Antibody microarray analysis identifies biomarkers associated with radioresistant breast cancer cell lines.
Authors: ELFadl D , Smith L , Qutob O , Watson MB , Beavis AW , Garimella V , Drew PJ , Lind MJ , Cawkwell L 
Published: December 2010

Abstract
Abstract #5072
Background: Resistance to radiotherapy may be a significant factor in the development of local recurrence following surgical resection and radiotherapy. In addition, if patients with radioresistant breast cancers can be identified, harmful side effects from exposure to unnecessary ionizing radiation could be prevented. We aimed to develop a novel in vitro model of radioresistance using breast cancer cell lines and to subsequently identify molecular biomarkers which may be associated with the radioresistant phenotype. Antibody microarrays offer a complementary approach for proteomic analysis in conjunction with standard screening methods such as two dimensional gel electrophoresis/ mass spectrometry and other quantitative proteomic techniques. We previously utilised the Panorama Cell Signalling Antibody Microarray Kit (Sigma-Aldrich) consisting of 224 antibodies (Smith et al Mol Cancer Ther 5:2115-20, 2006). In this study we utilised a novel high density 725 antibody microarray to screen for proteins associated with radioresistance.&#x2028; Material and Methods: We established novel breast cancer cell sublines which were significantly resistant to radiotherapy when compared with the parental cells (T47D; MCF-7). The radioresistant sublines were created by irradiating cells in fractionated doses of 2Gy up to a total dose of 40Gy. Sufficient time was allowed for the cells to recover between subsequent irradiations. A dose response curve was assessed at the end of treatment to demonstrate a statistically significant increase in radioresistance for the novel cell subline when compared with parental cells. The radioresistant/parental cell pairs were analysed using the Panorama Antibody Microarray XPRESS Profiler725 Kit (Sigma-Aldrich). The microarray comprised 725 different antibodies on nitrocellulose coated microscope slides. The antibodies were selected from a wide variety of pathways, including apoptotic and cell signalling pathways.&#x2028; Results: Utilising a Cy3/Cy5 labelling strategy the antibody microarray approach yielded a number of a total of 28 targets for further study. Of these, three proteins were identified independently from both of the radioresistant cell lines. These were GFI1 (Growth Factor Independence-1), DR4 (Death Receptor 4) and Importin a1. Immunoblotting and other proteomic approaches have confirmed the identities and differential expression of some candidate protein targets.&#x2028; Conclusion: High density antibody microarrays potentially offer a powerful new proteomic technique to allow the global analysis of many proteins simultaneously. This analysis has produced both complementary and confirmatory data in our proteomic screening for putative biomarkers associated with radiotherapy resistance. We successfully identified a number of protein targets which may be associated with a radioresistant phenotype. Further confirmatory and validation studies are ongoing.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5072.