Proteomic identification and validation of biomarkers associated with resistance to radiotherapy in breast cancer

Background:Breast cancer is an important health issue. The majority of patients present with early stage cancer and are therefore candidates for breast conserving surgery and radiotherapy. A proportion will suffer from local recurrence, which may be secondary to radiotherapy resistance. Though extensive research has been carried out into molecular markers of resistance, none has been applied to clinical practice, which suggests that the search for such markers is wanting.Materials and Methods:The principle of the biomarker discovery pipeline was applied and cancer cell lines were utilised for the first two phases of this project. Protein expression in radiosensitive and radioresistant cell lines was compared using, first antibody microarray technology (AbMA), as a screening tool, and secondly, western blot (WB) technique as a verification tool. The final stage was clinical validation. A clinical series of archival breast cancer tissue was identified; one representing a radiosensitive group, and a second representing a radioresistant group. Immunohistochemistry (IHC) was then employed to compare the differential protein expression between the two.Results:The AbMA technology was successfully utilised to yield 63 potential biomarkers of radioresistance. Of these, zyxin, PIASx and DR4 were confirmed using WB. Clinical validation showed no association between zyxin and radioresistance; this protein had been previously suggested to be associated with cellular stress. DR4 has been clinically validated using IHC, and has therefore been identified as a putative biomarker using all three techniques. In addition, the association between radioresistance and the 26S proteasome was clinically validated.Discussion:This work supports the role of zyxin as a stress associated protein. The underexpression of DR4, a pro-apoptotic factor, and 26S proteasome, a major effector in the protein proteolysis machinery and cell cycle has been proven. These two proteins present putative markers of radioresistance. The possibility of pre-treatment definition of the expected response to radiation therapy would improve patients’ outcome. Radiation can be offered only to those expected to respond to it, while others would be offered other treatment modalities