Cell Signalling in Cancer

Multiple signalling pathways mutated in cancer influence core metabolic processes and as a result, cancer is increasingly regarded as a metabolic disease. Metabolic reprogramming through the AKT-mTOR pathway can dysregulate the DNA damage response (DDR), contributing to cancer progression and resistance to ionising radiation (IR). The Macaulay lab has shown that depletion/inhibition of type 1 insulin-like growth factor receptor (IGF-1R), upstream of AKT, enhances sensitivity to IR and impairs double strand break (DSB) repair by non-homologous end joining (NHEJ) and homologous recombination repair (HRR), however the mechanism remains elusive. Previously it was assumed that all functions of the IGF axis require IGF-1R kinase activity, yet recently loss of unliganded IGF-1R and kinase-dead (KD) IGF-1R were shown to downregulate imprinted genes and alter thresholds for apoptosis, respectively. These roles of IGF-1R independent of its catalytic activity offer the possibility of a similar structural function of IGF-1R in the DDR.

My project focuses on assessing whether effects of the IGF axis on the DDR require IGF-1R kinase activity or structural properties of the receptor. I am using CRISPR/Cas9 genome editing to produce IGF-1R-null prostate cancer cells and novel CRISPR/Cas9 editing with single-stranded oligonucleotide templates to create IGF-1R ligand binding site and KD IGF-1R mutants. I will use three approaches to investigate consequences for the DDR. Firstly, using IGF-1R knockdown, IGF-1R-null and IGF-1R mutant cells, I will test basal and post-irradiation cell survival in 2D and 3D. If I find any differences in radiosensitivity, I will perform DSB reporter assays and immunofluorescence for foci associated with specific DNA repair pathways, and also test for general effects on chromatin structure. Secondly, I will assess the extent to which identified phenotypes can be rescued by re-introduction of IGF-1R, and/or by reconstitution of AKT-mTOR activity. Finally, I will use IGF-1R null cells and cells expressing wild-type or mutant IGF-1R to perform phosphoproteomic analysis and a compound screen, with the aim of identifying novel IGF-1R effectors, and vulnerabilities associated with IGF-1R loss. Altogether, this project is using hypothesis-led and unbiased approaches to further understand the metabolic regulation of the DDR with the hope of providing novel therapeutic targets for cancer treatment.