Understanding PI3K Signalling in Colorectal Cancer: from Function to Therapy
Total Page：16
File Type：pdf, Size：1020Kb
Understanding PI3K Signalling in Colorectal Cancer – From Function to Therapy Michelle Palmieri Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy March 2016 The Department of Medical Biology Walter and Eliza Hall Institute of Medical Research Faculty of Medicine, Dentistry and Health Sciences The University of Melbourne Produced on archival quality paper Abstract Aberrant phosphoinositide-3 kinase (PI3K) signalling is associated with the development and progression of several types of cancer including colorectal cancer (CRC) and accordingly a target for anti-cancer therapy. The canonical PI3K signalling cascade is initiated at the cell membrane where, upon receptor tyrosine kinase (RTK) activation by ligands such as EGF, PI3K is recruited and activated to convert phosphatidylinositol bisphosphate (PI(4,5)P2) to phosphatidylinositol trisphosphate (PI(3,4,5)P3), resulting in downstream signalling through phosphorylation and activation of AKT. This results in engagement of various downstream targets which are involved in the regulation of cellular processes such as proliferation, migration and apoptosis. PI3K signalling is negatively regulated by phosphatase PTEN, which removes the 3-phosphate from PI(3,4,5)P3. PIK3CA, encoding the catalytic subunit of PI3K, and PTEN are frequent targets of activating mutations or epigenetic silencing in CRC respectively. Furthermore, activating mutations of the MAPK pathway member KRAS has been implicated in oncogenic activation of PI3K signalling. Our knowledge of PI3K signalling derives from data assembled across a range of normal and cancer cell types however, a detailed survey of the downstream signalling cascade in CRC taking into account PI3K and MAPK pathway mutation status is presently lacking. Intriguingly, an alternate nuclear PI3K pathway has recently been identified in some cell types. Whether this nuclear PI3K cascade exists and is of importance in CRC remains unknown. This thesis aims to (i) map the molecular signature of EGF induced PI3K/MAPK signalling in CRC with respect to tumour PI3K/MAPK mutation status, (ii) to test for the potential involvement of nuclear PI3K signalling in CRC and (iii) to investigate the clinical and therapeutic significance of the respective canonical and nuclear PI3K pathways. 2 Declaration of Author This is to certify that, (i) The thesis comprises only my original work towards the PhD except where indicated in the Preface, (ii) Due acknowledgement has been made in the text to all other material used, (iii) The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Michelle Palmieri 3 Preface The work presented in this thesis involved a number of research collaborations as outlined below. Chapter 3 Reverse Phase Protein Array experiments were conducted in collaboration with Dr Scott Kopetz at the MD Anderson Cancer Center (Houston, Texas, USA). RPPA data processing and statistical analyses were performed with assistance from Dr Dmitri Mouradov. Tissue microarrays of patients with CRC were constructed by Mr David Byrne (Peter MacCallum Cancer Centre, East Melbourne). Immunohistochemistry assays for phospho-pS6 expression were performed at Ventana Medical Systems Inc. (Tucson, AZ, USA) and results interpreted by a pathologist Dr Shalini Singh (Ventana Medical Systems). My contributions to this chapter were the experimental design, sample preparation for RPPA analysis, validation of protein expression and phosphorylation by western blotting and univariate statistical analyses. I estimate my proportional contribution to the work presented in this chapter as 70%. Chapter 4 Super resolution microscopy was conducted at the Advanced Microscopy Facility with the help of Dr Paul McMillan (Bio21 Institute, Melbourne). Reverse Phase Protein Array experiments were conducted with Dr Scott Kopetz at the MD Anderson Cancer Center (Houston, Texas, USA) and analysed with assistance from Dr Dmitri Mouradov. RNA- Seq analysis was performed at the Australian Genome Research Facility (Melbourne) and differential gene expression analysis was conducted by Dr Christopher Love. Mass spectrometry was performed with the help of Dr Nick Williamson and Dr Ching-Seng Ang at the Mass Spectrometry and Proteomics Facility (Bio21 Institute, Melbourne). Protein identification was performed with Dr Eugene Kapp and protein modelling and bioinformatics motif searches were performed by Dr Robert Jorissen. The hypothesis regarding the presence of nuclear PI3K signalling in CRC and the mapping of this pathway was developed by myself with guidance from my supervisors Dr Oliver Sieber and Dr Bruno Catimel. My experimental contribution to this chapter were the immunofluorescence microscopy and subsequent image analysis, preparation of nuclear 4 and cytoplasmic lysates for Reverse Phase Protein Array, nuclear PI(3,4,5)P3 pulldown experiments, preparation of RNA samples for RNAseq, performing the RTK arrays, generating constructs for protein expression, expressing and purifying recombinant proteins and assessing kinetic binding to PI(3,4,5)P3 on the Biacore. I estimate my proportional contribution to the work presented in this chapter as 80%. Chapter 5 Tissue microarrays were constructed by Mr David Byrne (Peter MacCallum Cancer Centre, East Melbourne). Immunohistochemistry for PI3K expression was conducted in collaboration with Mr Cary Tsui and interpreted with support from anatomical pathologist Dr Michael Christie (Royal Melbourne Hospital). Immunohistochemistry for pS6, pMAPK, TP53, PTEN, EGFR and Ki67 expression was performed at Novartis Institutes for Biomedical Research (USA) and results interpreted by anatomical pathologist Dr Michael Christie. IC50 data for the CRC cell line panel was generated by Dr Walter Bodmer and colleagues (University of Oxford, UK). My experimental contribution to this chapter was assistance with the optimisation and interpretation of PI3K immunohistochemistry as well as the interpretation of all other immunohistochemistry data and IC50 data with respect to nuclear PI3K signalling. I estimate my proportional contribution to the work presented in this chapter as 60%. 5 Acknowledgments Firstly, I would like to thank my wonderful supervisors Dr Oliver Sieber and Dr Bruno Catimel. Their constant support and guidance has helped me complete this PhD and I thank them for all the opportunities they have provided. To all my fellow Sieber lab members, thank you so much for all your support both at the lab bench as well as outside of work. A very special thank you to Anu Sakthianandeswaren, Dmitri Mouradov and Marie Parsons for not only being wonderful colleagues, but also amazing friends. Outside of science, I would like to thank all my friends who have been so patient and supportive through this time, especially Connie Costantino, Bilal Sheik, Joanne Kennedy, Ben Jacob and Nicole Church. I really appreciate all the cups of coffee, skype sessions and being there to not only hear me vent but to also help me sometimes escape the lab bench. Last but not least I would like to thank my truly amazing family; my mum Maria, my dad Michael, my sister Rebecca and my partner Tristan. Without your constant love and support I could never have achieved all that I set out to and I thank you for your endless strength and patience. During the candidature I was supported by an Australian Rotary Health/Rotary District 9780 Bowelscan PhD Scholarship and I thank them for their funding. 6 Table of Contents Abstract ............................................................................................................... 2 Declaration of Author ......................................................................................... 3 Preface ................................................................................................................. 4 Acknowledgments ............................................................................................... 6 TABLE OF CONTENTS ............................................................................................... 7 LIST OF FIGURES ...................................................................................................... 11 LIST OF TABLES ........................................................................................................ 13 CHAPTER 1 : INTRODUCTION .............................................................................. 15 OVERVIEW OF COLORECTAL CANCER ................................................... 15 1.1 Colorectal Cancer Epidemiology ................................................................ 15 1.2 Colorectal Cancer Staging .......................................................................... 15 1.3 Molecular Pathology of CRC ...................................................................... 17 1.3.1 Classical Adenoma-Carcinoma Sequence.................................................... 18 1.3.1.1 APC ......................................................................................................... 19 1.3.1.2 KRAS ....................................................................................................... 19 1.3.1.3 TP53 ....................................................................................................... 20 1.3.2 Serrated Neoplasia Sequence ....................................................................... 20 1.3.2.1 BRAF ...................................................................................................... 21 1.3.2.2