Abstract
Skeletal metastasis occurs in around 75% of advanced breast cancers, with the disease incurable once cancer cells disseminate to bone, but there remains an unmet need for biomarkers to identify patients at high risk of bone recurrence. This study aimed to identify such a biomarker and to assess its utility in predicting response to adjuvant zoledronic acid (zoledronate). We used quantitative proteomics (stable isotope labelling by amino acids in cell culture-mass spectrometry; SILAC-MS) to compare protein expression in a bone-homing variant (BM1) of the human breast cancer cell line MDA-MB-231 with parental non-bone-homing cells to identify novel biomarkers for risk of subsequent bone metastasis in early breast cancer. SILAC-MS showed that dedicator of cytokinesis protein 4 (DOCK4) was upregulated in bone-homing BM1 cells, confirmed by western blotting. BM1 cells also had enhanced invasive ability compared with parental cells, which could be reduced by DOCK4-shRNA. In a training tissue microarray (TMA) comprising 345 patients with early breast cancer, immunohistochemistry followed by Cox regression revealed that high DOCK4 expression correlated with histological grade (p = 0.004) but not oestrogen receptor status (p = 0.19) or lymph node involvement (p = 0.15). A clinical validation TMA used tissue samples and the clinical database from the large AZURE adjuvant study (n = 689). Adjusted Cox regression analyses showed that high DOCK4 expression in the control arm (no zoledronate) was significantly prognostic for first recurrence in bone (HR 2.13, 95%CI 1.06-4.30, p = 0.034). No corresponding association was found in patients who received zoledronate (HR 0.812, 95%CI 0.176-3.76, p = 0.790), suggesting that treatment with zoledronate may counteract the higher risk for bone relapse from high DOCK4-expressing tumours. High DOCK4 expression was not associated with metastasis to non-skeletal sites when these were assessed collectively. In conclusion, high DOCK4 in early breast cancer is significantly associated with aggressive disease and with future bone metastasis and is a potentially useful biomarker for subsequent bone metastasis risk. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.