Clinical mutations in HUWE1: deficiency in the DNA damage response and links to cancer

This PhD position in the Cellular and Molecular Biology Programme provides a unique opportunity for a highly motivated individual to conduct clinically relevant research into the links between deficiencies in the DNA damage response and the molecular nature of tumorigenesis using state-of-the-art techniques.

Project Description

Lung cancer is one of the most common cancers in the UK and worldwide. Squamous cell lung cancer accounts for 20-25% of all lung cancers, for which the overall 5-year survival rate remains one of the poorest (about 15%). HUWE1 expression levels are elevated in a high percentage of squamous cell lung carcinoma, and mutations in huwe1 gene have been proposed to promote tumorigenesis [1].

HUWE1, also know as MULE, is a E3 ubiquitin ligase that regulates a number of proteins involved in DNA damage signalling, repair and oncogenic transformation (reviewed in [2]). Overexpression of HUWE1 leads to a significant delay in the p53 DNA damage response, defects in ATM-dependent repair of endogenous DNA lesions and MYC-driven transcriptional activation, establishing an
important role for HUWE1 in genome stability [3-5].

Recently, two individuals (< 10 years old) with symptoms indicative of DNA repair deficiencies were found to carry similar N-terminal mutations in HUWE1. It is possible that these mutations result in loss of function, leading to defects in the maintenance of genome stability. The aim of this PhD project is to understand the molecular consequences of N-terminal mutations in HUWE1 and their
links with genome instability and the development of squamous cell lung carcinoma. We will conduct functional studies to determine whether the mutations in HUWE1 lead to changes in 1) protein stability and/or activity, 2) the ubiquitination status of its relevant protein substrates, 3) the levels of endogenous DNA damage and its signalling and repair following exogenous insult, 4) cell
proliferation and apoptosis, and 5) its protein binding partners, all of which could contribute to HUWE1-driven tumorigenesis. In these studies we will use patient-derived lymphoblastoid cell lines (provided by Prof Andrew Wilkie, Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford) in comparison with appropriate controls. We will also use CRISPR/Cas9 genome editing to develop squamous lung cancer cell lines containing mutations in HUWE1. These studies are important, as they will provide further understanding into the role of HUWE1 in tumorigenesis.

The techniques that will be used include CRISPR/Cas9 genome editing, real-time qPCR, confocal microscopy, mass spectrometry, and methods of structural biology (in collaboration with Dr Luca Pellegrini, Department of Biochemistry, University of Cambridge).