RACK1 facilitates the activity and substrate specificity of PP2A in breast cancer cells

iii ABBREVIATIONS . v Chapter 1:General Introduction. 1 1.1 Breast Cancer . 2 1.1.1 Breast Cancer Incidence . 2 1.1.2 Breast Cancer Classification . 5 1.1.3 Current Therapeutic Strategies . 7 1.2 Receptor for Activated C Kinase 1 (RACK1). 10 1.2.1 RACK1: Gene and expression . 10 1.2.2 RACK1: Protein structure . 11 1.2.3 RACK1: Role as a scaffolding protein . 11 1.2.4 RACK1, the IGF-IR and PP2A . 13 1.2. 5 RACK1: Role in cancer . 14 1.3 Protein Phosphatase 2A (PP2A) . 17 1.3.1 Role of phosphatases in cellular signalling . 17 1.3.2 PP2A: Structure and assembly . 17 1.3.3 PP2A: A key regulator in Growth Factor signalling . 22 1.3.4 PP2A: Role in cancer . 23 1.3. 5 PP2A: Therapeutic strategies . 31 1.4 Aims and objectives . 35 Chapter 2:Materials and Methods . 37 2.1 Materials . 38 2.1.1 General Chemicals and Reagents . 38 2.1.2 Plasmid stocks and source. 38 2.1.3 Antibodies . 39 2.1.4 Cell Culture Reagents . 39 2.1.5 Cell lines . 39 2.2 Methods. 40 2.2.1 Cell adhesion, spreading and proliferation using the RTCA xCELLigence system. 40 2.2.2 Generation of stable cell lines. 42 2.2.3 Preparation of cellular protein extracts . 42 2.2.4 Immunoprecipitation of proteins from cellular extracts . 43 2.2. 5 SDS-PAGE and Western Blotting . 43 2.2. 6 Immunofluorescence to detect protein co-localisation in cells . 44 2.2. 7 PP2A Phosphatase Activity Assay . 45 2.2. 8 Adhesion Assay. 45 2.2. 9 Migration Assay . 46 2.2.10 Invasion Assay . 46 2.2.11 Plating Efficiency Assay . 46 2.2.12 Soft Agar Assay . 48 2.2.13 Spot Synthesis of peptides and Overlay analysis . 48 2.2.14 Alanine substitution array analysis . 49 2.2.15 3D Cell Culture . 49 2.2.16 Mass Spectrometry Analysis to identify proteins interacting with the RACK1/PP2A complex. 50 2.2.17 Gene expression . 51 2.2.18 Gene knockdown. 52 2.2.19 Statistical Analysis . 52 Chapter 3:Utilization of a real time cell analysis tool to analyse the anti-cancer effects of compounds on breast cancer cells. 54 3.1 Abstract . 55 3.2 Introduction . 56 3.3 Results . 62 3.3.1 Utilization of a real time cell analysis tool to analyse the anti-cancer effects of compounds on breast cancer cells. 62 3.3.1.1 Optimisation cell number to facilitate monitoring cell behaviour in real time. 62 3.3.1.2 VK2 has a significant inhibitory effect on the adhesion of breast cancer cells. 64 3.3.1.3 VK2 has a significant inhibitory effect on the proliferation of breast cancer cells. 64 3.3.1.4 A combination of low glucose media and VK2 has an added inhibitory effect on breast cancer cell adhesion. 67 3.3.1.5 A combination of low glucose media and VK2 has an added inhibitory effect on cell proliferation. 70 3.3.2 Utilizing the RTCA xCELLigence platform to investigate the effect of PRIMA-1 on breast cancer cells. 71 3.3.2.1 PRIMA-1 has no effect on the growth of MCF-7 breast cancer cells. 71 3.3.2.2 Hs578T(i8)2 breast cancer cells show resistance to PRIMA-1. 73 3.3.2.3 PRIMA-1 Met has an inhibitory effect on the growth of MDA-MB-468 breast cancer cells. 73 3.3.2.4 PRIMA-1 has an inhibitory effect on the growth of MDA-MB-453 breast cancer cells. 75 3.4 Discussion. 78 Chapter 4:Disruption of the RACK1/PP2A complex has implications for essential cell characteristics and PP2A activity. 84 4.1 Abstract . 85 4.2 Introduction . 86 4.3 Results. 89 4.3.1 RACK1 and PP2A C subunit are in a complex together in MCF-7 cells. 89 4.3.2 Identification of RACK1 interaction sites on PP2A C subunit peptide arrays. 89 4.3.3 Modelling of potential RACK1 interaction sites on the PP2A C subunit. 89 4.3.4 Generation of PP2A mutants that disrupt the RACK1/PP2A complex. 93 4.3. 5: Disruption of the RACK1/PP2A complex decreases PP2A activity. 95 4.3. 6 Characterisation of the stable PP2A mutant cell lines. 98 4.3. 7 Disruption of the RACK1/PP2A complex decreases