Bangor University DOCTOR OF PHILOSOPHY Identification and characterisation of novel cancer testis antigens in human cancer cells Alsiwiehri, Naif Award date: 2017 Awarding institution: Bangor University Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 08. Oct. 2021 Identification and characterisation of novel cancer testis antigens in human cancer cells Naif O. Alsiwiehri PhD Thesis 2017 NWCRFI School of Biological Sciences Bangor University Abstract Carcinogenesis is a multi-step process which involves genomic instability and abnormal cellular growth over long period of time which eventually develop tumour. Cancer testis antigens consistently reported in many types of cancer which suggest its oncogenic role. But, it’s functional role in cancer still unknown and need further investigation. Also, cancer testis antigens might be used as potential targets for cancer immunotherapy due to their main presence in normal testis cells and abnormally exist in several types of human cancer. Their aberrant expression in cancer makes them useful to target human malignancies by immunotherapy. TEX19 one of the novel CT gene was identified in this study. TEX19 show interesting expression pattern due to its confined in normal testis and expressed in many cancer tissues. This expression may indicate the oncogenic activity of TEX19 gene in somatic tissue. Thus, the possible approach to use this gene as target for cancer immunotherapy and cancer prognosis and diagnosis. In addition, TEX19 might play a role in stemness state, this was observed in TEX19 depleted cells, the stem cell markers genes such as OCT$, NANOG, SOX2 were affected after the knockdown of TEX19. However, Tex19 was not affected after differentiating NT-2 cancer cells using Retinoic acid and HMBA. We also studied the relation between TEX19 and transposable genetic elements by knockdown TEX19 in NT-2 and A2780 cancer cells. The qRT-PCR results show that human ERVK family gene expression was affected by TEX19 depletion. Moreover, our RNA sequencing data of TEX19 depleted sample and qRT-PCR analysis reveal the influence of TEX19 depletion on some differentially expressed genes. Interestingly, these genes were upregulated and down regulated after the depletion of TEX19 in four types of human cancer cell lines. Acknowledgment This work undergoes in NWCRFI at School of Biological Sciences of Bangor University, and funded by Taif University, MOHE of Saudi Arabia. My deep and sincere thanks to my supervisor Dr Ramsay MacFarlane for his help, continuous support and patient. This work couldn’t be achieved without his help, guidance and enriched knowledge. My thanks to all staff and colleagues within our group, for their help and support, thanks to Dr Khaled Alzeer for teaching me some techniques and methods in the laboratory. I would like to thank Mr Vicente Planells Palop for his help in RNA sequencing work. My thanks to Dr. Julia Feichtinger for her help in bioinformatics part of the work. My thanks to Dr Edgar Hartsuiker for helping me in DSB assay. Thanks to Dr Faisal Alzahrani for helping me with IPSCs. I would like to thank the lovely people of Felinheli for the good time and lovely memories that I will carry it back home with me. Thank to our locals in Tafarn Fic. and Plas Hotel Dinorwic. My great thanks and respect to my godfather Mr KennethWalker for all good times help and support. Finally, I would like to thank my parents for their influence on my education and career since I was young. Special thanks to my family particularly my wife Ejlal for her patient, support and devotion through the last years of my study. In addition, to my little angels Judy, Wajed, Hala and Yusur you are the one keep me going. Contents 1. Introduction ........................................................................................................................................ 1 1.1 Human malignancies ..................................................................................................................... 1 1.1.1 Carcinogenesis ........................................................................................................................... 1 1.1.3 Epigenetic origin of cancer ........................................................................................................ 3 1.1.3.1 DNA methylation: ................................................................................................................... 4 1.1.3.2 Histone Modifications: .......................................................................................................... 10 1.2.1 Meiotic homologous recombination ........................................................................................ 13 Initiation and repair of meiotic DSBs ............................................................................................... 15 1.3 Cancer Testis (CT) antigens ........................................................................................................ 17 1.3.2 Identification of CTAs ............................................................................................................. 18 1.3.3 Classification and expression of CTAs .................................................................................... 19 1.3.4 Function of CT genes ............................................................................................................... 20 1.4 Cancer immunotherapy ............................................................................................................... 22 1.4.2 Ipilimumab and melanoma....................................................................................................... 24 1.5 Transposable genetic elements.................................................................................................... 26 1.5.2 Classification of TEs ................................................................................................................ 26 1.5.3 Retrotransposons in cancer ...................................................................................................... 28 1.6 Stem Cells ................................................................................................................................... 28 1.6.2 Stem Cells Markers .................................................................................................................. 29 1.6.3 Stem Cells Differentiation Therapy ......................................................................................... 30 1.7 Aims and objectives .................................................................................................................... 31 2. Material and methods ...................................................................................................................... 32 2.1 Human cancer cell-lines source .................................................................................................. 32 2.2 Storage of cancer cell-lines ......................................................................................................... 32 2.3 Thawing of frozen cell-line storage ............................................................................................. 32 2.4.1 Western blot ............................................................................................................................ 33 2.4.2 Whole cell protein extraction .................................................................................................. 33 2.4.3 Nuclear and cytoplasmic protein extraction ............................................................................ 33 2.4.4 Western blotting protocol ....................................................................................................... 33 2.5 RNA isolation ............................................................................................................................... 34 2.6 cDNA synthesis ............................................................................................................................ 35 2.7 Polymerase chain reaction (PCR) ................................................................................................ 35 2.8 DNA purification and sequencing ............................................................................................... 37 2.9 Quantitative real time PCR.......................................................................................................... 38 2.10 Knockdown gene by RNA interference ..................................................................................... 38 2.11 Extreme limiting dilution analysis (ELDA) ................................................................................. 39 2.12 Detection of SPO11 covalent bound to DNA