Characterization and functional analysis of mutant p53 secretome in human cancer

5 DECLARATION . 8 AWARDS & SCHOLARSHIPS . 9 PUBLICATIONS . 10 ACKNOWLEDGEMENTS . 12 Chapter 1: Introduction . 16 Preface . 16 Tumour suppressor protein: p53 . 17 Mutations of p53 . 18 Mutant p53 gain-of-function . 20 The landscape of p53 mutations in human cancer . 22 Transcriptional activity of mutant p53.24 Mutant p53 and the p53 family members, p63 and p73 . 25 Biological effects /activities of mutant p53 in cancer.28 Targeting mutant p53 in human cancer . 30 Restoring wild-type p53 activity of mutant p53 . 31 Mutant p53 elimination or degradation . 38 Targeting mutant p53 induced downstream oncogenic effects . 41 Mutant p53 and the cancer cell secretome . 51 Research Gaps and Objectives of the thesis . 52 Thesis Outline . 54 Chapter 2: General Materials and Methods . 57 Cell lines and inducible expression system . 57 iTRAQ analysis by 2D nanoLC ESI MS/MS . 58 Sample collection . 58 Instruments used . 58 Sample preparation and digestion . 59 Strong cation exchange HPLC/ Chromatographic fractionation of peptides . 59 NanoLC-ESI-MS/MS analysis . 60 Data processing and criteria . 60 Data analysis . 61 Bioinformatics analysis . 61 Real-time PCR analysis . 62 Western blot analysis . 63 Immunofluorescence . 63 Conditioned medium collection . 64 Generation of knockdown cell lines . 64 Migration assay . 66 Inverted invasion assay.66 Chicken chorio-allantoic membrane (CAM) invasion assay.67 CAM immunohistochemistry (IHC) . 67 Lung tissue microarrays (TMAs) . 68 Immunohistochemistry . 69 Image capture and quantification of A1AT and p53 immunostaining . 70 In silico analysis of p53 and p63 response elements . 70 Chromatin Immunoprecipitation assays (ChIP) . 71 Soft Agar Colony Formation Assay . 72 Cell proliferation and cell cycle analysis . 72 Chapter 3: Characterization of mutant p53 secretome . 78 Preface . 78 3.2 Introduction . 79 3.3 Results . 83 3.3. 1 Mutant p53 drives the release of pro-invasive factors . 83 3.3.2 iTRAQ based quantitative analysis of mutant p53 secretome . 85 3.3.3 Bioinformatics analysis and profiling of the mutant p53 induced secreted proteins.92 3.3.4 Validation of Mutant p53 induced secreted proteins in experimental and array datasets . 102 3.3. 5 Identification of potential secreted target of mutant p53 . 108 3.4 Discussion . 111 Chapter 4: Alpha-1 Antitrypsin (A1AT) functions as a novel secreted mediator of mutant p53 gain-of-function . 119 Preface . 119 Introduction . 120 Results . 124 Sustained expression of mutant p53 is required for invasion of lung cancer cells . 124 A1AT induces cell invasion and migration . 126 A1AT specifically mediates mutant p53 driven cell migration and invasion . 128 A1AT drives lung cancer cell invasion in an in vivo chick chorio-allantoic membrane (CAM) model . 130 A1AT alters epithelial-mesenchymal transition (EMT) marker expression . 132 Secreted A1AT enhances mutant p53 induced cell migration and invasion . 134 Blockade of secreted A1AT using neutralizing antibody ablate mutant p53 induced cell migration and invasion . 137 Discussion . 140 Chapter 5: Mutant p53 regulated Alpha-1 Antitrypsin (A1AT) is a prognostic marker of lung adenocarcinoma . 145 Preface . 145 Introduction . 146 Results . 148 Analysis of A1AT expression in lung cancer cell lines . 148 A1AT expression is upregulated in lung ADC tumours . 150 Elevated p53 expression associates with high A1AT expression in lung ADC TMAs . 152 Elevated A1AT expression correlates with poor overall survival in human lung ADC as well as gastric cancer patients. 158 Discussion . 162 Chapter 6: Mutant p53 interacts with p63 to regulate SERPINA1 (protein: A1AT) expression.165 Preface . 165 Introductio