Abstract
Targeted therapies, including anti-endocrine agents and inhibitors of growth factor receptors and tyrosine kinases have significantly changed the treatment of breast cancer. However, clinical resistance to agents like tamoxifen poses a major problem. We therefore aimed to identify the molecular mechanisms behind drug resistance in breast cancer in order to reverse them. Here we provide an in-depth characterisation of acquired resistance to tamoxifen, fulvestrant and lapatinib using novel breast cancer cell line models (MCF7-TR, T47D-FR and SKBR3-LapR, respectively). We utilised methods such as cell viability assays, Western blotting, Real time quantitative polymerase chain reaction, Annexin V-FITC with propidium iodide assay, and confocol microscopy . Mefloquine (2 μM) or LY294002 (10μM) (autophagy inhibitors) were used to asses the role of autophagy in mediating drug resistance. Mechanisms of lapatinib-resistance were assessed by micro-RNA array. Oestrogen receptor-alpha (ERα) was significantly down-regulated in the drug-resistant sublines and p27kip1 trans-activation reduced, whilst HER1/2/3, PI3K, Akt and ERK were significantly up-regulated. Treatment with the dual HER1/2 inhibitor lapatinib indicated the presence of collateral sensitivity in both MCF7-TR and T47D-FR cells through reduced HER1 and HER2 signalling, re-induction of p27kip1 and reactivation of ERα. These data provided important evidence of the crosstalk between ERα and HER1/HER2 signalling pathways. Lapatinib stimulated cell death in tamoxifen- and fulvestrant-resistant cells when used as monotherapy and a synergistic reduction in cell viability was observed when used concomitantly with tamoxifen. Analysis of cell death pathways implied that apoptosis was impaired due to loss of caspase activity and that autophagy was activated as a survival mechanism. Both MCF7-TR and T47D-FR cells were substantially sensitized to tamoxifen treatment when mefloquine or LY294002 were co-administered. MiR-205 was up-regulated, indicating inhibition of epithelial-mesenchymal-transition, together with an apparent hyperactivation of the MET-receptor in lapatinib-resistant SKBR3 cells. Overall, our findings highlight the highly plastic and heterogeneous nature of endocrine-resistant breast cancer and demonstrate that drug combination regimens targeting different signalling and cell death pathways significantly could improve therapeutic outcome.