sensitizes to Temozoiomide Independent MGMT. 27 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozolomide a translation factor. Upon phosphorylation by AKT (Sutherland et al. 2005) or RSK (Stratford et al. 2008) at SeriO2 located in the DNA binding (Stratford et al. 2008) domain, YB-i translocates into the nucleus where it regulates transcription (Sutherland et al. 2005) by binding to Y-box (sequence motif CTGATTGG) in the promoter regions of growth promoting genes such as her-2 and egfr (Wu et al. 2006). YB-i has also been implicated in drug resistance by increasing the expression of multi-drug resistance gene (mdrl), multidrug resistance-related protein-i (mrpl) and major vault protein (mvp) (Wu et al. 2007). Much of what is known about YB-i was revealed through studies of adult cancers however very little is known about the functional role of YB-i in childhood tumors particularly GBM. It has also not been characterized in adult GBM. Recently, we reported that YB-i is over-expressed in pGBM (Faury et al. 2007). Subsequent studies showed that YB-i also referred to as NSEP-i is amongst the top genes that are differentially expressed in pGBM, as well as aGBM, as compared to control brain tissues [9, 10]. YB-i is detectable in 100% (14/14 cases) of primary pGBM tumor tissues based on qRT-PCR while at the protein level it is detectable in 8 i % (26/3 2 cases) of tumors at high levels [2, 9]. Thus YB-i is a fairly common molecular change in both primary pGBM and aGBM. We sought to characterize the role of YB-i in mediating GBM growth, invasion, tumorigenesis and response to TMZ in models of pGBM and aGBM. 2.2 Material and methods Cell Culture The SFi88 and U25i cells (pGBM and aGBM, respectively) were obtained from Dr. Nada Jabado, University of McGill and cultured in Minimum Essential Medium/Earle’s Balanced Salt Solutions (MEM/ EBSS) (Hyclone, Logan, Utah, USA) and Dulbecco’s Modified Eagle Medium (DMEM)/High Glucose (Hyclone) respectively, supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Burlington, ON, Canada) and cultured at 37°C , in a 5% CO2 incubator. shRNA Transfection(done in Dr. Jabado’s lab) Cells were transfected with the empty vector pSuper or the pSuper vector harboring the sequence 5’-GGTCATCGCAACGAAGGTT’fl’-3’ (OligoEngine, Seattle, WA) as a tail-to-tail tandem repeat of bp 285 to 305 of the human YB-i coding sequence. Stable transfections with liposomal preparation Fugene 28 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozolomide were performed in conjunction with G41 8 resistance plasmid pUHD 1 5C lneo (BD-Clontech, Heidelberg, Germany). Five micrograms of total plasmid DNA and 15 il of Fugene solution were mixed in 500 l of serum-free medium, incubated for 15 mm at room temperature, and added dropwise to culture medium (10 ml/plate). After 24 h, the medium was exchanged and selection with G418 at a concentration of 400 ,ug/ml was started. Within 2 wk, single-cell clones were apparent and selectively picked. Screening for the presence of pSuper plasmid DNA was performed, and changes of YB-i protein levels were performed by immunoblotting. The transfected cells were cultured in normal medium containing 200 ig/ml G418. Immunocytochemistry for YB-i and pYB1SlO2 SF188 and U25 1 cells (1.0 x 10) were seeded on glass coverslips, washed with phosphate-buffered saline (PBS), fixed with 2% formaldehyde for 20 mm, rinsed twice with PBS, and then incubated with PBS containing 0.1% Triton X-100 (Sigma) for 30 mm. Next, the coverslips were washed with PBS, incubated with rabbit anti-YB-i or (Cell Signaling Technology) antibody dissolved in buffer containing 10% BSA and 2% goat serum for 1 hour at room temperature in a humidified container. After washing three times with PBS, glass slides were incubated with Alexa 488 anti- rabbit antibody (Invitrogen, Molecular Probes) for 1 hour, washed three times and then mounted using Vectashield mount ing medium (Vector Laboratories, CA). DAPI was used for nuclear staining. Cells were observed using Olympus BX61 Fluorescent microscope and photographed using DP71 digital camera (Olympus, Japan). siRNA Transfection SF188 wild type (WT) cells were plated in 6-well culture plates (3 x i0 cells per well) 24 hours prior to transfection. Cells were transfected with either 10 nM control (oligonucleotide sequence UUCUC CGAACGUGUCACGU, Qiagen, Maryland, USA) or YB-i siRNA (oligonucleotide sequence CCACG CAAUUACCAGCAAA, Dharrnacon, Lafayette, CO, USA). siRNA and LipofectamineTMRNAiMAX (In vitrogen) were diluted in 500il serum-free Opti-MEM (Invitrogen) and the mixture was incubated at room temperature for 15 mm yielding a final concentration of 10 nM. MEMJEBSS were put into each wall to a final volume of 2m1. The cells were incubated at 37° C, in a 5% CO2 incubator for subsequent experiment. 29 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Thmozolomide Immunoblotting Harvested cells were washed with cold PBS and pelleted by centrifugation at 3,000 rpm for 3 minutes. After the supematant was aspirated, the pellets were resuspended in 4-packed-cell volumes of egg lysis buffer (ELB) and incubated on ice for 20-30 minutes. Proteins were isolated by centrifuging the samples at 13,000 rpm for 10 minutes at 4°C. The protein-containing supernatants were retained for the subsequent studies. The protein extracts (10-5 0 jtg) were mixed with 5 x sample loading buffer, and boiled for 5 minutes. These denatured protein samples and 5l pre-stained broad range standards (Bio-Rad, Hercules, CA, USA) were loaded on to a SDS-polyacrylamide gel. Antibodies were as follows: anti-YB-i (1:1500; Abcam, Cambridge, USA), anti-pH2AX’39(1:1000; Abcam), anti-MGMT (1:200; Abcam), anti-pYB 1SerlO2 (1:1000; Cell signaling Technology, Danvers, MA, USA) and anti-Vinculin (1:1000; Sigma). The primary antibodies were diluted in 5% bovine serum albumin (BSA) (Sigma, St Louis MO, USA) in PBS T. The membrane was incubated with appropriated secondary antibody (anti-mouse or anti-rabbit lgG horseradish peroxidase-linked antibody) in 5% non-fat milk in PBS-T for 1 hour at room temperature on a rocking platform, followed by 3 x 5 minutes washing in PBS-T. Invasion Assay Cellular invasion was evaluated by quantifying the number of cells that migrated through a polyethy lene terephthalate membrane coated with a layer of matrigel. Briefly, tumor cells (lx i0 cells in 100 l warmed 0.1% FBS MEMJEBSS) were seeded on the top of transwell inserts containing 40 il diluted matrigel (Matrigel: Serum-free medium=l:8). The lower chamber was filled with 600 il MEM/EBSS, supplemented with 10% FBS, as chemoattractants. Cells were cultured for 16 hours at 37°C, in a 5% CO2 incubator. The non-invaded cells in the upper chamber were subsequently scraped off. The membrane was fixed with 500 il ice-cold methanol (100%) for 5 minutes at room temperature. After 5 minutes air dry, membranes were washed with 500 il PBS for 5 minutes and stained with Hoechst (0.5 tg/ml in PBS) for 5 minutes. Following 3 x 5 mm PBS washes, membranes were placed on the glass slides with gelvatol on the surface. The cells that had migrated through the membranes were quantified in three randomly chosen visual fields under the microscope. 30 Chapter 2. Inhibition of YB-I Sensitizes Glioblastoma Multiforme to Temozolomide Soft Agar Assays The bottom layer was prepared by mixing 2x MEMIEBSS medium (Hyclone) with 1.2% agarose solution in 1:1 ratio. The mixture was added into the 6-well plates with 1 ml in each well. The upper layer was made by adding SF188 (5 x i04 cells per well) into a 1:1 mixture of 2 x medium and 0.6% agarose solution. For drug treatment study, the cell layer was composed of tumor cells, 1:1 mixture of 2 x medium and 1.2% agarose, and varying concentrations of temozolomide (British Columbia Cancer Agency, Canada). The assays were incubated in 37°C, in a 5% CO2 incubator for 28 days. The size and number of colonies were measured under a microscope (Leica DMIL). The experiment was performed in triplicate on three different occasions. Effect of Silencing YB-i in Xenograft Model SF188 cells have been shown to be tumorigenic in rats (Ma et al. 2002), however this has not been tested in mice. For model validation, we first conducted a pilot study to determine the conditions required for tumor formation in mice. The SF188 control cells (1 x 106) were injected subcutaneously into the lower hind flank of female 6-8 week old BalbC Nu/Nu mice (n=12); weight of mice and size of the tumor were then measured for 4 weeks. Tumors were harvested after that. After optimization of the conditions required for tumor formation was performed, both SF188 control and shYB-1 cells were injected subcutaneously into each side of lower hind flank of female Nu/Nu mice (SF188 control- left, SF188 shYB-1- right) (n=8). The weight of the mice and the size of the tumor on each side were measured using calipers for 4 weeks. In detail, cells were washed with Hanks Balanced Salt Solution (Invitrogen) twice and counted to prepare 1 x 106 cells, which were subsequently mixed with matrigel (BD Bioscience, MA, USA) in a 1:1 ratio with a total volume of 150 il. The tumor cells/matrigel was injected subcutaneously into the left or right lower hind flank of female Nu/Nu mice using a 26-gauge needle. Weight of mice and size of the tumor on each side were measured using calipers for 4 weeks. Mice were terminated with CO2. Tumors were frozen in liquid nitrogen at the time of harvest. Tumor volumes were calculated from the equation (4ir/3) (a/2) x (b/2) x (c/2) where a, b and c were the width, length and depth of the tumors, respectively. All studies were conducted in accordance with the University of British Columbia Animal Care and Use Guidelines. Differences in tumor size was determined using a Student’s T test where significance was determined if p<O.OS. 31 Chapter 2. Inhibition of YB-I Sensitizes Glioblastoma Maltiforme to Temozolomide Apoptosis assays SF188 control and shYB- 1 cells were treated with various concentrations of TXL or TMZ and incu bated for 24 hours before being stained with Annexin V-PE (Promega, Madison, WI, USA). Staining was done according to manufacturer’s protocol as previously described by us (Lee et al. 2008). In brief, the harvested cells were washed once with cold PBS, followed by 15 minutes incubation with lx binding buffer containing Annexin V-PE and 7AAD in the dark. Then 250 jil of 1 x binding buffer was added into each sample and flow cytometry analysis was performed within 1 hour. The experiment was repeated on three separate occasions. Following this, we confirmed that cells were undergoing apoptosis by evaluating changes in chromatin condensation. SF188 control and shYB-i cells (3,000 cells per well) were seeded in a 96-well plate and treated with TMZ and TXL with various concentrations for 24 hours. After the medium was aspirated, 100 il of PBS containing 2% paraformaldehyde and Hoechst dye (1igIml) was added to each well and the cells were kept at room temperature for 20 minutes. The plates were analyzed and the images were taken on the ArrayScan VTI Reader (Cellomics, Pittsburgh, PA, USA). Analysis of YB-i Expression in aGBM GEO (www.ncbi.nlm.nih.gov/geo) data were mined to examine YB-i expression in aGBM (Barrett et al. 2007). GSE4290 provided the largest dataset for a comparison containing 23 samples from epilepsy pa tients used as nontumor samples and 81 grade TV adult glioblastoma samples (Sun et al. 2006). Expression and clustering analysis was performed as previously described (Cheung et al. 2008). We used probe sets previously described as segregating nontumor from tumor samples based on known functions in general and alternative splicing, RNA export, RNA degradation, miRNA processing and nonsense-mediated de cay. Hierarchical clustering was performed with the EBI Profiler tool using a Euclidean distance measure with the complete linkage algorithm (www.ebi.ac.uklexpressionprofiler). 32 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozolomide 2.3 Results and Discussion Characterization of YB-i in the pediatric glioblastoma cell line SF188. Initially we characterized the SF188 pGBM cell line to show that it expresses high levels of YB-i and P-YB- iSb02 by immunofluorescence and immunoblotting (Figure 2. iA and 2. iB). We also noted that most of 1S2 was in nuclear, which may be linked to drug resistance as has been reported in other types of cancer (Wu et al. 2007). It therefore served as a model to silence YB-i stably and subsequently study the ramification on tumor growth and sensitivity to chemotherapy. By stably expressing a shYB- 1 plasmid, the levels of YB-i inhibition were confirmed to be reduced by >80% (Figure 2. lB left). As expected, PYBislo2was also decreased. Further, YB-i was transiently silenced using siRNA and an 8-day time course was conducted. YB-i was reduced by nearly 100% one day after transfection and this prominent knock-down was sustained at least 4 days post-transfection (Figure 2. lB right). While siRNA still showed a minimal effect 6 days posttransfection, the YB-i level returned to the normal level at day 8. Thus, levels of YB-i were manipulated either by stable or transient inhibition using siRNA for the studies that follow. Inhibition of YB-i perturbed the tumorigenic potential of SF188 cell in vitro and in vivo To understand how YB-i may contribute to pGBM progression, we examined cellular invasion in vitro by matrigel invasion assay using a cell line in which YB-i was stably knocked-down (shYB-i cells). The number of shYB- i cells that have migrated through the matrigel was 50% less than that of the control cells (Figure 2. iC). To confirm that silencing YB-i inhibits the invasion of SF188 cells, we transiently knocked- down YB-i with siRNA and performed the same study one day after transfection. The siYB- i cells de creased invasion by >70% compared with the control cells (Figure 2. iD). Together, these results strongly suggest that attenuated YB-i expression significantly inhibited SFi 88 cell invasiveness. These data are consistent with reports indicating that it regulates invasion proteases such as matrix metalloproteinase-2 (MMP-2) (Mertens et al. i998). Loss of YB-i also significantly reduced the size of colonies that formed in soft agar (Figure 2.2A). Bigger colonies (> 70km) were present in much greater number in the control than in shYB-i cells, yet there was no difference in the total number of colonies that developed (data not shown). The difference in the number of big colonies (> 70tm) is shown in the photomicrographs and the bar chart in Figure 2.2A 33 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Muitiforme to Temozolomide left. In addition, the average size of the colonies in the control and shYB-1 cells was statistically different (Figure 2.2A right). In order to characterize the tumorigenicity of SF188 cells in mice, we conducted a study by injecting 1 x 106 SF188 control cells to the lower right hind flank of female Nu/Nu mice (n=12). The weight of mice and the size of tumors were measured for 4 weeks. By the first week, 70% of the mice developed palpable tumors. At the end of this 20-day study, all of the mice developed tumors. Tumors size of each mouse was measured after sacrifice (Figure 2.2B). The result of this study indicates that SF188 cells are highly tumorigenic in mice. Therefore, we sought to determine if silencing YB-i inhibited tumor growth in this xenograft model. SF188 control and shYB- 1 cells were bilaterally injected into the hind flank of female Nu/Nu mice. One week after injection, 83% of the mice developed tumors on the side where control cells were injected while only 40% of the mice showed palpable tumors on another side where shYB-1 cells were injected (Figure 2.2C). The difference of tumor incidence in the first week indicated knocking-down YB-i delayed the onset of tumors in vivo. However, all mice eventually developed tumors on both sides in the following week (Figure 2.2C). During the course of this 4-week experiment, we measured tumor size and showed that tumors arisen from the shYB-1 cells were significantly smaller than those derived from the control cells. Clear differences in tumor size were observed in harvested tumors (Figure 2.2D). This study serves to further support the idea that YB-i is possibly a molecular target for the treatment of aggressive forms of cancer. We recently reported that inhibiting YB-i with siRNA suppressed the growth of aggressive breast cancer cells in vitro and in vivo (Lee et al. 2008). Given the data presented here, where pGBM display YB-i dependency we expect that it may also be a viable molecular target for brain tumors. Inhibition of YB-i increased the sensitivity of SF188 cells to chemotherapeutic agents Paclitaxel and Temozolomide While we have demonstrated that targeting YB-i alone can perturb tumor growth. In the clinical set ting, inhibiting this target would likely involve combinations with current chemotherapies such as TMZ or perhaps Taxanes. These agents have been used in patients but their success is limited. Previous studies in dicate that TMZ insensitivity can be attributed to high levels of the DNA repair enzyme06-methylguanine- DNA methyltransferase (MGMT) (Hegi et al. 2005). SF188 cells do not express MGMT (Figure 2.3A, inset) yet they relatively resistant to TMZ (Figure 2.3A and 2.3B). The cells were treated with iOO, 500 or 1000 M TMZ for 24 hrs and then apoptosis was assessed by flow cytometry using Annexin V-PE. 34 Chapter2. Inhibition of YB-i Sensitizes Gliobiastoma Multiforme to Temozoiomide Overall, the cells were relatively insensitive to TMZ as it took 1000 M to induce cell death that only occurred in 4% of the cell population (Figure 2.3A). In addition, we treated the cells with TMZ and evalu ated anchorage independent growth. In this study, we also found that TMZ was not very effective against SF188 cells as it had no inhibitory effect up to 500 jsM (Figure 2.3B). We concluded that SF188 cells were not very sensitive to TMZ and perhaps this is because they express high levels of YB-i. This is in keeping with a previous report indicating that the IC50 of TMZ was 426±216 uM for SF188 cells. In that case, cell growth was assessed in monolayer after incubating the cells for 96 hours with the drug (Ma et al. 2002). We questioned whether or not silencing YB-i rendered cancer cells more sensitive to TMZ by evalu ating apoptosis through the induction of histone 2AX (H2AX). Phosphorylation of H2AX occurs at sites of DNA strand breaks during programmed cell death and therefore serves as an early apoptosis marker in our studies. In a recent study reported by us, we showed that inhibiting YB-i sensitized breast cancer cells to Paclitaxel (Lee et al. 2008). Thus, Paclitaxel (TXL) was used as a positive control for the induction of apoptosis in SF188 cells. We first examined the level of PH2AXsl39 in SF188 control and shYB-1 cells treated with increasing concentrations of TXL (0 nM, 1 nM, 10 nM and 100 nM) or TMZ (0 JLM, 10 tM, 100 iM and 500 jiM) for 24 hours. We noted a dramatic increase in the PH2AXsl39 in SF188 shYB-1 cells compared to the control cells (Figure 2.3D) following exposure to either of these chemotherapeutic agents. In addition, we also stained the cells with Annexin V-PE and analyzed the results by flow cytom etry to quantify whether apoptosis resulted from drug treatments. SF188 control and shYB-i cells were treated with TXL or TMZ for 24 hours, and stained with Annexin V-PE. The cells were subjected to flow cytometry. The cells in the lower right quadrant were Annexin-V-PE positive and 7AAD negative, which are the early apoptotic cells that we focus on. The result showed that SF188 shYB-1 groups had TXL 2-fold and TMZ 5-fold more Annexin V-PE positive cells, compared to the SF188 control cells (Figure 2.3C). To further substantiate our finding that silencing YB-i enhanced cell sensitivity to chemothera peutic agents, we employed the ArrayScan VTI high content screening (HCS) instrument and performed Hoechst staining. Using the ArrayScan instrument, we examined intensity of Hoechst staining in the cells treated with TXL or TMZ for 24 hours as a measure of cell number and chromatin condensation. Hoechst dye specifically binds the nuclei, which becomes fragmented and condensed, resulting in stronger signal in cells undergoing apoptosis (Figure 2.3E). Therefore, silencing YB-i may cause the cells to become more likely to go chemotherapeutic agent-induced apoptosis. Thus, we have demonstrated that silencing YB-i in SF188 cells enhanced TXL and TMZ-induced apoptosis. Given these intriguing findings, we next addressed whether YB-i is highly expressed in primary 35 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozolomide aGBM. Previous studies indicated the YB-i was among the top differentially expressed RNA regulatory factors in a comparison of 10 primary adult GBM and 10 nontumor control brain tissue samples (Cheung et al. 2008). To expand these studies we performed a similar analysis using data mined from GEO where 21 normal and 81 aGBM were evaluated. Interestingly, in this larger data set YB-i expression remained among the top 25 differentially expressed genes (data not shown) and its expression was consistently higher in the tumors compared to normal tissues (Figure 2.4). These data suggest that YB-i is commonly over-expressed in primary aGBM that implies they may serve an important function for the way in which these tumors respond to therapy. In further support of this finding, the aGBM cell lines U87 and U25 1 both express YB-i as do the pediatric cell lines SJ-G2 and SF188 (Figure 2.5A). In the aGBM cell line U25 1, P-YB-i5102 was readily detectable and mainly found in the nucleus (Figure 2.5B) similar to the pattern found in SF188 cells. We therefore developed a stable shYB-i cell line where >90% of YB-i was lost (Figure 2.5C). Importantly, co-targeting YB-i remarkably sensitized the U25 1 cells to TMZ (Figure 2.5E) and this was again independent of MGMT expression (Figure 2.5D). These findings reinforce the idea that co-targeting YB-i with standard-of-care chemotherapy may improve the treatment of cancer. We also propose that YB-i may be a novel mechanism for TMZ resistance independent of MGMT. Thus, future studies on the use of YB-i as a biomarker of therapeutic resistance may have clinical utility. This is further supported by the demonstration that this system is operative in GBM.We conclude that inhibiting YB-i has the potential to improve the treatment of GBM that arise in children and adults. 36 Chapter 2. Inhibition of YB-i Sensitizes Giioblastoma Multiforme to Teniozoloniide Figures Figure 2.1: Cellular invasion through Matrigel was inhibited by transient and stable YB-i knock down A) YB-i and pYBiS2expression in pediatric glioblastoma cell line SF188 wild type (WT) cells. B) Expression of YB-i in transient and stable YB-i knock-down SF188. Left: SF188 were transfected with control or shYB- 1 plasmids targeting YB-i. The protein expression of YB-i and pYBiSlO2was examined in SF188 wild-type (WT), control (control) and YB-i knocked-down (shYB-i) cells. Right: SF188 WT cells were transfected with 1 OnM control or siRNA oligos targeting YB-i and the whole cell extracts were prepared 1, 2, 3, 4, 6 and 8 days post-transfection. Vinculin was measured as a loading control. C) Cell invasiveness of SF188 control and shYB-i cells was assessed using matrigel invasion assay (Magnification 100 and 400-fold). The cells that had migrated through the membranes were quantified by determination of the cell number in three randomly chosen visual fields under the microscope. D) SF188 WT cells were treated with lipofectamine, control or siRNA oligo targeting YB-i. One day after transfection, the cells were re-plated in matrigel assays to assess changes in cellular invasion. The transient loss of YB-i caused a marked suppression of invasion. Representative images of invasive cells (200-fold magnification). 37 Chapter 2. Inhibition of YB-I Sensitizes Glioblastoma Multiforme to Temozolomide 140 120 100 80 = 60 o 40 20 0 (A) (B) (C) SF188 WT contro’ shYB-1 .- — -- - YB-i pYB-i S102 SF188 WT vinculin LP CO 1 2 3 YB-1 —, ._.._ VII IIUIII I 4 6 8 Days * 35 30 C) 25 20 C 15 C10 5 0 SFid control SF188 shYB-1 (D) LP * Co YB-i sIRNA Figure 2.1 38 chapter 2. Inhibition of YB-i Sensitizes GiiobiastomaMuitiforme to Temozolomide Figure 2.2: Knock-down of YB-i reduced cologemcity and tumor formation ability of SFi88 cells A) SF188 control and shYB-1 cells were plated in soft agar containing regular growth medium for 28 days. Colonies 70 m were counted under the microscope. Left: Number difference of the colonies formed by control & shYB-1 cells. Right: Size difference of the colonies formed by control & shYB-1 cells. B) Evaluation of tumorigenicity of SF188 cells in xenograft model. SF188 control cells were unilat erally injected into the left hind flank of female Nu/Nu mice. The percentage of tumor baring mice was monitored for 4 weeks. Tumor size was measured after the mice were sacrificed. C) SF188 control and shYB-1 cells were bilaterally injected into the hind flank of female NufNu mice (control-left, shYB-1-right). Position of injection of the control and shYB-1 cells is shown. Tumor incidence was monitored for two weeks. D) Tumor growth was monitored for four weeks. Tumor volume was measured for the control and shYB-1 groups using callipers. Differences in tumor size were determined using a Student’s T test, p<0.05. Right: example of differences observed in tumor volume. 39 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozolomide C E100% 0. .2 80% 60% 40% 0 20% — 0% 0 Weeks after injection SF188 control SF188 shYB-1 0 E z 0 > 12.0 .210.0 8.0 o 6.0 o 4.0 2.0 < 0.0 (A) 1 0 .0 E 35r > 3nfg 25 20 15 (B) 100% 80% 60% 0 40% 0 E 20% — 0%g 0 (C) (D) * SF188 shYB-1 5 10 15 20 Days after injection 25 30 beontrol . shYB.1 Tumor volume in mice 2 (mm3) 1 8f 0 161 141 121 80 60 40 20 control shYB-1 U Wek0 Weeki Week2 Week3 Week4 Figure 2.2 40 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozoloinide Figure 2.3: Induction of apoptosis after Paclitaxel and Temozolomide treatment A)SF188 control cells were treated with increasing concentrations of temozolomide (TMZ) for 24 hours. The levels of Annexin V-PE were then evaluated by flow cytometry. Levels of MGMT were also assessed by immunoblotting. B) In soft agar, the SF188 cells were resistant to TMZ up to 500 1iM. C) Annexin-V-PE Assay (early apoptosis detection) using SF188 control and shYB-1 treated with TXL and TMZ for 24 hours. D) The level of P-H2AX5139 phosphorylation (early apoptosis maker) in SF188 control & shYB-1 treated with TXL & TMZ for 24 hours was evaluated by western blotting. Vinculin was measured as loading control. E) After SF188 control and shYB-1 cells were treated with TXL and TMZ for 24 hours, the cells were fixed and stained with Hoechst dye. The signal intensities and pictures of the cells were obtained by ArrayScan VTI Reader. 41 Chapter 2. Inhibition of YB-i Sensitizes Gliobiastoma Multiforme to Temozolomide (E) MGMT14 > 12 0 >6 DMSO control I shYB-1 25 f 20 __ IT __ 015 ________ __________ I I1DMSO L0 IThtb0nM .1000. I- I - 0 Figure 2.3 SF188 control SF188 shYB-1 (B) SF188 TMZ(pM) DMSO 10 100 50Q Jurkat WI control shYB I . . 1 ‘o I ___________ - ‘ vinculin ::. . .0 35 z 30 25 DMSO .2 20 •TMZIO 15 IDTMZIO ,io DTMZ5O <0 (A) (C) (D) lOOuM 500uM l000uM TMZ dose DMSO TXL lOnM TMZ I000IIM >. I I ‘ .14 SF188 ‘°i L.. I I’ I I 12 controlS 1PF 1 ________ _ _ _ __ . I 10’ 1 I flQ4 S io I 5 5 5 i iS SF188 control SF188 shYB-1 pH2AXSI39 pH2AX’3 vinculin vul TXL(nM) 0 1 10 100 0 1 10 100 —. OMSO TXL lOnM TMZ 1000pM SF188 control SF188 shYB-1 - -., — — —— ————— ——— 0 10 100 500 0 10 100 500 42 chapter 2. Inhibition of YB-I Sensitizes Glioblastoma Multiforme to Temozolomide Figure 2.4: Hierarchical cluster analysis revealed elevated YE-i expression association with aGBM. Shown is the clustering analysis for 21 nontumor samples (NT) and 81 aGBM samples (GB). The expression levels are indicated by a gradient from low (blue) to high (red). 43 p 0 I I < < < < < < (< < < < C C C < < C C < < (C < < < < < < < < < < < < (C C < < C C C < (< < < < < < < < < < < < < < (( < < < < C < < < < < < < < C C C C C C C p. U. .• ‘• Tl CD IN ) Chapter 2. Inhibition of YB-i SensitizesG1iob1astoma Muitiforme to Temozolomide Figure 2.5: Inhibition of YB-i in aGBM increases sensitivity to TMZ A) YB-i protein is also readily detectable in the aGBM cell lines U25 1 and U87 as well as pGBM cell lines SJ-G2 and SF188. B) In U251 cells, YB-i and P-YBi5102 is readily detectable. C) A stable shYB-i expressing U251 cell line was developed where YB-i and P-YB-1S102 were decreased by at least 80%. D) Similarly to SF188 cells, the U25 1 cell line does not express MGMT. Jurkat extracts were used as a positive control for MGMT. E) Inhibition of YB-i sensitizes these cells to TMZ and TXL following exposure for 24 hrs. Apoptosis was monitored by P-H2AX’39and equal loading was accounted for using vinculin. 45 Chapter 2. Inhibition of YB-i Sensitizes Glioblastoma Multiforme to Temozolomide (A) (B) YB-i pYB-i S102 aGBM pGBM U87 U251 SJ-G2 SF188 YB-i — — vinculin U251 Jurkat control shYB-i MGMT vinculin TMZ (uM) TXL mM) TMZ (uM) TXL (nM) DMSO 100 500 1000 10 25 DMSO 100 500 1000 10 25 — — — ———__ — —— — — —I— — — — — U25i CO LJ251 shYBi U25i control shYB-i (D)(C) (E) pYB-1 S102 YB-i vinculin pH2AXS139 vinculin Figure 2.5 46 Chapter 2 Inhibition of YB-i Sensitizes Glioblastoma Muitiforme to Temozolomide References Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, Evangelista C, Kim IF, Soboleva A, Tomashevsky M, Edgar R. NCBI GEO: Mining tens of millions of expression profiles-database and tools update. Nucleic Acid Res 2007; 35D:760-765. Cheung HC, Baggerly Ka, Tsavachidis S, Bachinski LL, LNeubauer VL, Nixon TJ, Aldape KD, Cote GJ, Krahe R. Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays. BMC Genomics 2008; 9(216):1-16. Donson AM, Addo-Yobo SO, Handler MH, Gore L, Foreman NK. MGMT promoter methylation corre lates with survival benefit and sensitivity to pediatric glioblastoma.Pediatric Blood Cancer 2007; 48(4):403- 407. Faury D, Nantel A, Dunn SE, Guiot M, Haque T, Hauser P, Garami M, Bognar L, Hanzely Z, Liberski PP, Lopez-Aguilar E, Valera ET, Ton LG, Carret A, Del Maestro RF, Montes J, Gleave M, Albrecht S. Jabado N. Molecular profiling identifies prognostic subgroups of pediatric glioblastoma.J Clin Onc 2007; 25(10): 1196-1208. Forsyth P, Cairncross G, Stewart D, Godyear M, Wainman N, Eisenhauer E. Phase II trial of docetaxel in patients with recurrent malignant gliioma: A study fo the National Cancer Institute of Canada Clinical Trials Group. Invest New Drugs 1996; 14(2):203-206. Friedmann HS, Kerby T, Calvert H. Temozolomide and treatment of malignant glioma. Clin Cancer Res 2000; 6:2585-2597. Gottardo NG, Gajjar A. Chemotherapy for malignant brain tumors of childhood. I Child Neurol 2008; 23(10):1149-1159. Haque T, Faury D, Albrecht 5, Lopez-Aguilar E, Hauser P, Garami M, Hanzely Z, Bognar L, Del Mae stro R, Atkinson J, Natel A, Jabado N. Gene expression profiling from formalin-fixed paraffin-embedded tumors of pediatric glioblastoma. Clin Cancer Res 2007; 13(21):6284-6292. Hegi ME, Diserens AC, Gorlie t, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfeliner JA, Mason W, Mariani I, Bromberg JEC, Hau P, Mirimanoff RO, Cairncross JG, Janzer RC, Stupp R. MGMT gene silencing and benefit from temozolomide in glioblastoma. NJEM J 2005; 352( 10):997- 1003. Lee C, Dhillon 1, Wang M, Zhao Y, Leung 5, Park E, Hu K, To K, Stratford AL, Hung MC, Nielsen TO, Huntsman DG, Dunn SE. Targeting YB-i in Her-2 overexpressing breast cancer cells induces apoptosis via the mTORISTAT3 pathway and suppresses tumor growth in mice. Cancer Res 2008; 68(21):8661-8666. Ma I, Murphy M, O’Dwyer P1, Berman E, Reed K, Gallo JM. Biochemical changes associated with a 47 Chapter 2. Inhibition of YB-fr Sensitizes Glioblastoma Multiforme to Temozolomide mulidrug-resistant phenotype of a human glioma cell line with temazolomide-acquired resistance. Biochem Pharm 2002; 63(7):i219-1228. Mertens PR, Alfonso-Jaum MA, Steinmann K, Lovett DH. A synergistic interaction of transcription fac tors AP2 and YB-i regulates gelatinase A enhancer-dependent transcription. The Journal of biological chemistry 1998; 273(49):32957-32965. Stratford AL, Fry CJ, Desilets C, Davies AH, Cho YY, Li Y, Dong Z, Berquin TM, Roux PP, Dunn SE. Y-box binding protein-i serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells. Breast Cancer Res. 2008; iO(6):99. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJB, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairn- cross JG, Eisenhauer E, Mirimanoff RO. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. NJEM 1 2005; 352:987-996. Sun L, Hui AM, Su Q, Vortmeyer A, Kotliarov Y, Pastorino S, Passaniti A, Menon 1, Walling 1, Bailey R, Rosenblum M, Mikkelsen T, Fine HA. Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain. Cancer Cell 2006; 9(4):287-300. Sutherland BW, Kucab JE, Wu 1, Lee C, Cheang MCU, Yorida E, Turbin D, Dedhar 5, Nelson CC, Pollack M, Grimes HL, Miller K, Badve S, Huntsman D, Gilks B, Chen M, Pallen CJ, Dunn SE. Akt phosphory lates the Y-box binding protein-i at SeriO2 located in the cold shock domain and affects the anchorage- independent growth of breast cancer cells. Oncogene 2005; 24:428i-4292. Wu J, Lee C, Yokom D, Jiang H, Cheang MC, Yorida E, Turbin D, Berquin TM, Mertens PR, Iftner T, Gilks CB, Dunn SE. Disruption of the Y-box binding protein-i results in suppression of the epidermal growth factor receptor and HER-2. Cancer Res 2006; 66:4872-4879. Wu J, Stratford AL, Astanehe A, Dunn SE. YB-i is a transcription/translation factor that orchestrates the oncogenome by hardwiring signal transduction to gene expression. Translational Oncogenomics 2007; 2:i-i7. 48 Chapter 3 Discussion 3.1 The Effect of YB-i Knock-down on Cell Growth and Cellular Functions We have previously demonstrated there was an almost 90% YB-i protein decrease in both transient siYB-i and shYB-i stable knock-down models.The growth of SF188 and U25i cells was compared fol lowing transient and stable knockdown. YB-i siRNA treatment in SF188 cells greatly disturbed cell proliferation. After inhibiting YB-i by siRNA in SF188 and U25 1 cells for 8 days, growth was inhibited by 70% and 80% respectively (Figure 3.1). Since YB-i promotes cell proliferation by up-regulating sev eral growth-related genes as mentioned in section 1.3.3, its depletion by RNA interference may therefore inhibit tumor cell growth by altering proliferation signals. Consistent with this, Lee et al. (2008) reported that knocking-down YB-i by siRNA perturbed breast cancer cell growth in monolayer (Lee et al. 2008). In that report, tumor cell growth was inhibited in 6/7 breast cancer cell lines by between 40-80%. However, the stable loss of YB-i using shRNA did not perturb the cell growth in monolayer(Figure 3. i). Consistent with this observation, silencing YB-i by shRNA in gastric carcinoma and pancreatic carcinoma cell lines also showed no sign of growth inhibition despite a 90% decrease in YB-i mRNA level (Kaszubiak et al. 2007). This is consistent with our finding that SF188 and U25i shYB-i cells haven’t shown different cell proliferation rate from control cells. It is highly possible that these shYB-i cells have activated alternative signaling pathways to compen sate for the loss of YB-i. After examining the signaling pathway in both stable and transient knock-down models using immunoblotting, we find that expression of EGFR and mTOR were perturbed by transient knock-down YB-i, while they regained in the stable knock-down model. It suggests the long-time sup pression of YB-i in GBM cells might induce compensatory activations of P13K or MAPK pathway which are crucial to cell proliferation, resulting in higher cell proliferation rate in the stable YB-i knock-down 49 Chapter 3; Discussion cells than the transient knock-down cells. Similarly, compensatory activation of P13K and MAPK pathway had been determined after exposure of cells to a variety of stresses (Dent et al. 2003). On one hand, I be lieve it is better to employ the transient knock-down model to evaluate the cellular functions and signalling pathways in vitro to avoid the compensatory effect rising in the long-term YB-i knock-down cells. On the other hand, it is unreliable to assess the tumor initiation ability in vivo with transient knock-down model since the inhibition is not sustained long-term. Stable YB-i knock-down cells should be the better choice to carry out the in vivo assay. In addition to monolayer growth studies, we have also shown in colony formation assays that knock ingdown YB-i disturbed anchorage-independent growth of SF188 cells. Since anchorage-independence correlates strongly with tumorgenicity and invasiveness, soft agar assay is widely used to evaluate the malignant properties of tumor cells. In addition, this assay provides a three-dimensional culture system for tumor cells to manifest certain cell behaviors which would not be otherwise captured. For instance, Kolesnikova et al. reported that the expression of EWI-2, a cell surface immunoglobulin protein, in GBM cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar (Kolesnikova et al. 2009). In our studies, shRNA targeting YB-i altered cell proliferation rate in soft agar assays (Figure 2.2A) and it is independent from proliferation rate in monolayer(Figure 3.1). However, silencing YB-i in SF188 cells did not completely cease anchorage-independent growth but suppressed colony formation in vitro. Consistent with this, silencing YB-i by siRNA also perturbed colony formation in breast cancer cells SUM149 and BT474-mi by 70%-90% (Stratford et al. 2007; Lee et al. 2008). The decrease in colony size suggests an indispensable role of YB-i in cell proliferation in three-dimensional culture. YB-i may thus be important in tumor development in vivo. Cell invasion is a prerequisite for metastasis. GBMs tend to infiltrate into the surrounding normal brain, which confounds therapeutic attempts at local control. Cancer cell invasiveness is often apparent in GBMs implying that this phenotype is a significant parameter for GBM tumorigenesis (Louis et al. 2006). To gain a deeper understanding of the functional role of YB-i in driving GBM oncogenesis, we employed matrigel invasion assay to evaluate the effect of YB-i knock-down on cell invasiveness. The matrigel in this assay is composed of basement membrane containing collagens and growth factors thereby mimicking the in vivo environment in which cells can demonstrate their invasive ability. In the YB-i stable knock down cell lines, there was approximately a 50% decrease in invasion compared to the control (Figure 2.iC). Compared with the control, siYB-1 cells exhibited a 70% decrease in invasion (Figure 2.iD). There is a growing body of literature suggesting the role of MMPs on GBM migration and invasion. Inhibitors of 50 Chapter 3. Discussion MMPs have subsequently been developed and have demonstrated in vitro efficacy in GBM (Demuth and Berens 2004). YB-i is implicated in the regulation of MMP-2, MMP-i2 and MMP-13, as mentioned in section 1.3.3. The suppression of cell invasion after YB-i siRNA treatment might therefore be attributed to a decrease in MMP expression. Similar results were obtained in a carcinoma cell line established from rabbit keratinocytes which was transfected with siRNA targeting YB-i. These cells showed decreased cell invasion in matrigel assays (Huber et al. 2004). The role of YB-i in invasion was further illustrated by a recent study from Schittek et al. (2007) reporting that stable knock-down of YB-i reduced cell invasion, accompanied by a decrease in MMP-2 in metastatic melanoma cells (Schittek et al. 2007). More recently, our laboratory observed that YB-i is central to the regulation of urokinase plasminogen activator (uPA), another key in cellular invasion (under review). Further to this effect, YB-i is known to be a potent inducer of CXCR4 (Basaki et al. 2007). Thus, together there are several possible explanations for YB-i’s role in mediating GBM invasion however the precise mechanism warrants further investigation. These in vitro studies ascertain the role of YB-i in driving cell growth and oncogenesis in pGBM and provides much needed tools that will prove valuable in pre-clinical trials targeting this factor. 3.2 Inhibition of YB-i Suppressed Xenograft Growth in Nude Mice Following our intriguing studies in vitro, we have shown for the first time using a pGBM model that, silencing YB-i delayed the onset of tumor formation and inhibited tumor growth in vivo. We injected SF188 control and shYB- 1 cells bilaterally into each side of the hind flank of nude mice. In the shYB- 1 side, palpable tumors were detected later than the control side. Although at the end of the study, 100% tu mor development was observed on both sides of the mouse, the tumors arising from the shYB-i cells were significantly smaller in size. This observation is consistent with our in vitro soft agar studies showing that knock-down of YB-i inhibited colony formation. It appears that silencing YB-i in GBM cells suppressed cell growth and therefore resulted in a delay in tumor formation in vivo. Querying on the accuracy of tumor size, one may argue that there remained differences between the recorded tumor volume during the course of the study and the final tumor volume measured after sacrifice. Two causations are proposed to explain this discrepancy- i) It was unavoidable to include the thickness of skin and possibly fatty tissue appended around the tumors when measuring tumor size in a live animal. The measured size may therefore be larger than the actual size of a tumor. 2) Since the tumors were 51 - Chapter 3. Discussion measured by two researchers before and after the animals were sacrificed, individual measuring habits could confer such a difference. Despite this minor discrepancy, results from either measurement clearly showed that silencing YB-i suppressed tumor development in vivo. In support of this, Lee et al. have recently demonstrated an inhibition of tumor growth and a delay in tumor formation in breast cancer cells pre-treated with YB-i siRNA before injection (Lee et al. 2008). To avoid inter-animal variations, we injected both the control and shYB-i cells bilaterally into the same mice. Presumably tumor take rates should be the same, however this may not be the case.To avoid false positive results, one could either alter the side of injection to warrant against a favored site, or alternatively individual mice could be treated. 3.3 Silencing YB-i Improved the Sensitivity of SF188 and U251 Cells to Temozolomide and Paclitaxel Treatment As mentioned in section 1.3.4, YB-i is implicated in drug resistance. Elevated nuclear YB-i level has been reported in cancers of the breast (Bargou et al. i997), prostate (Gimenez-Bonafe et al. 2004), bone (Oda et al. 2008), ovary (Kamura et al. 1999) and muscle (Oda et al. 2003). By immunocytochemistry, we detected a high level of PYB1sehio2in the nucleus of pGBM SFi88 and aGBM U25i cells. This is of particular interest to us because nuclear YB-i may help confer drug resistance to chemotherapeutic agents in these cells. To investigate the role of YB-i in GBM drug resistance, we first examined the drug-sensitivity of SF188 cells to chemotherapeutic agents. Temozolomide (TMZ) is a lead chemotherapeutic agent in GBM treatment yet drug-resistance develops in patients with high MGMT expression. In monolayer studies, only 4% of the total cell population underwent apoptosis at the maximum TMZ dose tested (1000 MM). In anchorageindependent conditions, colony formation was not inhibited even at a relatively high dose of drug treatment (500 M) (Figure 2.3A and 2.3B). We thus questioned what factors could possibly contribute to TMZresistance in SF188 cells. A recent study indicated that MGMT could mediate TMZ sensitivity in pGBM patients (Donson et al. 2007). MGMT is a DNA repair enzyme which could negate TMZ induced methylations in DNA, as mentioned in section 1.2.3.3. GBM patients with unmethylated MGMT promoter (resulting in MGMT high expression) barely benefit from TMZ treatment. With this evidence in mind, we examined MGMT expression in SF188 cells. To our surprise, the level of MGMT was undetectable in 52 Chapter 3. Discussion these cells by immunoblotting, suggesting that MGMT may not be the prime factor for TMZ-resistance in certain pGBM models. We then hypothesized YB-i to be a novel factor contributing to TMZ-resistance. Stable YB-i knock down cells showed a marked increase in pH2AxSerl39,an early apoptosis marker (Bonner et al. 2008), compared with the control after TMZ treatment (Figure 2.3D). An increase in Hoechst intensity (Figure 2.3E) and Annexin V-PE stained cells (Figure 2.3C) additionally supported the result that the combina tion of YB-i knock-down and TIVIZ confers an improved cytotoxic effect compared to single agent alone. Paclitaxel (TXL) is one of the most well-known broad spectrum anti-cancer drugs, however thus far it provides only limited clinical benefit in GBM treatment owing to its inability to effectively cross the BBB because of its large molecular weight. Many studies had been carried out to improve TXL delivery through BBB into brain tumors, as mentioned in section 1.2.3.1. Therefore there still remains value for TXL in future treatment of this disease. From our results, it seemed that silencing YB-i rendered SF188 and U25 1 cells to be more sensitive to TMZ and TXL. This is a rather exciting result in that for the first time, YB-i was shown to be related to TMZ sensitivity. There have been studies demonstrating the role of YB-i in conferring drug resistance to other anti-cancer drugs. Decreased resistance to cisplatin and etoposide was observed following YB-i knockdown in melanoma cells (Schitteck et al. 2007). Moreover, we have recently shown that silencing YB-i improved growth suppression with Herceptin in soft agar assays (un published data). There is mounting evidence suggesting the role of YB-i in multi-drug resistance. It is well accepted that the cells with higher proliferation rate are more susceptible to anti-cancer drugs. According to the Figure 3. i, we found there was little difference between the control and shYB-i cells proliferation rates. Thus, we believe there might be other underlying mechanisms could help explain our results. In the following sections, a few hypotheses of how YB-i may contribute to TMZ and TXL in SFi 88 cells will be discussed with support from current literature (Figure 3.3). 3.3.1 YB-i Modulates Drug-sensitivity via Regulation of MDRiIP-glycoprotein Expression One of the widely studied mechanisms mediating chemoresistance is the expression of efflux pump protein that selectively transports substances out of cells. The multidrug resistance gene (mdrl) encodes for P-glycoprotein (Pgp), a drug efflux pump, whose over-expression has been primarily observed in human cancers resistant to chemotherapy (Schaich et al. 2009). Pgp has been found to be expressed in the 53 Chapter 3. Discussion majority of brain tumors, including glioblastoma (Demeule et al. 2001), indicating an intrinsic resistance to anticancer drugs in GBM. In addition, Pgp functions as a component of the BBB, limiting the penetration of drugs into brain (Schinkel et al. 1994). Interestingly, YB-i was reported to induce the expression of Pgp (Goldsmith et al. 1993). The Y box is one of the key elements in the mdrl promoter to which YB-i binds (Ohga et al. 1998). The detail transcriptional control of Pgp by YB-i is still under investigation. Recently, Chattopadhyay et al. (2008) reported human AP-endonuclease (APE1) stably interacts with YB-i and enhances its binding with mdri promoter, leading to the activation of Pgp transcription. The association between nuclear YB-i and Pgp expression has been suggested in cancers of the prostate (Gimenez-Bonafe et al. 2004), bone (Oda et al. i998), ovarian (Oda et al. 2007), muscle (Oda et al. 2008), colon (Vaiman et al. 2007), and breast (Saji et al. 2003). It is evident that YB-i plays a central role in enhancing drug resistance of tumor cells by activating Pgp. Both YB-i (Haque et al. 2007; Cheung et al. 2008) and Pgp (Demeule et al. 2001) are expressed in GBM. Although to date, there has not been study showing an association between YB-i and Pgp in GBM, based on the reported association in a broad range of cancers, it would not be surprising to find such a relationship in GBM in the future. The fact that TXL often encounters undesirable clinical side-effects and multidrug resistance (MDR) may be due to over-expression of P-glycoprotein (Pgp), which was reported to transport TXL (Sparreboom et al. i997). Moreover, Pgp expression in the brain tumor neovasculature limits TXL penetration into the tumor as a component of BBB (Gallo et al. 2003). TXL was delivered into brain tumor more efficiently in mice in which Pgp was knocked-out (Fellner et al. 2002). The combination of TXL and a Pgp blocker, valspodar, suppressed the intracerebral GBM xenograft in mice much more effectively than TXL alone (Fellner et al. 2002). It is reasonable to hypothesize YB-i as a multi-drug resistant protein contributing to TXL resistance by up-regulating Pgp expression in GBM. Interestingly, Fujita et al. (2005) had proposed that increased nuclear YB-i and a corresponding up-regulation of Pgp was the mechanism by which breast cancer cells acquire resistance to TXL (Fujita et al. 2005). Therefore YB-i may confer both intrinsic and acquired resistance to TXL by modulating Pgp. In our studies, GBM cells did not seem to respond well to TMZ despite an undetectable level of MGMT. In the clinical setting, some GBM patients with aberrant MGMT promoter methylation still can not benefit from TMZ (Finlay et al. i995). Results indicated that methylation status is unlikely to be the only molecular mechanism affecting patient outcome after TMZ treatment. We therefore wondered 54 Chapter 3. Discussion whether Pgp would be involved in modulating the response to TMZ. Research has been underway to investigate whether Pgp alters the delivery of TMZ into the brain (Jelinek et al. 1999). Schaich et al. recently confirmed TMZ to be a substrate of Pgp. MDR- 1 negative cells displayed enhanced sensitivity to TMZ and in vitro analysis revealed that the cytotoxic effect of TMZ is related to Pgp expression (Schaich et al. 2008). Furthermore, genotypes of mdrl predicted outcome of TMZ treatment independent of MGMT methylation status in GBM patients (Schaich et al. 2008). These findings were exciting to us as we observed a relationship between YB-i and TJvIZ-resistance in our studies. It is probable that in our GBM model, YB-i transcriptionally up-regulates Pgp expression, thereby conferring resistance to TMZ. Further studies will be required to examine Pgp level after YB-i knock-down. 3.3.2 YB-i May Mediate DNA Repair as a Possible Explanation for It Associated TMZ-Insensitivity As mentioned in section 1.3.4, temozolomide induces methylation on 06 position of guanine (8%), N7 position of guanine (70%) and N3 position of adenine (10%). These adducts lead to either cell death or genetic mutations in the DNA helices. Three DNA repair systems can help cells overcome the harmful effects of TMZ- 1)6-alkylguanine-methyltransferase (MGMT) removes small aykyl adducts from the most toxic lesion, 06 methylguanine (06-MG), 2) Mismatch repair (MMR) recognizes base mismatches and attempts to repair the damage and/or 3) Base excision repair (BER) removes lesions and repairs N7 methylguanine (N7-MG) and N3 methyladenine (N3-MA) caused by TMZ (Marchesi et al. 2007). Since the main cytotoxic effect of TMZ is caused by06-MG, MGMT, a specific 06-MG methyltransferase, becomes particularly important in the prediction of TMZ efficacy in GBM patients. However, we were unable to detect MGMT protein in pGBM SF188 and aGBM U25 1 cells by immunoblotting (Figure 2.3A and figure 2.5D). MGMT is therefore unlikely to be crucial in mediating TMZ-resistance in our models. Mismatch repair (MMR) recognizes base mismatches, cuts the nucleotide sequence containing the lesion, and restores the correct base sequence. MMRis believed to be involved in resistance to DNA damaging drugs. However, the system does not suppress, but instead promotes the cytotoxic effects of TMZ. MMR is unable to repair the lesion caused by TMZ. The “futile repair”, along with lethal double strand breaks, results in apoptosis (Bignami et al. 2000). TMZ induced apoptosis in MMR-proficient but not MMR-deficient malignant cells (Meyers et al. 2004). Interestingly, YB-i is implicated in the MMR system. YB-i was found to be complexed with the proliferating cell nuclear antigen (PCNA) (Ise et al. 55 Chapter 3. - Discussion 1999), the MutS homologue 2 (MSH2) and DNA polymerase 6 (Gaudreault et al. 2004), all of which are involved in mismatch repair. The mechanism by which YB-i influences the MMR is still largely unexplored. There remains possibility for YB-i to mediate TMZ-resistance via regulation of the MMR system, which is an area that warrants further investigations. Another pathway involved in the DNA repair in response to TIvIZ treatment is base excision repair (BER). As previously mentioned, TMZ induces the formations of N7 methylguanine (N7-MG) and N3 methyladenine (N3-MA). N7-MG is not a particularly cytotoxic biochemical whereas N3-MA is highly lethal if unrepaired by the BER system (Johannessen et al. 2008). As a result, the DNA repair activity of the BER pathway in GBM is at least partially involved in provoking resistance towards TMZ. Trivedi et al. (2008) recently reported an imbalance in BER system leading to enhanced cellular sensitivity to the cell- killing effects of TMZ. Overall BER capacity may therefore help predict drug sensitivity. Interestingly, YB-i was found to bind BER-related Ku antigen and to co-localized with it (Gaudreault et al. 2004). The activity of human endonuclease III (hNthi), a BER initiation protein, could be potentially up-regulated by YB-i (Marenstein et al. 200i). A recent study suggested that mammalian AP-endonuclease (APE1), a major player in the BER pathway, could stably interact with YB-i and form distinct complexes (Bhakat et al. 2008). Therefore, it would be interesting to determine if YB-i could mediate TMZ-sensitivity via interacting with BER proteins in future. In addition to binding DNA repair proteins, YB-i directly interacts with abnormal DNA strands, in dicating its potential role in DNA damage detection which is crucial to TMZ resistance. YB-i has been shown to bind depurinated DNA (Hasegawa et al. 1991), mismatched nucleotide pairs (Gaudreault et al. 2004) and preferentially cisplatin-modified strands (Torigoe et al. 2005). These results collectively sug gest a role of YB-i in mediating drug resistance by interacting with the components of the different DNA repair systems. 3.3.3 Interaction Between YB-i and Microtubules Might Perturb TXL-sensitivity YB-i was recently reported to interact with tubulin and microtubules to promote their assembly in vitro (Chernov et al. 2008). This result triggered our interest in examining the effect of silencing YB-i on paclitaxel sensitivity in GBM cells. The use of paclitaxel in clinical setting has become prevalent since its approval by the FDA in 1992 56 Chapter 3. Discussion in treatment of ovarian cancer (www.fda.org). However drug resistance still presents a major obstacle to improving the overall response and survival of cancer patients (Off et al. 2003). Paclitaxel achieves its cy totoxic effect by disturbing microtubule dynamics and functions. In eucaryotes, microtubules are involved in a diverse range of cellular functions including mitosis and meiosis, motility, as well as maintenance of cell shape. In vitro, paclitaxel binds to microtubule polymers, enhancing the polymerization of tubulins and thus suppressing microtubule dynamics. Consequently, the dynamics of mitotic spindle are disrupted, resulting in inhibition of tumor cell proliferation (Fojo and Menefee 2007). Paclitaxel as a microtubules stabilizer interferes with microtubule dynamics in tumor cells. Interest ingly, Chemov et al. (2008) determined that YB-i also regulates microtubule dynamics by stimulating their assembly in vitro. Paclitaxel binds along microtubules and increases inter-tubulin affinity in an un known manner (Nogales et al. 2001). In contrast, YB-i promotes the formation of normal microtubules and probably stabilizes the microtubule by coating the microtubule wall (Chemov et al. 2008). Thus we hypothesized that YB-i over-expressing cancers have a shift toward more stable microtubule complexes, resulting in resistance to paclitaxel-induced cell death. Indeed, our results have shown an increased sen sitivity to paclitaxel in YB-i knock-down GBM cells. However, as these studies are still preliminary in understanding the interaction between YB-i and microtubule, further investigations will be needed to decipher the mechanism. 3.4 Preliminary Study of YB-i Potential Role in GBM Cancer Stem Cells Properties Recent studies suggest that the progression of brain tumors is driven by a small subset of cancer cells that have stem-cell properties to self-renew, proliferate and differentiate into multiple neural lineages. Dicks and colleagues subsequently named this subpopulation of tumorigenic cells ‘cancer stem cells’ (CSC) (Singh et al. 2003). According to the CSC hypothesis, this subpopulation in the bulk of a tumor is responsible for tumor initiation, recurrence and progression. Although the idea of targeting CSC seems to be an attractive strategy for cancer treatment, it is still hindered by technical difficulties in obtaining a complete and successful separation and enrichment of CSC in tumor cells. To date, the gold standard for assessing brain CSC is tumorigenic assays in vivo. For In vitro studies, there are at least two methods that help determine brain CSC and they are the prospective sorting with stem-cell markers (nestin and 57 Chapter 3. Discussion CD133) and culturing the CSC in defined media using a 3D system (neurosphere assay). With the concept of tumor initiating cells in mind, we believe that the SF188 xenograft tumors established in immunode ficient mice may have arisen from a small population of CSC that were responsible for tumor formation and maintenance. In fact, research has shown CD133+ GBM cells to be more resistant to a panel of chemotherapeutics (including TMZ and TXL) compared with the rest of the tumor cells (Liu et al. 2006). GBM CSCs cultured in neurosphere assays exhibited significantly enhanced chemo-resistance to various anti-tumor agents, including TMZ (Eramo et al. 2006). This chemo-resistant phenotype may very well be conthbuted by a small fraction of cells that have the ‘stem-like’ properties, and this hypothesis was supported by experimental evidence showing a subpopulation enrichments in stem-cell markers, including CD1 33, in GBM cells survive BCNU treatment. These cells demonstrated tumor-initiating ability in vivo (Kang et al. 2007). Thus, CSCs subpopulation seem to be responsible for the drug-resistance. This may explain why current therapies, in spite of being extremely cytotoxic to the bulk of highly proliferative tumors cells, still fail to obliterate the relatively resistant CSC compartment (Das et al. 2008). However, there remains opposed evidence that CSCs were more likely to be depleted by anti-tumor drug (Beier et al. 2008). Thus, how CSCs play roles in drug resistance still need further investigation. Silencing YB-i suppressed tumor growth in mice and increased drug sensitivity of cancer cells to TMZ and TXL, we therefore questioned whether this protein is related to CSC properties. Although to date there is still a lack of direct evidence for the role of YB-i in regulation of GBM CSC cellular functions, studies have indicated that YB-i might cooperate with factors important for CSC maintenance, proliferation and differentiation. In our lab, chromatin immunopreciptitation (ChIP)-on-chip (COC) analyses with promoter arrays revealed a panel of putative YB-i target genes including Bmi-1 and several members of the Notch and WNT family, which are associated with stem-cell signatures (Finkbeiner et al. 2009). CSCs studies indicated Notch (Radtke et al. 2003) and WNT (Clark et al. 2007) pathways positively regulate stem- cell self-renewal and proliferation. Also, Bmi- i is involved in self-renewal of stem cells (Molofsky et al. 2003). Furthermore, c-Myc is required for maintenance of glioma CSCs (Wang et al. 2008) and it is positively regulated by YB- 1 (Kolluri and Kinniburgh 199i). EGF participates in mitogenic regulation of glioma CSCs (Soeda et al. 2008) and YB-i is determined to regulate EGFR signaling (Stratford et al. 2007). The fact that YB-i is associated with some of these proteins suggests its potential role in regulation of CSC properties. With the given information, we hypothesized that knock-down of YB-i silencing may have an impact on GBM CSC properties; therefore, drug-sensitivity and tumorigenicity of cells may be altered in vivo. 58 Chapter 3. Discussion CD133 sorting assay and neurosphere assay were used to assess the potentially different CSC signa tures in SF188 control and shYB-i cells in vitro. In cell sorting assays, only less than 0.1% of the total cell population was CD133-positive (Figure 3.2A). In neurosphere assays, the colony forming frequency was around 2% in SF188 (Figure 3.2B). While Das et al. isolated 29% CD133-positive cells with 31% neurosphereforming frequency from fresh primary GBM cells (Das et al. 2008), we obtained much less CD133-positive cells from SF188 and U25 1 cancer cell lines. Consistent with the results we have, Kondo et al. reported only a 1% CSC subpopulation from an established glioma cell line (Kondo et al. 2004). The relatively small fraction of CSC in established tumor cell lines can be explained in part by the con dition used to culture these cells. Fetal bovine serum supplemented in the media may cause the cells to lose ‘stem-like’ properties (Setoguchi et al. 2004). It is reasonable to see much fewer cells formed neu rospheres in our established GBM cell lines than primary GBM cells reported by other groups (Das et al. 2008). In order to gain a better examination of YB-i effect on GBM CSC properties in vitro, we might have to employ primary GBM cells in future. Although the CD133-sorting and neurosphere studies with or without drug treatment did not indicate a difference between the control and shYB-1 cells (Figure 3.2A & B), we are still unable to conclude definitively whether YB-i modulates CSC properties since theses assays merely enrich or partially separate CSC in vitro (Dirks et al. 2008). In fact, recent study has shown that CD 133 is not the best marker identifying GBM CSC because CD1 33-negative cells also formed tumors in vivo (Wang et al. 2008). In addition, neurosphere formation should not be equated with being derived from a stem cell. Since non CSC, such as progenitor cells, also have the ability to form neurospheres (Galli et al. 2004). Therefore improved methods and assays in vitro will be required for future studies. According to gold standard CSC assay in vivo, I might inject various numbers of control and shYB-1 cells in mice and try to examine the least number of cells needed for tumor initiation in each group. Since I injected 106 cells in this study, I could not assess differences in tumor initiation in a manner that would indicate differences in the CSC population in each group. In future, I would therefore propose to inject 10, 100, 1000 and 10,000 or 100,000 cells of each cell line into the mice to gauge differences in tumor-initiating ability. 59 Chapter 3. Discussion 3.5 Summary and Future Directions In our studies, I have determined the functional role of YB-i in GBM cell lines. Both transient and stable knock-down of YB-i suppressed cell invasiveness and colony formation in soft agar. I further examined the tumorigenic potential of the shYB-i cells in vivo. Inhibition of YB-i not only delayed the onset of tumor formation but also suppressed tumor growth by 30% in immunodeficient mice. These results together indicate a potentially crucial role of this protein in GBM progression. To gain a deeper understanding of the mechanism by which YB-i contributes to the GBM invasion and metastasis, I will need to examine the alterations in MMPs, uPA and/or CXCR4 expression in the YB-i knocked-down cells. Cell proliferation markers should also be evaluated in in vitro and in vivo studies to ascertain the significance of YB-i in growth control of cancer cells. I further investigated the response of YB-i knocked-down cells to chemotherapeutic agents in mono layer and anchorage-independent conditions. Intriguingly, knocking-down YB-i significantly enhanced sensitivity of SF188 and U25 i cells to TXL and TMZ, suggesting YB-i might be a novel target in combi nation therapies. I plan to examine YB-i expression in the drug-sensitive GBM cells and patient samples to further confirm YB-i roles in the drug-resistance. And it will be promising to examine the drug sensi tivity in the shYB-i xenograft model in vivo. As for investigating the underlying mechanisms, YB-i might i) regulate Pgp expression, 2) implicate in the MMR and BER pathway and 3) associate with CSC prop erties, all of which are related to GBM drug-resistance. It would be of interest to closely study how these players interact to contribute to disease aggressiveness. Also, I might alter our YB-i silencing method from shRNA to siRNA to assess the cell sensitivity to chemotherapeutic agents, since evidences indicated that shYB-i cells have activated alternative signaling pathways to compensate for the loss of YB-i. YB-i siRNA treatment might provide more promising enhanced drug-sensitivity and might be more accessible in clinical application. Realizing the importance of YB-i in cancer pathogenesis, I have recently developed inhibitory cell permeable peptides and peptidomimetics targeting YB-i. The peptide sequence resembles that of YB-i in the cold shock domain around SeriO2 site. These peptides compete with YB-i for binding with RSK, PKC and AKT, inhibit YB-i phosphorylation and subsequently nuclear translocation. Peptidomimetics were designed to have similar structure and function with the YB-i peptide but with reduced cost and enhanced stability. These inhibitors have additional advantages over the peptide in that they are small in molecular weight which is important for drug penetration through the blood-brain-barrier. Rigorous 60 Chapter 3. Discussion studies have been carried out in our lab to evaluate the effectiveness of these YB-i inhibitors in different cancer models. With improved understanding of the functions of YB-i, novel therapeutic approaches developed to target this oncogenic factor are expected to provide promising therapeutic benefit in treatment of GBM. 61 Chapter 3. Discussion Figures Figure 3.1: Cell proliferation of transient and stable YB-i knock-down cells In order to investigate the capability of self-renewal and proliferation potential of YB-i silenced cells, we employed ArrayScan VTI Reader to obtain the cell number information. A) SF188 WT and U25 1 control cells were pre-seeded in 96-well plate. After siRNA treatment for 8 days, the cell proliferation rate difference between control oligo and siRNA targeting YB-i was examined using ArrayScan VTI Reader. B) SF188 control/shYB- 1 and U25 1 controllshYB-i cells were seeded in 96-well plate. 8 days after seeding the cell proliferation rate difference was examined using ArrayScan VTI Reader. 62 Chapter 3. Discussion 11 *1 control shYB-1 control shYB-1 SF188 U251 (A) 140 120 —. 100 80 o 60 1 0 40 20 0 (B) 180 160 140 120 100 o 80 60 40 20 0 Control oligo siRNA SF188 WT Control oligo siRNA U251 control Figure 3.1 63 Chapter 3. Discussion Figure 3.2: CD133 expression and neurosphere formation of SF188 shYB-1 cells In order to investigate the capability of self-renewal and clonogenic potential of GBM cells, CSC marker CD 133 sorting was used to assess CSC like subpopulations and a defined serum-free neurosphere medium containing normal growth medium containing bFGF and of EGF was employed for neurosphere assay. A) Both SF188 control and shYB-1 had a few CD133 positive cells, and they were partly depleted by TMZ 100 M treatment for 24 hours. B) SF188 control and shYB-1 cells were harvested and re-seeded at 1.0 x i04 cells in ultra-low attach ment surface 6-well plates containing various concentration of TMZ. Representative microphotographs and quantification of neurosphere numbers were taken 5 days later. 64 Chapter 3. Discussion (A) SF188 control SF188 shYB-1 CD133- r _________ ________ io 101 102 1o io Tho° ;12 3 ‘; CD133 CD133 ,JJ TMZ treatment for 24 hours,JJ. 0, 0• o 0 <cJ <CJ <0F... C o - 100 101 io2 i3 CD133 CD133 (B) DMSO TMZ lOOuM TMZ 500uM : SF188 control •.. .1 .5 _ 3 . 1m .. SF188 ) shYB-1 &:. S , . .4 250 .0 E 200 • control 150 L shYB-1 0. 100 z5:T _i, TMZ lOOuM TMZ 500uM Figure 3.2 0, 0 CD133- • 0.07% o_. CD133• CDI 33- 133+ C0133- 0.03% CDI 33. 65 -- Chapter 3. Discussion Figure 3.3: YB-i mediated chemo-sensitivity mechanism model In cytoplasm, YB-i might interact with microtuble thus interfere its binding with TXL. In nucleus, YB-i may confer drug-resistant by 1) recognizing DNA damage site induce by TMZ, 2) promoting Pgp expression to dilute TMZ & TXL concentration in cells, 3) getting involved in DNA repair pathways, 4) interacting with microtubule. 66 Chapter 3. 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PLoS ONE. 2008; 3(1 1):3769. 73 Appendix BUFFERS AND REAGENTS Egg Lysis Buffer (ELB) 100mM HEPES (pH7.4) 500m1v1 NaC1 10mM EDTA (pH8.0) 0.2% NP-40 1mMDTT 3% Stacking Gel (SDS-PAGE) 4% bis-acrylamide 0.1% SDS 0.125M Tris (pH6.8) 0.05% ammonium persulfate Western Blot Sample Loading Buffer 0.5M Tris-HC1 (pH6.8) 0.8m1 glycerol 10% SDS 1% bromophenol blue 3.8m1 distilled water 0.4m1 of 14.3M 2-/3-mercaptoethanol 12% Seperating Gel (SDS-PAGE) 12% bis-acrylamide 0.1% SDS 0.375M Tris (pH8.8) 0.1% TEMED 0.05% ammonium persulfate Phosphate Buffered Saline (PBS) 8g NaC1 0.2g KCI 1.44g Na2HPO 0.24g KH2PO4 Make up to 1L with distilled water Adjust pH to 7.4 lOx Transfer Buffer 140g glycine 30.3g Tris Make up to 1L with distilled water 74

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