The Development and Characterisation of Everolimus Resistant Breast Cancer Cells

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonThe mTOR inhibitor and rapalogue everolimus was approved use in 2012, in HR+, HER-2-, post-menopausal patients, who had previously failed aromatase inhibitor treatment. mTOR pathway activation has been associated with resistance to breast cancer therapies, but how cells may become resistant to mTOR therapies themselves in breast cancer is currently not well explored, due to the relative recentness of everolimus approval. Drug resistance across all areas of cancer research is a major clinical issue, often leading to the spread of a patient’s cancer. This project set out to create in vitro breast cancer models that were resistant to everolimus, and thus explore any changes that had developed in these models, help determine the mechanisms behind resistance and discover drugs/drug combinations that could overcome resistance. Cell lines T47D and MDA-MB-361 were subsequently developed into everolimus resistant lines (EveR) over the course of 4-6 months using an on/off exposure routine. The exact mechanism behind the everolimus resistance was not fully determined but EveR cells did show multiple intriguing characteristics. An increase in dormancy and stem-cell like phenotype was noted, as revealed by a decrease in cell cycle progression and an increase in increase ALDH activity. mTORC2 components and signalling was up-regulated although siRNA down-regulation of PKCα did not decrease everolimus resistance, suggesting other mTORC2 targets may be involved. The rapalogue ‘receptor’, FKBP12, was up-regulated which was accompanied by an increased growth inhibition by the rapalogue, temsirolimus, possible due to temsirolimus lower binding affinity for FKBP12 compared to everolimus. No resistance to the dual mTOR/PI3K inhibitor BEZ-235 was observed, in line with similar published work. The combination of vitamin D/calcitriol and everolimus had no added effect compared to everolimus alone, in parental cells, but the addition of 1μM calcitriol did drastically lower EveR cell resistance to everolimus. Future work focusing on the exact nature of calcitriol’s interaction with the mTOR pathway is required to advance calcitriols role as a breast cancer therapeutic. Research with everolimus resistant breast cancer patients has not yet been published on, but the work presented here aims to help guide such studies, when they are carried out in the future