THE EFFECTS OF SELECTIVE INHIBITORS OF N-GLYCOSYLATION AND ENDOPLASMIC RETICULUM STRESS INDUCERS ON NEUROBLASTOMA CELL PROLIFERATION AND APOPTOSIS by Ebtehal Elkamel M. Eshiak Student Number: 3467609 A thesis submitted in fulfillment of the requirements for the degree: Magister Scientiae (MSc) Department of Medical Biosciences Faculty of Natural Sciences University of the Western Cape Supervisor Co-Supervisor Prof Donavon Hiss Dr Okobi Ekpo 13 July 2017 ©University of the Western Cape All Rights Reserved DECLARATION DECLARATION I, Ebtehal Elkamel M. Eshiak, declare that “The Effects of Selective Inhibitors of N- Glycosylation and Endoplasmic Reticulum Stress Inducers on Neuroblastoma Cell Proliferation and Apoptosis” is my original work and that all the sources that I have used or cited have been indicated and acknowledged by means of complete references, and that this document has not been submitted for degree purposes at any other academic institution. Ebtehal Elkamel M. Eshiak : Student Number: 3467609 Date Signed : 13 July 2017 ii http://etd.uwc.ac.za/ DEDICATION DEDICATION This study is dedicated to my family. iii http://etd.uwc.ac.za/ ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS I would like to convey my gratitude to the following persons and institutions for making this research undertaking a realization: My supervisor, Prof Donavon Hiss, for his guidance throughout the process. My co-supervisor, Dr Okobi Ekpo, for his involvement in the project. Prof Antonio Serafin, Department of Medical Imaging & Clinical Oncology, Faculty of Medicine and Health Sciences, University of Stellenbosch, for the gift of the neuroblastoma cell lines. Dr Jelili Abiodun Badmus, for his tolerance, encouragement and commitment to my cause, especially with regard to coaching and developing my skills in tissue culture procedures and laboratory assays. Mr Ahmed Eldud for his expertise and helpful insights into the statistical analysis of the results of the project. Prof Thomas Monsees, Prof Gerhard van der Horst and Dr Liana Maree, for their kind assistance with fluorescence microscopy. Mr Beynon Abrahams, Mr Leeshan Pillay, Ms Greshon Oliver, Mr Hamza Abouhamraa, Mr Dean Solomons, Mr Keenau Pearce, Ms Rabia Isaacs, Ms Maymoena Sablay and Dr Wejdan Husein, for their help and enthusiasm throughout the execution of this project. All the students and staff in the Department of Medical Bioscences, University of the Western Cape, for their advice, assistance and motivation. The South African National Research Foundation and the University of the Western Cape, for funding the research in the Molecular Oncology Laboratory. The Libyan Embassy, for financial assistance. iv http://etd.uwc.ac.za/ ABSTRACT ABSTRACT Among all childhood cancers diagnosed, neuroblastoma (NB) is the most prevalent during infancy. In patients younger than 15 years, it accounts for more than 7% of malignancies and about 15% of all paediatric mortalities. A family history of NB is considered a genetic predisposition and risk factor. In 1-2% of inherited cases, the molecular causative factors linked to the disease are germline mutations, including anaplastic lymphoma kinase (ALK, gain of function), PHOX2B (loss of function) and MYCN (gain of function). NB differs from most solid tumours because of its heterogeneity in both pathobiologic and clinical behaviour, ranging from spontaneous regression to highly-aggressive metastatic disease resistant to conventional and investigational anticancer drugs. Hence, NB embodies a spectrum of disease. Moreover, disease progression (uncontrolled cellular proliferation), a recognizable signature of NB, may be set off by inadequate targeting of resistant subpopulations (e.g., side-population cells or cancer stem cells/tumour-initiating cells) in the tumour microenvironment (TME). The complex molecular heterogeneity responsible for the disparate responses to treatment of NB provides a platform for discovering molecules and pathways for specific biologically-targeted interventions. In spite of contemporary improvements in intensive multimodal therapeutic approaches to NB (surgery, local radiation and differentiation therapy with 13-cis-retinoic acid, and myeloablative cytotoxic therapy with autologous haematopoietic stem cell transplantation), the overall survival of high risk patients has persistently been less than 40%. Since NB is a heterogeneous paediatric tumour that arises from the embryonic neural crest, it is not surprising that cancer stem cells (CSCs) or tumour initiating cells (TICs) have been implicated in tumour relapse and resistance to chemotherapy in high-risk neuroblastoma (HR- NB) patients. In 40-50% of the HR-NB cases, amplification of the MYCN gene remains the major key predictor of poor clinical outcomes. HR-NB patients with MYCN amplification generally show the development of resistance to induction chemotherapy. Thus, novel effective v http://etd.uwc.ac.za/ ABSTRACT therapies are urgently needed to improve clinical outcomes of HR-NB patients. Remarkably, coordinated expression of the MYC gene and those that encode for the ABC multidrug resistance transporters has been confirmed in a larger number of NB patient samples, experimental NB models and NB cell lines, but more work is needed to clarify this poorly understood prosurvival signature. Earlier work has found that high levels of ABCB1(P- glycoprotein/P-gp/MDR1) RNA are frequently associated with resistance to chemotherapy in NB. Recent studies have unequivocally provided evidence that MYCN enhances P-gp/MDR1 gene expression in the human metastatic NB IGR-N-91 model. CSCs in the heterogeneous NB TME exhibit high resistance to standard therapy, a cancer hallmark that correlates with overexpression of some members of the ABC family of MDR transporters, increased expression of anti-apoptotic molecules, increased DNA repair capacity and activation of specific stem cell survival signals. In recent years, the NB glycome has emerged as an important research paradigm that aims to show the significant impact of NB glycans in the TME on tumour progresssion, MDR and patient clinical responses to anticancer therapy. The role of protein N-glycosylation in neural transmission is widely recognized, but less well understood. Glycosylation is the multifaceted post-translational modification of proteins that takes place in the endoplasmic reticulum (ER) and offers a landscape for developing novel targeted approaches to CSC eradication and anticancer chemotherapy. Altered glycans on tumour- and host-cell surfaces and constituents of the extracellular matrix in the TME mediate key events in cancer pathogenesis, progression and survival. Glycan-based therapeutic rationales have been applied to cancers of the liver, pancreas, breast, lung, gastrointestinal system, melanomas, and lymphomas, but rarely to NBs. The success of anti- disialoganglioside (GD2, a glycolipid antigen) antibodies support glycan-based therapies for NB and also implies a proof-of-concept for protein glycosylation-based therapies for NB. N- glycosylation determines protein conformation, stability and function, degradation of misfolded proteins, programmed cell death (apoptosis), cell survival and immunity. vi http://etd.uwc.ac.za/ ABSTRACT Any disruption of this function of the ER to coordinate and maintain proper protein folding can lead to the ER stress (ERS) response. Prolonged ERS triggers the unfolded response (UPR) and eventually apoptosis. However, cancer cells and their prosurvival TME usually adapt through various mechanisms, including epithelial-mesenchymal transition (EMT), to evade ERS-induced apoptosis, and so continue to proliferate disproportionately, leading to relapse. P-gp and many other ABC drug transporters are associated with resistance to chemotherapy in NB. P-gp protects drug-resistant tumour cells from multiple forms of caspase-dependent apoptosis. As a glycoprotein, P-gp is also subject to protein N-glycosylation. It has been reported that inhibition of N-linked glycosylation of P-gp results in a reduced MDR phenotype. Inhibitors of N-glycosylation have been widely used to delineate the significance of glycosylation in MDR and the malignant phenotype. Targeting of the ERS and UPR has been the subject of intense investigation in recent years. A fundamental tenet of the present study is that, transcriptionally, MYCN (a hallmark of NB) directly regulates the ABC transporters, and their upregulation correlates with poor prognosis. Most aggressive NBs exhibit MDR, attributable to p53 mutations and/or a loss of function induced during chemotherapy, which promote relapse. p53 is a stress protein activated in response to various stresses and, in different contexts, the activation of p53 can either keep cells alive (the survival pathway) or kill the cells (the death pathway). SK-N-BE(2) NB cells, selected for this study, also overexpress P-gp. In the current study, selected glycosylation inhibitors and ERS inducers—tunicamycin (an antibiotic that blocks the rate-limiting step of protein N-glycosylation and induces ERS and apoptosis in cells), deoxynojirimycin (inhibits Golgi mannosidases and thus maturation of glycoproteins, causing ERS), celecoxib (a potent cyclooxygenase 2 inhibitor, known to impede sarcoplasmic/endoplasmic reticulum calcium ATPase/SERCA) and so triggering relentless Ca2+ dysregulation that can induce ERS-mediated apoptosis), swainsonine (a glycosidase inhibitor resulting in the accumulation of glycoproteins, hence causing ERS and initiating the vii http://etd.uwc.ac.za/ ABSTRACT UPR and apoptosis),