HER Family Receptor Activation and Dimerisation in Colorectal Cancer and Cancer-Derived Exosomes
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HER family receptor activation and dimerisation in colorectal cancer and cancer-derived exosomes Fangfei Gao A thesis submitted to the University College London for the degree of Doctor of Philosophy December 2019 Department of Oncology UCL Cancer Institute University College London 72 Huntley Street, London, WC1E 6DD, UK 1 DECLARATION I, Fangfei Gao confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in this thesis. 2 ABSTRACT The EGFR pathway is upregulated in several human cancers including colorectal and head and neck cancers. The anti-EGFR antibodies cetuximab and panitumumab are currently widely used in the management of metastatic colorectal cancer with limited duration of response and almost inevitable development of resistance. HER receptors are able to form alternative dimers and can therefore compensate the loss of function of one receptor during targeted therapies. Usage of pan-HER inhibitor such as AZD8931 has been trialed and may represent one way of overcoming resistance to anti-EGFR therapy. However the effects of anti-HER therapy in patients cannot be easily measured without tissue biopsy. Analysis of exosomes offers a potential platform for the longitudinal monitoring of HER signalling in the form of a liquid biopsy by examining their protein and miRNA contents. Foster Resonance Energy Transfer (FRET) assay using fluorescence lifetime imaging microscopy (FLIM) was used for quantitative analysis of HER dimerisation in colorectal cancer cells. Treatment with cetuximab resulted in increase in HER3 phosphorylation and HER2-HER3 heterodimerisation in the sensitive cell line LIM1215, but not in resistant line DLD1 (KRAS WT). Treatment with AZD8931 resulted in reduced phosphorylation in HER1, HER2 and HER3 in both cell lines, and similar HER dimerisation changes as with treatment with cetuximab. HER3 activation and HER2- HER3 dimer rewiring upon anti-HER therapy could contribute to treatment resistance in colorectal cancer cells. Cell-derived and circulating exosomes were isolated using differential ultracentrifugation, and characterised using Nanosight and immunostaining. Reverse phase protein array provided an alternative high-throughput platform to dot blot for exosomal protein analysis but requires further optimisation. Exosomal HER protein quantity reflected HER expression in vitro. In a cohort of patients with advanced colorectal cancer patients undergoing first line systemic therapy, circulating exosomal HER3 changes predicted treatment failure prior to radiological progression. In another cohort of patients receiving first line AZD8931, early changes in exosomal HER2-HER3 dimer may be predictive of response to anti-HER therapy. Furthermore, levels of exosomal miR-21 could be used for monitoring of treatment response in patients with colorectal cancer. 3 IMPACT STATEMENT Colorectal cancer is the third most common cancer in the world. Despite recent therapeutic advances the disease still causes more than 650,000 deaths worldwide each year. While at early stages surgery is curative, the prognosis for patients with advanced disease and metastatic spread remains bleak with five-year survival rates of 5%. The standard first line systemic therapy for patients with metastatic colorectal cancer without RAS mutation consist of combination chemotherapy with anti-EGFR therapy such as cetuximab or panitumumab. Resistance to anti-EGFR therapy develop due to a number of mechanisms, one of which is through oncogenic receptor signal rewiring. The selection of patients who are likely to benefit from treatment with anti- HER therapy remains controversial. CEA is the only circulating biomarker used in clinic for monitoring colorectal cancer burden but has low sensitivity and specificity. There is increasing demand for better prognostic and predictive biomarkers that can be obtained non-invasively for monitoring treatment response. In this project, we demonstrated the successful use of FRET/FLIM to examine HER dimer interaction in colorectal cancer cells and circulating exosomes, and found HER2- HER3 heterodimer can be modulated upon anti-HER treatment, suggesting the importance of HER dimerisation status in early tumour evolution. We optimised techniques for proteomic and genetic analysis of cell-derived and circulating exosomes, and identified several exosomal protein and miRNA biomarkers correlating to HER signalling that could be predictive of treatment failure. The identification of exosomal protein, miRNA and dimer biomarkers that are predictive of treatment response will allow better stratification of patients to therapy and early switch to alternative treatment prior to later radiological detection of cancer progression. The validation of these biomarkers will enable longitudinal monitoring of patients through therapy in the form of liquid biopsies rather than current practice of invasive tissue biopsies. The techniques we optimised to analyse HER dimerisation and exosomal contents could be adopted by the research community to address other research questions such as protein interactions within the exosomes, whether certain proteins affect an outcome in a certain group of patients. As exosomes are implicated in many physiological and pathological processes including cancer, cardiac diseases, neurodegenerative disorders, the potential application could be arched through different specialities. Refinement of our methods on exosome works could lead to 4 commercialisation of blood tests that give hints to various disease status or prognosis stratification. 5 ACKNOWLEDGEMENTS I would like to thank my supervisors Prof. Daniel Hochhauser and Prof. Tony Ng for their guidance and support during this long journey and for giving me the opportunity to carry out my PhD studies in their laboratory. I wish to show appreciation to Prof. John Hartley for his encouragement along the way and identifying me as part of the group. This study would not been possible without the generous funds from the Comprehensive Cancer Imaging Centre and Cancer Research UK. I would like to thank all members of the CRUK Drug-DNA interactions research group at UCL Cancer Institute, and the Dimbleby department and Randall Division at KCL for their endless help and support, with special thanks to Dr Paul Barber, Dr Gregory Weitsman, Dr Giovanna Alfano, Dr Juanjo Garcia Gomez, Dr Jose Miguel Vicencio Bustamante, Dr James Monypenny, Dr Valenti Gomez, Dr Sylwia Jones, Dr Arola Fortian Bernabeu, Dr Arman Esfandiari, Dr Oana Coban, Dr Miguel Esteras, and Dr Valeria Santoro, for their scientific support and patience. Thanks to Dr Shih-hsin Chen, Dr Myria Galazi, Avigayil Chalk, Simon Corbett, Jo Clancy, Dr Ruhe Chowdhury, Dr Kenrick Ng for their friendship and advice sharing this PhD journey. Thanks to Prof Neil Carragher and Mr Kenny McCleod from Edinburgh University for their help with setting up the Reverse Phrase Protein Array. Finally, thanks to my family, my husband, my parents, and my baby girl, for their unconditional love and everlasting support. 6 Contents DECLARATION 2 ABSTRACT 3 IMPACT STATEMENT 4 ACKNOWLEDGEMENTS 6 TABLE OF CONTENTS 7 LIST OF FIGURES 12 LIST OF TABLES 16 LIST OF ABBREVIATIONS 17 Chapter 1............................................................................................................................... 22 Introduction .......................................................................................................................... 22 1.1 Colorectal cancer overview ................................................................................... 23 1.2 Pathogenesis of colorectal cancer and molecular subtypes ....................... 24 1.3 Chemotherapy and combination regimens in colorectal cancer ................ 26 1.3.1 5-Fluorouracil .................................................................................................... 26 1.3.2 Oxaliplatin ........................................................................................................... 27 1.3.3 Irinotecan ............................................................................................................ 28 1.3.4 Combination chemotherapy and sequencing ........................................... 29 1.3.5 Other cytotoxic agents .................................................................................... 29 1.4 Biological targeted agents in colorectal cancer .............................................. 30 1.4.1 Anti-EGFR therapies ........................................................................................ 30 1.4.2 Anti-VEGF strategies ....................................................................................... 31 1.5 The Human Epidermal Growth Factor Receptor Family ................................ 31 1.6 The epidermal growth factor receptor (EGFR) ................................................. 35 1.7 HER receptors as biological targets in colorectal cancer ............................ 37 1.7.1 Cetuximab ........................................................................................................... 38 1.7.2 Panitumumab ..................................................................................................... 40 1.7.3 Clinical activities of cetuximab and panitumumab in mCRC ................ 40 7 1.8 Mechanisms of resistance to current anti-EGFR therapy ............................. 41 1.8.1 RAS status in predicting response to anti-EGFR agents ...................... 41