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Hippo and Sonic Hedgehog Signalling Pathway Modulation of Human Urothelial Tissue Homeostasis

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Hippo and Sonic Hedgehog Signalling Pathway Modulation of Human Urothelial Tissue Homeostasis Hippo and Sonic Hedgehog signalling pathway modulation of human urothelial tissue homeostasis Thomas Crighton PhD University of York Department of Biology November 2020 Abstract The urinary tract is lined by a barrier-forming, mitotically-quiescent urothelium, which retains the ability to regenerate following injury. Regulation of tissue homeostasis by Hippo and Sonic Hedgehog signalling has previously been implicated in various mammalian epithelia, but limited evidence exists as to their role in adult human urothelial physiology. Focussing on the Hippo pathway, the aims of this thesis were to characterise expression of said pathways in urothelium, determine what role the pathways have in regulating urothelial phenotype, and investigate whether the pathways are implicated in muscle-invasive bladder cancer (MIBC). These aims were assessed using a cell culture paradigm of Normal Human Urothelial (NHU) cells that can be manipulated in vitro to represent different differentiated phenotypes, alongside MIBC cell lines and The Cancer Genome Atlas resource. Transcriptomic analysis of NHU cells identified a significant induction of VGLL1, a poorly understood regulator of Hippo signalling, in differentiated cells. Activation of upstream transcription factors PPARγ and GATA3 and/or blockade of active EGFR/RAS/RAF/MEK/ERK signalling were identified as mechanisms which induce VGLL1 expression in NHU cells. Ectopic overexpression of VGLL1 in undifferentiated NHU cells and MIBC cell line T24 resulted in significantly reduced proliferation. Conversely, knockdown of VGLL1 in differentiated NHU cells significantly reduced barrier tightness in an unwounded state, while inhibiting regeneration and increasing cell cycle activation in scratch-wounded cultures. A signalling pathway previously observed to be inhibited by VGLL1 function, YAP/TAZ, was unaffected by VGLL1 manipulation. In MIBC, overexpression of VGLL1 was observed in a subset of differentiated tumours associated with significantly reduced survival, indicative of dysregulated VGLL1 function. This study reveals a novel Hippo pathway-independent function of VGLL1, with the protein observed to play an important role in regulating urothelial tissue homeostasis. Consequently, these findings contribute to the current understanding of how dysregulation of tissue homeostasis can facilitate MIBC progression. 2 Contents Abstract……………………………………………………………………………………………....2 Table of contents………………………………………………………………………………....3 List of figures……………………………………………………………………………….. …...15 List of tables…………………………………………………………………………………. ……26 Acknowledgements………………………………………………………………………. ……29 Author declaration………………………………………………………………………………30 1. Introduction………………………………………………………………………………………..31 1.1 The urinary system……………………………………………………………………………..31 1.2 Urothelium………………………………………………………………………………………….33 1.2.1 Urothelial morphology and function………………………………………..33 1.2.2 Molecular markers of urothelial cells in situ…………………………….36 1.3 Normal Human Urothelial (NHU) cells in vitro…………………………………….37 1.3.1 NHU cell phenotype…………………………………………………………………37 1.3.2 NHU cytodifferentiation…………………………………………………………..38 1.3.2.1 Induction of cytodifferentiation in vitro……………………….38 1.3.2.2 PPARγ…………………………………………………………………………..39 1.3.2.3 Other transcription factors involved in urothelial differentiation………………………………………………………………40 1.3.3 NHU cell growth regulation……………………………………………………..45 1.4 Bladder cancer……………………………………………………………………………………49 1.4.1 Epidemiology…………………………………………………………………………..49 1.4.2 Pathology………………………………………………………………………………..49 1.4.3 Molecular subtyping of muscle-invasive bladder cancer…………51 1.4.4 Molecular targets in muscle invasive bladder cancer………………55 1.5 Thesis aims…………………………………………………………………………………………60 3 2. Materials and methods……………………………………………………………………….62 2.1 General………………………………………………………………………………………………62 2.2 H2O…………………………………………………………………………………………………….62 2.3 Ethical approval………………………………………………………………………………….62 2.4 Tissue culture……………………………………………………………………………………..63 2.4.1 General……………………………………………………………………………………63 2.4.2 Tissue sample collection………………………………………………………….63 2.4.3 Cell isolation……………………………………………………………………………64 2.4.3.1 Urothelial cell isolation………………………………………………..64 2.4.3.2 Stromal cell isolation…………………………………………………...64 2.4.4 Cell culture……………………………………………………………………………….66 2.4.4.1 Maintenance of NHU cell lines……………………………………..66 2.4.4.2 Maintenance of stromal cell lines…………………………………66 2.4.4.3 Culture of established cell lines…………………………………….67 2.4.4.4 Pharmacological agonists and antagonists……………………68 2.4.4.5 ABS/Ca2+ differentiation of NHU cells……………………………68 2.4.4.6 Measurement of Transepithelial Electrical Resistance (TEER)…………………………………………………………………………..70 2.4.4.7 Scratch wounding of cell cultures grown on membranes………………………………………………………………….71 2.4.4.8 Lifting of cell sheets by dispase treatment……………………72 2.4.4.9 TZ/PD differentiation of NHU cells……………………………….72 2.4.4.10 Mycoplasma spp. testing……………………………………………..72 2.5 Cell viability assay………………………………………………………………………………73 2.6 Cell cycle analysis……………………………………………………………………………….74 2.7 Gene expression analysis……………………………………………………………………75 2.7.1 General……………………………………………………………………………………75 2.7.2 RNA extraction…………………………………………………………………………75 2.7.3 DNAse treatment of RNA…………………………………………………………76 2.7.4 Sodium acetate precipitation of RNA……………………………………….77 2.7.5 cDNA synthesis…………………………………………………………………………77 2.7.6 Primer design……………………………………………………………………………78 4 2.7.7 Primer optimisation………………………………………………………………….78 2.7.8 Reverse Transcription Polymerase Chain Reaction (RT- PCR)………………………………………………………………………………………….79 2.7.9 Gel electrophoresis…………………………………………………………………..79 2.7.10 Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)…………………………………………………………………………………80 2.7.11 RNA sequencing……………………………………………………………………….80 2.8 Molecular biology……………………………………………………………………………….81 2.8.1 General…………………………………………………………………………………….81 2.8.2 Generation of overexpressing retroviral vectors………………………81 2.8.2.1 PCR amplification of gene of interest……………………………81 2.8.2.2 Purification of PCR product…………………………………………..82 2.8.2.3 Ligation of PCR product into plasmid…………………………….82 2.8.2.4 Bacterial transformation……………………………………………….84 2.8.2.5 Colony PCR……………………………………………………………………84 2.8.2.6 Plasmid DNA extraction………………………………………………..85 2.8.2.7 DNA sequencing……………………………………………………………85 2.8.2.8 Restriction digest of subcloned DNA…………………………….85 2.8.2.9 Gel extraction and purification of plasmid DNA……………86 2.8.2.10 Dephosphorylation of plasmid……………………………………..87 2.8.2.11 Production of bacterial glycerol stocks…………………………88 2.8.3 Generation of shRNA constructs………………………………………………88 2.8.3.1 Design of shRNA oligonucleotide sequences………………..88 2.8.3.2 Annealing and ligation of shRNA oligonucleotides into pSIREN-RetroQ…………………………………………………………….88 2.8.3.3 Bacterial transformation and colony PCR…………………….90 2.8.3.4 MluI restriction analysis……………………………………………….90 2.8.4 Genetic manipulation of NHU and MIBC cell lines……………………90 2.8.4.1 Transfection of retroviral packaging cells……………………..90 2.8.4.2 Transduction of cell lines with retroviral vector…………..91 2.8.4.3 Transfection of NHU cells with siRNA……………………….....92 5 2.9 Protein analysis…………………………………………………………………………………93 2.9.1 Immunohistochemistry……………………………………………………………93 2.9.1.1 Paraffin embedding and sectioning……………………………..93 2.9.1.2 Immunolabelling of sections………………………………………..94 2.9.2 Indirect immunofluorescence labelling of cells……………………….97 2.9.2.1 Preparation of fixed adherent cells on slides…..............97 2.9.2.2 Immunocytochemistry…………………………………………………98 2.9.3 Western blotting…………………………………………………………………….102 2.9.3.1 Preparation of protein lysates………………………..............102 2.9.3.2 Protein quantification………………………………………………...102 2.9.3.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)…103 2.9.3.4 Electroblotting……………………………………………………………103 2.9.3.5 Immunoblotting………………………………………………………….104 2.10 Bioinformatics……………………………………………………………………………..108 2.10.1 General…………………………………………………………………………………108 2.10.2 RNAseq analysis pipeline………………………………………………………108 2.10.2.1 Preprocessing of sequencing reads……………………………108 2.10.2.2 Mapping of reads to reference transcriptome…………..108 2.10.2.3 Differential gene expression analysis…………………………108 2.10.3 Other bioinformatics tools……………………………………………………109 2.11 Statistical analysis……………………………………………………………………….110 3. Characterisation of VGLL1 and Hippo pathway signalling in normal human urothelial cells………………………………………………………………………………….111 3.1 Introduction……………………………………………………………………………………..111 3.1.1 Hippo signalling pathway……………………………………………………….111 3.1.2 YAP/TAZ function in non-malignant mammalian cells……………113 3.1.3 Vestigial-like protein family……………………………………………………114 3.2 Aims and hypothesis…………………………………………………………………………117 3.3 Experimental approach……………………………………………………………………..119 3.3.1 Transcriptomic analysis of Hippo pathway genes…………………..119 3.3.2 Characterisation of Hippo pathway component protein expression in differentiated urothelium…………………………………119 6 3.3.3 Induction of VGLL1 expression by urothelial transcription factors……………………………………………………………………………………120 3.3.4 Effect of active EGFR and downstream signal transduction pathways on VGLL1 expression………………………………………………121 3.3.5 Modulation of NHU cell phenotype using VGLL1 overexpression and knockdown constructs…………………………………………………….122 3.4 Results………………………………………………………………………………………………124 3.4.1 RNAseq analysis of urothelium in situ and NHU cells…………….124 3.4.1.1 Expression of differentiation-associated genes in urothelial cells…………………………………………………………….124
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