Title: Understanding proliferation-quiescence decisions in response to DNA damage
Funder: Cancer Research UK

Abstract: Background Mutation drives tumorigenesis, cancer evolution and resistance. Yet cancer cells must preserve a degree of genome stability compatible with survival and proliferative potential. Therefore, cancer cells balance proliferation with DNA repair. Many therapies tip this balance towards excessive DNA damage to promote cancer cell death, yet we do not understand how such a balance is achieved at the molecular level. In response to intrinsic DNA damage, non-transformed cells can enter a reversible p21(Cip1/Waf1)-dependent quiescent state. p21-dependent quiescence maintains genome stability in normal cells that inherited DNA damage from the previous generation. p21 is rarely mutated (0.35%) in non-small cell lung cancer (NSCLC). Whether p21-dependent quiescence affects genome stability, proliferative potential and therapy response in NSCLC is unknown. Aims 1. Characterise the mechanism(s) by which p21-dependent quiescence is maintained in normal cells and how cells re-enter the cell cycle; 2. Define the proteomic and phospho-proteomic signatures of p21-dependent quiescence versus G1 and senescent normal cells; 3. Determine if p21-dependent quiescence maintains relative genome stability and proliferative potential in NSCLC and how it impacts chemotherapy response. Methods Aim 1. I will study proliferation-quiescence decisions at the molecular level by quantitative, single-cell, time-lapse imaging. New fluorescent biosensors will be generated, including a CDK4/6 activity reporter. Imaging new and existing reporter cell lines, where Skp2 and Cdc25A have been fluorescently-labelled by CRISPR/Cas9 gene-editing, will determine the role of these proteins in proliferation-quiescence decisions. These data will be used to parametrise new mathematical models to generate mechanistic insight. Aim 2. Quantitative mass spectrometry will be used to identify proteins whose expression and phosphorylation status changes between p21-dependent quiescent versus proliferating G1 and senescent cells. To determine which of the identified proteins regulate proliferation-quiescence transitions, I will use image-based CRISPR/Cas9 and siRNA screens and characterise candidate protein function in molecular detail. Aim 3. I will test the role of p21-dependent quiescence in maintaining relative genome stability and proliferative potential in NSCLC cell lines using siRNA, CRISPR/Cas9 and single-cell, time-lapse imaging. I will generate new fluorescent-reporter NSCLC cell lines to use time-lapse imaging to determine how p21 affects chemotherapy response. How the results of this research will be used This research will provide a molecular basis for how normal and NSCLC cells transition between proliferative and non-proliferative states in response to DNA damage. These mechanisms will open-up new therapeutic avenues for targeting these transitions to tip cancer cells towards excessive DNA damage and cell death.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS;GENERIC HEALTH RELEVANCE