Mapping the intracellular molecular mechanisms of chemokine signalling within cancer Gerald Roman Sion Keil A thesis presented for the degree of Doctor of Philosophy at the University of East Anglia, School of Pharmacy March 2019 "This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived therefrom must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution.” Abstract Chemokines are extracellular signalling molecules which function as chemoattractants for leukocytes by directing their migration towards sites of inflammation as part of the immune response. On epithelial cells chemokine receptor expression is normally low or absent. However during cancer progression, chemokine receptors can become overexpressed on cancer cells, whilst chemokines are frequently present at sites of metastasis. Consequently aberrant chemokine signalling is associated with both metastasis and a poor prognosis in cancer patients. The chemokine signalling network is therefore consider a potential therapeutic target for cancer treatment. The aim of the research undertaken in this thesis was to identify novel therapeutic targets involved in chemokine downstream signalling in cancer cells. To investigate the chemokine downstream signalling pathway, a number of different chemokines and small molecules were used with their effects on cellular intracellular calcium signalling and migration of different leukemic and carcinoma cells assessed. The findings from the screen identified a role for CCL2 and CCL3 signalling in the migration of PC-3 and MCF-7 cells respectively. In MCF-7 cells, CXCL12 intracellular calcium signalling was shown to be dependent on Gαi, Syk/Src, c- Raf, DOCK1/2/5 and Arp2/3. With DOCK1/2/5 also shown to be essential for CCL3 and CXCL12 intracellular calcium signalling in both MCF-7 and THP-1 cells, as well as for CXCL12 chemotaxis of Jurkat cells. For Arp2/3 its importance in chemokine signalling was specific to MCF-7 cells, whilst the roles of the microtubules and FAK were dependent on both the chemokine and cell type. In this thesis DOCK1/2/5 was identified as a novel target for blocking leukemic T-cell migration (Jurkats) in response to CXCL12. In addition, Arp2/3 and the microtubules were implicated in chemokine signalling and therefore would warrant further investigation to establish their importance in cancer cell migration. 2 Contents Abstract………………………………………………………………………….....2 List of Figures..............................................................................................10 List of Tables……………………………………………………………………..18 Acknowledgements…………………………………………………….............19 Chapter 1.0 Introduction……………………………………………………......20 1.1 Cellular Signalling .................................................................................. 20 1.2 G-protein Coupled Receptors ................................................................. 21 1.2.1 Structure .......................................................................................... 21 1.2.2 Classification.................................................................................... 23 1.2.3 Heterotrimeric G-protein Signalling .................................................. 23 1.3 Chemokine Receptors ........................................................................... 25 1.4 Chemokines ........................................................................................... 26 1.4.1 Chemokine Families ........................................................................ 27 1.4.2 Chemokine Structure ....................................................................... 28 1.5 Gαi\βγ Downstream Signalling ............................................................... 29 1.6 Biased Signalling ................................................................................... 30 1.7 Chemokine and Chemokine Receptor Oligomerisation ......................... 32 1.8 Chemokine Biological Activity ................................................................ 33 1.9 Cell Migration ......................................................................................... 35 1.9.1 Cell Migration Modes ....................................................................... 35 1.9.2 Actin Polymerisation ........................................................................ 37 1.9.3 Leading Edge (Extension) ............................................................... 39 1.9.3.1 Filopodia .................................................................................... 39 1.9.3.2 Lamellipodia .............................................................................. 40 1.9.3.3 Cellular Polarity ......................................................................... 40 3 1.9.4 Focal Adhesions (Traction) .............................................................. 42 1.9.4.1 Integrins ..................................................................................... 43 1.9.4.2 Focal Adhesion Kinase .............................................................. 43 1.9.4.3 Focal Adhesion Maturation ........................................................ 45 1.9.5 Trailing Edge (Retraction) ................................................................ 46 1.10 Cancer Metastasis ............................................................................... 47 1.11 Chemokines and Cancer Metastasis ................................................... 51 1.12 Chemokine Signalling: A Therapeutic Target for Cancer Treatment…………………………………………………………………………..53 1.12.1 Small Molecules Targeting Chemokine Signalling in Cancer Patients…………………………………………………………………………..53 1.12.2 Antibodies and Peptides Targeting Chemokine Signalling in Cancer Patients…………………………………………………….. ........................... 54 1.12.3 Challenges in Targeting Chemokine Signalling ............................. 56 1.13 Research Aim ...................................................................................... 60 Chapter 2.0 Materials and Methods……………………………….………….62 2.1 Tissue culturing ...................................................................................... 62 2.1.1 Reagents ......................................................................................... 62 2.1.2 Cell culture ....................................................................................... 62 2.1.3 Cell culture protocol ......................................................................... 63 2.1.4 Description of cell lines and subculture methods used .................... 64 2.1.4.1 MCF-7 cells ............................................................................... 64 2.1.4.2 MDA-MB-231 cells ..................................................................... 64 2.1.4.3 MIA PaCa-2 cells ....................................................................... 64 2.1.4.4 PC-3 cells .................................................................................. 65 2.1.4.5 CHO-CCR5 cells ....................................................................... 65 2.1.4.6 THP-1 cells ................................................................................ 66 2.1.4.7 Jurkat cells ................................................................................ 66 4 2.1.4.8 Summary of cell lines and subculture methods used................. 67 2.2 Chemokines ........................................................................................... 68 2.3 Antibodies .............................................................................................. 79 2.4 Cellular migration experiments ............................................................... 80 2.4.1 The wound healing assay ................................................................ 80 2.4.2 Agarose spot assay ......................................................................... 81 2.4.3 Transwell migration assay ............................................................... 82 2.4.3.1 Suspension cells........................................................................ 82 2.4.3.2 Adherent cells ............................................................................ 83 2.4.4 Time-lapse assay ............................................................................. 84 2.5 Cellular signalling experiment ................................................................ 84 2.5.1 Intracellular calcium flux assay ........................................................ 84 2.6 Cellular proliferation experiment ............................................................ 85 2.6.1 MTS cytotoxicity assay .................................................................... 85 2.7 Cellular imaging experiments ................................................................. 86 2.7.1 Immunofluorescence ....................................................................... 86 2.7.2 Phalloidin Staining ........................................................................... 87 2.7.3 Phalloidin Staining: ImageJ analysis ................................................ 88 2.8 Data and Statistical Analysis .................................................................. 88 Chapter 3.0 Screening for chemokine signalling pathways involved in carcinoma metastasis……………………………………………….………….89 3.1 Introduction ............................................................................................ 89 3.2