Identification of proteins associated with radiotherapy resistance in breast cancer cells: a combined proteomic and microarray screening approach

Background

Radiotherapy is one of the major modalities in breast cancer treatment. However, 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 novel in vitro models of radio-resistance using breast cancer cell lines and to subsequently identify molecular biomarkers that may be associated with the radioresistant phenotype. We used a combined proteomic (two-dimensional gel electrophoresis/mass spectrometry) and transcriptomic (expression microarrays) screening approach.

Methods

We established three novel breast cancer cell sublines that were significantly resistant to radiotherapy when compared with the relevant parental cells (MCF-7, T47D, MDA-MB-231). Radioresistant sublines were created by irradiating cells in fractionated doses of 2 Gy up to a total dose of 40 Gy. 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 sublines when compared with parental cells. Each radioresistant/parental cell pair was analysed using two-dimensional gel electrophoresis. The protein profiles were compared and differentially expressed proteins were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Immunoblotting was used to confirm the identities of a subset of proteins. A 3 k cancer-related oligonucleotide microarray was also used to identify targets that were differentially expressed between each novel radioresistant derivative and its parental cell line. Real-time quantitative PCR was used to confirm the difference in expression of a subset of genes that demonstrated significant (at least twofold) differential expression.

Results

Using the proteomic approach, 47 differentially expressed proteins were identified from one or more cell line pair. These proteins include glutathione S transferase mu 3 (GSTM3), proteasome activator subunit 1 (PSME1), PSME2, PSMA7, L-plastin, cytokeratin 17, TRAP-1 and aldolase A. The differential expression of GSTM3, PSMA7, L-plastin, cytokeratin 17, TRAP-1 and aldolase A have so far also been confirmed by western blotting. Using expression microarray analysis, the expression of 69 genes was found to be significantly altered in one or more radiotherapy-resistant cell sublines. Real-time quantitative PCR expression was also used to confirm the differential expression of GSTM3, PSME1 and PSME2.

Conclusion

The development of these novel radiotherapy-resistant breast cancer cell sublines and a combined proteomic/transcriptomic complementary approach has identified candidate biomarkers that may be associated with radiotherapy resistance. In particular, proteasome activator subunits and GSTM3 appear to be of interest from both screening approaches. Further validation, functional and clinical evaluation is required, but this complementary screening approach has identified candidate biomarkers that may be involved in radioresistance and may reveal novel therapeutic targets in breast cancer.