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Regulation and Implication in Breast Cancer

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Regulation and Implication in Breast Cancer Tumour promoting HER2 splice variant Δ16HER2: Regulation and implication in breast cancer Anna-Lena Dittrich 2/22/2017 A thesis submitted for the degree of Doctor of Philosophy i Abstract Results from high-throughput studies of the last decade have shown an important connection between regulated pre-mRNA splicing and tumorigenesis as well as therapy resistance. To date only relatively few such interactions have been studied in depth. The human epidermal growth factor receptor 2 (HER2) is an important biomarker in cancer, especially in 20-30% of breast cancer cases. This subset of breast cancer patients are treated with HER2-targeting therapies in addition to chemotherapy. Although these therapies are very successful, in a subset of patients with primary tumours as well as in advanced metastatic breast cancer resistance occurs. The underlying mechanisms are not well understood; one potential cause is the alternative splicing of HER2 pre-mRNA yielding protein variants which can evade or prevent action of these therapeutics. This PhD thesis details progress in understanding the mechanisms regulating the production of the HER2 variant Δ16HER2, which has been shown to have high oncogenic potential. Four key splicing factors that regulate this splicing event were identified as well as a specific RNA:protein binding site in the Δ16HER2 splicing region. In addition, a potential novel HER2 mRNA transcript was identified involving retention of intron 25 (termed I25HER2). I25HER2 was shown to be expressed in both normal human tissues and breast cancer tissue samples. Interestingly, the expression level of I25HER2 is not proportional to wild type HER2 in all tissues. Although, I25HER2 would be expected to undergo nonsense-mediated decay, this study indicated that I25HER2 mRNA has similar stability to Δ16HER2 mRNA. An ongoing challenge in both clinical and non-clinical studies is the inability to distinguish different HER2 protein variants. Initial results presented in this thesis, from a comparative study between immunohistochemistry (using three different HER2 antibodies, including the clinically used HercepTestTM) and HER2 gene amplification (using HER2 CISH pharmDxTM) in a cohort of primary human ductal carcinomas showed a subset of discordant cases. In a few of these tumours elevated levels of HER2 splice variants could be detected. The ii possible implication for successful treatment of these patients’ highlights the importance of linking the basic processing of HER2 transcripts and protein variants with pathological information from patients. iii Acknowledgement "A journey of a thousand miles begins with a single step" by Lao Tsu (604 BC - 531 BC) I came across a poster with this quote in the first weeks of my PhD and it has kept me company throughout these three years. Step by step I completed this journey and with every step came new challenges, but also help from many wonderful new people. First I would like to thank my supervisors, Dr Alison Tyson-Capper for giving me this opportunity, encouraging me and giving me the chance to explore my own ideas, and my second supervisor Dr Hannah Gautrey for her hands on help with my research and encouragement. This work would not have been possible without the breast tumour samples provided by the Breast Cancer Now Tissue Bank and the help of Dr Helen Kourea, with her experience in clinical pathology. My thanks also go to Dr John Brain for letting me use his microscope. Next I have to thank my annual review team Dr Jeremy Brown and Dr Xiao Wang for ripping my work apart to find any weakness, to challenge and encourage me to bring the best out of my work. I couldn't imagine what this time would have been without the other members of our lab. First of all, Marco Silipo who started and finished his PhD with me. He was always there to bounce ideas off, to give a helping hand and to cheer me up. Helen Lawrence and Soulaf Kakel who were great colleagues and friends, as well as all our BSc and MSc students who brought in fresh air, some challenges but also some really good fun. I had the most amazing time not last due to the John Kirby lab who basically adopted me. Letting me borrow reagents and equipment, inviting me to their Christmas parties as well as the lab drinks. You all were great colleagues to work with and amazing friends: Bea, Ben, Cat, Irene, Jose, Laura, Nina, Rachel, Rishab, Shameem and Tom. The people that can't be forgotten for the lovely company they were and the great source of knowledge, the wonderful team of technicians on our floor: Barbara, Grant, Julie, Kate, Nikki and Rachel. iv Halfway along this journey I found a wonderful man who has stayed with me through all my ups and downs. Thank you for being there Mike! Even across 1000s of miles I have been supported by my best friend. Thanks a ton Tita! But nothing, not the first and surely not the last step on this journey to my PhD would have been possible without my family. Mama, Papa, Alex, Ihr seid die Besten! v Table of contents Abstract ............................................................................................................... ii Acknowledgement .............................................................................................. iv Abbreviations .................................................................................................... xii List of Figures ................................................................................................... xv List of Tables ........................................................................................................ i Chapter 1: Introduction .................................................................................. 1 1.1. The Human Breast ................................................................................. 1 1.2. Breast cancer ......................................................................................... 3 1.2.1. Breast cancer epidemiology ............................................................ 3 1.2.2. Breast cancer risk factors ................................................................ 3 1.2.3. Breast cancer categorisation ........................................................... 5 1.3. Oestrogen receptor ................................................................................ 9 1.4. Progesterone receptor ......................................................................... 11 1.5. Human epidermal growth factor receptor 2 .......................................... 13 1.5.1. The regulation of HER2 gene expression ...................................... 13 1.5.2. HER2 transcript and protein variants ............................................. 15 1.5.3. The protein structure of HER2 ....................................................... 16 1.5.4. HER2 signalling pathways ............................................................. 20 1.5.4.1. Co-receptors of HER2 ................................................................ 22 1.5.4.2. The RAS/MAPK signalling pathway ........................................... 23 1.5.4.3. The PI3K-AKT signalling pathway .............................................. 24 1.5.5. The crosstalk between HER2 and the hormone receptors ............ 26 1.5.6. Function of nuclear HER2 ............................................................. 28 1.6. Breast cancer therapy .......................................................................... 29 1.6.1. HER2 targeting therapy ................................................................. 31 vi 1.7. Eukaryotic mRNA processing .............................................................. 34 1.7.1. The importance of pre-mRNA splicing ........................................... 35 1.7.2. Basic mechanisms of splicing........................................................ 37 1.7.3. Assembly and function of the spliceosome ................................... 38 1.7.4. Regulators of alternative splicing ................................................... 40 1.8. Alternative splicing and cancer ............................................................ 43 1.9. HER2 splice variants and their role in cancer ...................................... 44 1.10. Anti-HER2 therapy resistance: Focus on HER2 splice variants ........ 48 1.11. Rationale, hypotheses and aims ....................................................... 51 1.11.1. Study rationale ........................................................................... 51 1.11.2. Hypotheses ................................................................................ 51 1.11.3. Aims .............................................................................................. 52 Chapter 2: General materials and methods ................................................. 53 2.1. Cell lines .............................................................................................. 53 2.2. Cell culture ........................................................................................... 53 2.2.1. Cell line maintenance .................................................................... 54 2.2.2. Mycoplasma testing ....................................................................... 54 2.2.3. Cryopreservation of cells ............................................................... 55 2.2.4. Cell counting.................................................................................. 55 2.2.5. Transient gene knockdown by siRNA transfection ........................ 56 2.3. Human breast tissue ...........................................................................
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