4 Year Wellcome Trust PhD Programme: How growth signals regulate replication licensing and cell division in normal and cancerous oesophageal cells

In order to maintain genetic stability and prevent the amplification of chromosome segments, the process of DNA replication occurs in two strictly non-overlapping phases. In late mitosis and G1, replication origins are ‘licensed’ for replication by being loaded with double hexamers of MCM2-7 proteins. Then, during S phase, replication forks initiate at these licensed origins. Defects in regulation of the licensing system are associated with a variety of diseases including cancer and growth disorders [1].
In collaboration with Prof Inke Nathke we have recently shown that intestinal stem cells reside predominantly in a previously uncharacterised unlicensed G1 state [2]. This is completely different from the situation in cancer-derived mammalian tissue culture cells, where licensing starts during late mitosis. Significantly, the unlicensed state is lost in pre-cancerous APC-mutant stem cells, likely due to lack of a functional CDK4/6-dependent restriction point, causing licensing to occur in late mitosis. Using intestinal organoids we also showed that EGFR inhibition caused intestinal cells to reversibly exit the cell cycle into a G0 state. Our results suggest that EGFR signalling puts cells in a proliferative state and subsequent CDK4/6 activity promotes replication licensing and drives entry into S phase. This regulation becomes defective at an early stage in intestinal tumorigenesis.
The PhD project will to extend this work to a oesophageal systems. The development of oesophageal cancer is poorly understood. Oesophageal cancers are difficult to treat and commonly have mutations in receptor kinase signalling (such as EGFR) or CDK4/6 regulation, which directly affect replication licensing. In this project we will investigate the role of EGFR or CDK4/6 signalling in controlling replication licensing and cell division in oesophageal systems, including oesophageal cancer cell lines, organoid culture and patient biopsies. We will determine whether primary (non-cancerous) oesophageal cells delay replication licensing until the restriction point, and whether this restriction point control is lost during the development of oesophageal cancer. We will also use cutting-edge high content imaging approaches [2] to investigate the effect of these pathways on cell behaviour in a physiological 3-dimensional tissue context. We will collaborate with Prof Russell Petty, a Medical Oncologist with expertise in oesophageal cancer, to apply our results to patient material and to put our new insights into a clinical context. This will provide important new information into the therapeutic potential of inhibiting CDK4/6 and EGFR signalling pathways in oesophageal cancer.