The proteomic analysis of resistance to anticancer therapy in human breast cancer

Resistance to anticancer therapy represents a major barrier in the successful management of human breast cancer. The identification of novel biomarkers that correlate with treatment response would increase our understanding of the resistance mechanisms and may allow therapy to be tailored on an individual patient basis. Those patients unlikely to respond to a particular treatment strategy would be spared the serious life-threatening side effects for no therapeutic gain. The immediate implementation of a more appropriate treatment strategy may also prevent disease progression and such biomarkers would also suggest new drug targets and/or sensitising agents for future therapeutic intervention.The primary aim of this thesis was to establish and optimise two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and to exploit this global proteomics approach to identify molecular markers associated with resistance to anticancer therapy in human breast cancer cells. This technique was applied to compare the MCF7 cell line with novel derivatives displaying significant resistance to cisplatin (MCF7CR) and radiotherapy (MCF740Gy). Differentially expressed proteins were identified by MALDI-TOF MS analysis. Several molecular markers have been identified, which may play a role in resistance to anticancer therapy. Western blotting was used to confirm the expression of selected targets and to ensure the accuracy of protein identification. Examples of those targets which were confirmed by western blotting include GST-M3, cytokeratin 17, peroxiredoxin 4 and HSP27 (which were associated with cisplatin resistance) and L-Plastin, GST-M3 and cytokeratin 17 (which were associated with radio-resistance). These provide interesting targets and are worthy of further studies.Although 2DE-MALDI-TOF MS remains the gold standard for the global analysis of protein expression this technique is associated with several drawbacks. The antibody microarray is a powerful new technique designed to overcome many of these drawbacks and provides complementary information. Antibody microarrays were applied to identify further molecular markers associated with resistance to anticancer therapy in human breast cancer cells. The MDA-MB-231 cell line and a novel derivative (MDAMB- 231DR) displaying significant resistance to doxorubicin were analysed using Panorama Cell Signalling antibody microarrays (Sigma-Aldrich) comprised of 224 antibodies. Several proteins were associated with resistance to doxorubicin and some proteins were confirmed by western blotting. These targets included p-ERK, cyclin D2 and cyclin B 1. The technique was subsequently extended using the Labvision Antibody Microarray Kit (Neomarkers) to analyse the MCF7 parental cell line and the derivative (MCF740Gy) displaying significant resistance to radiotherapy. The Labvision antibody microarrays were comprised of 720 antibodies. Results indicated that this kit requires further optimisation and a preliminary analysis of the data revealed that further developmental and statistical work is required for interpretation of the results.This thesis has generated a list of potential candidate proteins, which may play a role in the development of resistance to anticancer therapy. Some of these targets overlap between different treatment modalities and have been confirmed by western blotting. Further work is now required in order to determine the functional relevance of these interesting targets. Ultimately, overcoming resistance to both chemotherapy and radiotherapy would represent a major advance in the effective management of breast cancer today