University of Dundee

China Scholarship Council PhD Programme: How growth signals regulate replication licensing and cell division in normal and cancerous oesophageal cells

Primary Supervisor: Prof Julian Blow, Second Supervisor: Prof Inke Nathke, Third Supervisor: Prof Russell Petty (School of Medicine)

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.

References

[1] Alver, R.C., Chadha, G.S. and Blow, J.J. (2014). The contribution of dormant origins to genome stability: from cell biology to human genetics. DNA Repair 19, 182-189

[2] Caroll, T., Newton, I., Chen, Y., Blow, J.J. and Näthke, I. (2018). Lgr5+ intestinal stem cells reside in an unlicensed G1 phase. J Cell Biol 217, 1667-1685