Port, Jennifer Lynne Forbes (2018) Investigating the therapeutic potential of NUAK1 for the treatment of colorectal cancer. PhD thesis. https://theses.gla.ac.uk/9125/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Investigating the Therapeutic Potential of NUAK1 for the Treatment of Colorectal Cancer Jennifer Lynne Forbes Port This thesis is submitted to the University of Glasgow in fulfilment of the requirements of the Degree of Doctor of Philosophy. Institute of Cancer Sciences College of Medical, Veterinary, and Life Sciences University of Glasgow The Beatson Institute for Cancer Research Garscube Estate Glasgow September 2017 Abstract NUAK1 (aka ARK5) is a member of the AMPK-related kinase family and has been associated with many essential cellular processes that are often perturbed in cancer. Elevated NUAK1 expression has been observed in advanced stages of colorectal cancer (CRC) and is further enriched in liver metastasis in human patients. Therefore, NUAK1 is a novel tumour progression-associated factor, however, its primary function in this context and its use as a potential therapeutic target remains unclear. NUAK1 was previously identified as synthetic lethal in MYC-overexpressing tumour cells. This study shows that high NUAK1 levels correspond to poor patient survival in human CRC, and that increased NUAK1 RNA correlates with advanced tumour stages in a human TMA of CRC. Using mouse models of CRC, it is demonstrated that Nuak1 deletion inhibits colon tumour initiation, and acute depletion of Nuak1 by shRNA in established tumours significantly reduces tumour burden after just 7 days of shRNA activation. Interrogation of tumours acutely depleted of Nuak1 showed decreased cell proliferation concurrent with increased ROS and cell death. Interestingly, the requirement for Nuak1 did not extend to healthy wildtype intestine; depletion of Nuak1 in mouse intestine had no impact on cell death, proliferation or differentiation, and wildtype 3D organoids were resistant to Nuak1 inhibition. Using human CRC cell lines and transformed 3D organoid cultures, the study confirms that the NRF2 oxidative stress response is compromised in NUAK1 depleted cells, and treatment with a ROS scavenger can rescue the detrimental consequences of this in vitro, ex vivo and in vivo. Mechanistically, it was found that NUAK1 is necessary for the nuclear accumulation of NRF2 by counteracting negative regulation of this process by GSK3β, and that direct inhibition of GSK3β is able to restore NRF2 nuclear accumulation in NUAK1 deficient cells. Furthermore, it is shown that ROS-dependent activation of NUAK1 by cysteine oxidisation leads to the phosphorylation of MYPT1. Activation of MYPT1 results in the suppression of PP1β activity, which in turn inhibits dephosphorylation of GSK3β thus allowing NRF2 to accumulate in the nucleus and upregulate the anti-oxidant response pathway. In summary, this thesis is proposing a fascinating, new and conserved mechanism of redox signal transduction in which activation of NUAK1 coordinates PP1βMYPT1 inhibition, with AKT activation in order to suppress GSK3β-dependent inhibition of 2 NRF2 nuclear import. Exploiting the heightened sensitivity of tumour cells to ROS is emerging as a plausible strategy for cancer therapy and is implicated in the resistance to chemotherapy. Therefore, inhibiting the anti-oxidant response via transient inhibition of NUAK1 may offer a new strategy for improving therapeutic outcomes in cancer. 3 Table of Contents List of Tables ........................................................................................................................ 7 List of Figures ....................................................................................................................... 8 Acknowledgements ............................................................................................................. 10 Abbreviations ..................................................................................................................... 13 Chapter 1 Introduction ...................................................................................................... 18 1.1 Colorectal Cancer ................................................................................................................. 18 1.2 CRC clinical manifestations................................................................................................. 18 1.3 Clinical diagnosis and staging.............................................................................................. 18 1.3.1 TNM Classification of Malignant Tumours (TNM) .............................................................. 19 1.3.2 The Dukes’ staging system ............................................................................................................ 22 1.4 Current treatment of CRC .................................................................................................. 22 1.5 Normal Intestinal Homeostasis ............................................................................................ 23 1.6 Molecular characterization of CRC .................................................................................... 25 1.6.1 Adenomatous polyposis coli (APC), β-Catenin and Wnt signalling .............................. 26 1.6.2 MYC deregulation in CRC ............................................................................................................... 27 1.6.3 The ‘adenoma-to-carcinoma’ sequence ................................................................................... 28 1.6.4 KRAS ....................................................................................................................................................... 31 1.6.5 Other common mutations in CRC ............................................................................................... 33 1.6.6 The consensus molecular subtypes of CRC ............................................................................ 34 1.7 Approaches to modelling CRC ............................................................................................ 35 1.7.1 Human cell lines ................................................................................................................................ 35 1.7.2 Intestinal organoids ......................................................................................................................... 36 1.7.3 Genetically engineered mouse models (GEMMs) ................................................................ 37 1.8 AMPK-Related kinases ........................................................................................................ 43 1.8.1 AMPK ...................................................................................................................................................... 43 1.8.2 AMPK-related kinases ..................................................................................................................... 44 1.8.3 NUAK1 and NUAK2: structure, expression and a common target ................................ 46 1.8.4 NUAK2: function and regulation................................................................................................. 49 1.9 NUAK1 .................................................................................................................................. 50 1.9.1 NUAK1: function and regulation................................................................................................. 50 1.9.2 NUAK1 mouse models .................................................................................................................... 51 1.9.3 NUAK1 and cancer ............................................................................................................................ 52 1.9.4 NUAK1 and synthetic lethality .................................................................................................... 54 1.10 Aims of the project ............................................................................................................. 58 Chapter 2 Materials & Methods ....................................................................................... 60 2.1 Mouse experiments and analyses ........................................................................................ 60 2.1.1 Colony Maintenance ........................................................................................................................ 60 2.1.2 Genotyping ........................................................................................................................................... 60 2.1.3 Experimental Cohorts ..................................................................................................................... 60 2.1.4 Tissue preparation and scoring .................................................................................................. 64 2.1.5 Tissue stains and immunohistochemistry