Abstract
IntroductionmTOR is a serine/threonine kinase forming two distinct complexes, mTORC1 and mTORC2. Over-active mTOR signalling is implicated in many cancers including breast cancer, and can be inhibited through the clinical use of Everolimus. Resistance to this drug is poorly studied in breast cancer, with our aim was to develop everolimus resistant cell-lines as models of resistance.Material and methodsEverolimus resistant (EveR) cells were developed by exposure to the drug at the GI60 concentration, on a week on/off basis. Overall responsiveness to everolimus was determined using a sulforhodamine B (SRB) staining assay, where cells were deemed resistant once a stable GI60 had been achieved. EveR cells were characterised and compared to their parental counter parts. Western blotting was used to assess relative levels of mTOR pathway proteins and activation of downstream targets. To compare growth rates, growth curves and cell cycle analysis were used. Antibody arrays were used to compare shifts in global signalling patterns. The responsiveness of these cells to mTOR inhibitors rapamycin, temsirolimus and BEZ-235 was also studied with drug concentrations in the range of 0.1 nM–10 µM.Results and discussionsTwo EveR breast cancer cell lines were made; T47D-EveR and MDA-MB-361-EveR. Cells showed differential resistance to other rapalogues with both EveR cell lines significantly resistant to rapamycin after 1 week treatment but were far less resistant to temsirolimus; no resistance to BEZ-235 was observed. Combination therapy with everolimus and vitamin D was seen to increase the effectiveness of treatment in EveR lines, shifting responsiveness towards pre-resistant levels. Both EveR cell lines showed decreased levels of cell proliferation with cell cycle analysis also showing an increase of the proportion of cells in G1 phase. Antibody arrays revealed wide decreases in cell signalling across multiple pathways. T47D-EveR cells also showed up-regulation of mTORC2 related proteins including phospho-PKCα, although siRNA knockdown of PKCα failed to reverse resistance.ConclusionEveR cells displayed key differences compared to parental cells, including resistance to everolimus and rapamycin but not temsirolimus; suggesting mutations may have occurred in either FKBP12 or mTOR, with current work to sequence these genes on-going. Observed decreases in p.
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