Luận án Tiến sĩ về nọc ong và melittin trong điều trị ung thư vú

Honeybee venom and melittin for the treatment of HER2-enriched and triple-negative breast cancer Ciara Evelyn Lilly Duffy Bachelor of Science (Honours) This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia School of Human Sciences 2020 Abstract Breast cancer is the most commonly occurring cancer in women worldwide. There are currently no effective targeted therapies available for triple-negative breast cancer (TNBC), which is aggressive, proliferative and associated with poor prognosis. The epidermal growth factor receptor (EGFR) is commonly overexpressed in TNBCs, and is important for cell proliferation and survival, conferring oncogenic signalling often dependent on the PI3K/Akt pathway downstream. To date, blocking EGFR signalling in TNBC has demonstrated limited clinical efficacy and the development of drug resistance.

Although therapies targeting human epidermal growth factor receptor 2 (HER2) in HER2-enriched breast tumours have dramatically improved median survival in the metastatic setting, resistance is almost inevitable for this subtype as well. Clearly, the discovery of more effective and selective therapeutic strategies for these cancers is an area of prime importance in clinical oncology. Venom from the European honeybee (Apis mellifera) and melittin, the major peptide in the venom, possess anti-cancer activity particularly by disrupting the plasma membrane. However, the precise molecular mechanisms explaining the anti-cancer activity of these agents against malignant breast cells are unknown.

The objective of this PhD was to determine the therapeutic potential of honeybee venom and melittin to target and kill aggressive breast cancer cells. The first aim was to determine the molecular mechanisms of breast cancer cell death due to honeybee venom and melittin. The second aim was to enhance the breast cancer selectivity of melittin and assess the interaction of melittin with growth factor receptors overexpressed in breast cancer cells. The final aim was to combine melittin with docetaxel in vivo to assess whether there is a synergistic interaction.

The anti-cancer properties of both honeybee venom and melittin, and selectivity towards TNBC and HER2-enriched breast cancers, were assessed by investigating the cellular response in a panel of breast cancer and normal-like cell lines. The results revealed that honeybee venom and melittin significantly and selectively reduce the viability of TNBC and HER2-enriched breast cancer cells and induce cell death through membrane disruption and caspase 3-mediated apoptosis. Melittin modulates oncogenic Akt and MAPK signalling in breast cancer cells by suppressing the activation of EGFR and HER2. Finally, melittin is synergistic with docetaxel both in vitro and in vivo, reducing cancer cell proliferation and consequent tumour growth in an aggressive TNBC allograft model.

Overall, this PhD demonstrates that honeybee venom and melittin potently and rapidly reduce breast cancer cell viability and can be used with chemotherapies or other small molecules to treat highly aggressive subtypes of breast cancer. Table of contents CHAPTER 1: General introduction and literature review .1 The intrinsic molecular subtypes of breast cancer .2 Limitations of current treatments for TNBC .3 Honeybee venom and melittin .4 The anti-cancer properties of bee venom and melittin .5 Targeting melittin, combination with chemotherapy and the use of nanotechnology to deliver melittin to cancer cells .6 Purpose and significance of this research .7 Aims and location in thesis .12 CHAPTER 2: Honeybee venom and melittin induce selective, potent, and rapid breast cancer cell death .1 Honeybees and bumblebees .2 Composition of honeybee venom compared to bumblebee venom .3 The application of bee venom and melittin in cancer .4 The cancer cell selectivity of honeybee venom and melittin .1 Chemical reagents and antibodies .2 Bee venom collection .4 Cell viability assays .5 Production of the primary monoclonal antibody against melittin .6 Enzyme-linked immunosorbent assay (ELISA) .7 Anti-melittin antibody competition experiments .8 Western blot for the detection of Cleaved Caspase-3 .10 Live cell microscopy .11 Scanning electron microscopy .1 Honeybee venom and melittin reduced tumour cell viability in triple-negative and HER2-enriched breast cancer cells .2 Honeybee venom reduced breast cancer cell viability with high affinity compared to bumblebee venom .3 Melittin was present in the same relative abundance in the venom of different honeybee populations, and mediated most of the anti-cancer effects .4 Honeybee venom and melittin induced caspase 3-mediated apoptosis, rapid cell death, and membrane disruption in breast cancer cells .5 Honeybee venom and melittin induced membrane disruption and nuclear condensation in breast cancer cells. 52 CHAPTER 3: Enhancing the breast cancer selectivity of melittin with RGD, and assessing the mechanisms of the suppression of EGFR and HER2 phosphorylation by melittin .1 Altering the charge of melittin to modulate the lytic effect .2 Increasing the cancer selectivity of melittin .3 Suppressing the phosphorylation of EGFR and HER2, and modulating downstream oncogenic signalling pathways in breast cancer cells .4 Utilising bioluminescence resonance energy transfer to determine the interaction of melittin with growth factor receptors .2 Chemical reagents and antibodies .3 Bee venom collection .5 Cell viability assays .6 Production of the primary monoclonal antibody against melittin .7 Enzyme-linked immunosorbent assay (ELISA) .8 Western blot for the detection of Cleaved Caspase-3 .10 Western blot for the detection of receptor phosphorylation and downstream pathway analysis .11 Bioluminescence resonance energy transfer (BRET) .1 The positive charge of melittin was essential for the anti-cancer effect, and the breast cancer selectivity of melittin was enhanced with RGD .2 The anti-melittin antibody recognised a conformational epitope of melittin that was not disrupted by the engineered RGD motif .3 Melittin was localised in the plasma membrane of breast cancer cells overexpressing EGFR or HER2 growth factor receptors .4 Honeybee venom and melittin suppressed the ligand-induced phosphorylation of EGFR and HER2 in breast cancer and modulated downstream signalling pathways in a time dependent manner .5 Melittin interacted with EGFR in the plasma membrane in a time and concentration dependent manner .6 Melittin indirectly interfered with RTK phosphorylation. 100 CHAPTER 4: Honeybee venom and melittin combined with docetaxel synergistically reduced breast cancer growth in vitro and in vivo .1 The mechanism of action of docetaxel and cisplatin .2 Limitations of current chemotherapy treatment .3 Combining honeybee venom and melittin with chemotherapy .4 Immune system modulation by melittin.1 Chemical reagents and antibodies .2 Bee venom collection .4 Cell viability assays .5 Production of the primary monoclonal antibody against melittin .6 Analysis of combined drug effects .7 Animal model and treatments .8 Immunohistochemical analysis of the tumours .1 Honeybee venom and melittin were synergistic with docetaxel in treating highly aggressive claudin-low breast cancer cells in vitro .2 The combination of melittin and docetaxel significantly reduced tumour growth in a highly aggressive claudin-low breast cancer model in vivo .3 The combined treatment of melittin and docetaxel reduced proliferation and induced apoptosis in claudin-low breast cancer in vivo.

128 CHAPTER 5: General discussion and future directions .1 Summary of results and significance. A1 Appendix A: Peer-reviewed publications. A1 Appendix B: List of conferences, symposiums, awards and courses. A2 Abbreviations Akt Protein kinase B ANOVA Analysis of variance BRET Bioluminescence resonance energy transfer BSA Bovine serum albumin CERI Centre for Entrepreneurial Research and Innovation CI Combination Index CIBER Center for Integrative Bee Research CO2 Carbon dioxide CPP Cell penetrating peptide CTG CellTiter-Glo DMEM Dulbecco’s Modified Eagle’s Medium DNA Deoxyribonucleic acid EGF Epidermal growth factor EGFR Epidermal growth factor receptor EMBL European Molecular Biology Laboratory F-12 Ham’s F-12 Nutrient Mixture FBS Fetal bovine serum FHS Fetal horse serum FITC Fluorescein isothiocyanate GLM Generalised linear model HER2 Human epidermal growth factor receptor 2 IC50 50% inhibitory concentration JAK2 Janus Kinase 2 MAPK Mitogen-activated protein kinase MEM α Minimal Essential Media α MMP2 Matrix metalloproteinase-2 mTOR Mammalian target of rapamycin NF-κB Nuclear factor-kappa B NSCLC Non-small-cell lung carcinoma PBS Phosphate-buffered saline PI3K Phosphatidylinositol 3-kinase PLA2 Phospholipase A2 RGD Arginine-glycine-aspartic acid motif RNA Ribonucleic acid RPMI Roswell Park Memorial Institute STAT3 Signal Transducer and Activator of Transcription 3 SV40 Simian virus 40 TBST Tris-buffered saline with Tween 20 TNBC Triple-negative breast cancer TRAIL Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand uPA Urokinase plasminogen activator USGS United States Geological Survey VEGF Vascular endothelial growth factor VEGFR-1 Vascular endothelial growth factor receptor 1 VEGFR-2 Vascular endothelial growth factor receptor 2 Acknowledgements Thank you to everyone who has been a part of my PhD.

This has been an incredible journey and a transformational time of my life. To my supervisors, Associate Professor Pilar Blancafort, Professor Boris Baer, Dr Anabel Sorolla, and Professor Killugudi Swaminathan Iyer; I am tremendously thankful for all that you have taught me, and all of the opportunities you gave me. Thank you for enabling me the freedom to pursue my own scientific ideas and avenues of research. I would like to thank everyone who I have had the pleasure of working alongside in the Blancafort Laboratory at the Harry Perkins Institute of Medical Research.

In particular, I would like to acknowledge Anabel Sorolla and Edina Wang; I have learnt a great deal from both of you. Thank you to Colette Moses, for your constant guidance and support throughout my research journey. Thank you to the Centre for Integrative Bee Research (CIBER) team, especially Tiffane Bates, for providing training and access to the honeybees at the UWA apiary. I would also like to thank my wonderful mentors; Mark Cregan, Miriam Borthwick, Kevin Pfleger, Matt Oldakowski, Intan Oldakowska, Carolyn Williams and Charlie Bass.

Thank you for your invaluable training, advice and encouragement. Thank you to my international research collaborators; Professor Jane Stout (Trinity College Dublin) and Professor Mark Brown (Royal Holloway University of London). I would like to sincerely thank all of the following researchers and/or beekeepers who helped me to access samples of different bee populations in Ireland and England; Saorla Kavanagh, Keith Pierce, Marcus Phelan, Susie Bioletti, David Morris, Terry Meakin, Martina Halpin, Edward Hill, Fabio Manfredini and Callum Martin. Thank you to those who assisted in the venom transport across the world, including Sylvia Mathiasen, Siobhán McNamee and Rob Prouse.

Thank you to Alison Boyce and Steve Portugal for assisting me with accessing and setting up the carbon dioxide for bee venom collection. This research was supported by an Australian Government Research Training Program (RTP) Scholarship, and a PhD Top Up Scholarship from the Cancer Council of Western Australia. Technical assistance was kindly provided by Kathleen Davern for the development of the anti-melittin antibody (Chapter 2); Diwei Ho, in assisting with setting up the scanning electron microscopy experiment (Chapter 2); Emily Golden, in assisting with the peptide modelling (Chapter 3); Elizabeth Johnstone, in helping set up the BRET assay (Chapter 3); and Anabel Sorolla and Edina Wang, for assisting with the in vivo experiment (Chapter 4). I would also like to thank Paul Rigby (CMCA) and Kevin Li (FACS Facility) at the Harry Perkins Institute of Medical Research.

Lastly, thank you to all of my friends for your unwavering encouragement, to my beautiful family, and to Quentin, for your unconditional support throughout this journey. Mum and Dad, I truly cannot thank you enough. It is to you I dedicate my thesis. Chapter 1 General introduction and literature review 1.1 The intrinsic molecular subtypes of breast cancer Breast cancer is a devastating disease caused by a combination of environmental and genetic factors that trigger damage to the cellular DNA 1, is the second most common cancer in the world, and the most commonly occurring cancer in women 2,3.

In Australia, one in eight women will be diagnosed with breast cancer. Genome-wide profiling has identified several intrinsic molecular subtypes of breast cancer using patient samples to correlate the characteristics of the tumours to clinical outcome. These molecular subtypes include luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, basal-like, claudin-low, and normal-like 4–6. These subtypes differ greatly in terms of their transcriptional profiles, receptor expression, unique DNA methylation patterns, response to therapy and patient survival.

Targeted therapies have yet to be established with clinical success for basal-like and claudin-low tumours, which are immunohistochemically characterised as triple- negative breast cancer (TNBC), lacking the expression of estrogen and progesterone receptors, as well as HER2 6.