A Novel Approach for the Discovery of Biomarkers of Radiotherapy Response in Breast Cancer

Abstract

Radiotherapy (RT) is an important treatment modality for the local control of breast cancer (BC). Unfortunately, not all patients that receive RT will obtain a therapeutic benefit, as cancer cells that either possess intrinsic radioresistance or develop resistance during treatment can reduce its efficacy. For RT treatment regimens to become personalised, there is a need to identify biomarkers that can predict and/or monitor a tumour’s response to radiation. Here we describe a novel method to identify such biomarkers. Liquid chromatography-mass spectrometry (LC-MS) was used on conditioned media (CM) samples from a radiosensitive oestrogen receptor positive (ER+) BC cell line (MCF-7) to identify cancer-secreted biomarkers which reflected a response to radiation. A total of 33 radiation-induced secreted proteins that had higher (up to 12-fold) secretion levels at 24 h post-2 Gy radiation were identified. Secretomic results were combined with whole-transcriptome gene expression experiments, using both radiosensitive and radioresistant cells, to identify a signature related to intrinsic radiosensitivity. Gene expression analysis assessing the levels of the 33 proteins showed that 5 (YBX3, EIF4EBP2, DKK1, GNPNAT1 and TK1) had higher expression levels in the radiosensitive cells compared to their radioresistant derivatives; 3 of these proteins (DKK1, GNPNAT1 and TK1) underwent in-lab and initial clinical validation. Western blot analysis using CM samples from cell lines confirmed a significant increase in the release of each candidate biomarker from radiosensitive cells 24 h after treatment with a 2 Gy dose of radiation; no significant increase in secretion was observed in the radioresistant cells after radiation. Immunohistochemistry showed that higher intracellular protein levels of the biomarkers were associated with greater radiosensitivity. Intracellular levels were further assessed in pre-treatment biopsy tissues from patients diagnosed with ER+ BC that were subsequently treated with breast-conserving surgery and RT. High DKK1 and GNPNAT1 intracellular levels were associated with significantly increased recurrence-free survival times, indicating that these two candidate biomarkers have the potential to predict sensitivity to RT. We suggest that the methods highlighted in this study could be utilised for the identification of biomarkers that may have a potential clinical role in personalising and optimising RT dosing regimens, whilst limiting the administration of RT to patients who are unlikely to benefit.