CAPG and GIPC 1 Breast Cancer Biomarkers for Bone Metastasis Development and Treatment
Background: Bone is the predominant site of metastasis from breast cancer, and recent trials have demonstrated that adjuvant bisphosphonate therapy can reduce bone metastasis development and improve survival. There is an unmet need for prognostic and predictive biomarkers so that therapy can be appropriately targeted. Methods: Potential biomarkers for bone metastasis were identified using proteomic comparison of bone metastatic, lungmetastatic, and nonmetastatic variants of human breast cancer MDA MB 231 cells. Clinical validation was performed using immunohistochemical staining of tumor tissue microarrays from patients in a large randomized trial of adjuvant zoledronic acid (zoledronate) (AZURE ISRCTN79831382) We used Cox proportional hazards regression, the Kaplan Meier estimate of the survival function, and the log rank test to investigate associations between protein expression, clinical variables, and time to distant recurrence events. All statistical tests were two sided. Results: Two novel biomarker candidates, macrophage capping protein (CAPG) and PDZ domain containing protein GIPC1 (GIPC1) were identified for clinical validation. Cox regression analysis of AZURE training and validation sets showed that control patients (no zoledronate) were more likely to develop first distant recurrence in bone (hazard ratio [HR] 4.5, 95% confidence interval [CI] 2.1 to 9.8, P .001) and die (HR for overall survival 1.8, 95% CI 1.01 to 3.24, P .045) if both proteins were highly expressed in the primary tumor. In patients with high expression of both proteins, zoledronate had a substantial effect, leading to 10 fold hazard ratio reduction (compared with control) for first distant recurrence in bone (P .008) Conclusions: The composite biomarker, CAPG and GIPC1 in primary breast tumors, predicted disease outcomes and benefit from zoledronate and may facilitate patient selection for adjuvant bisphosphonate treatment. The skeleton is the predominant site for metastasis in breast cancer, providing a fertile microenvironment for survival and growth of disseminated tumor cells. Bone targeted agents such as bisphosphonates and denosumab, which disrupt the destructive interactions between cancer and bone cells, are widely used to prevent skeletal complications of bone metastasis but have also been investigated as adjuvant agents to prevent or delay bone metastasis. Several large trials of This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:/creativecommons.org/licenses/by/3.0/ which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. D ow naded rom http/academ ic.p.com /jnci/article ab108/4/djv360/2412407 by gest on 10 April 2019 2 of 10 JNCI J Natl Cancer Inst, 2016, Vol. 108, No. 4 a r t ic le adjuvant bisphosphonate therapy in early breast cancer have now reported (1 3) including the open label, multicenter, phase III AZURE trial (BIG01/04 ISRCTN79831382) which recruited 3360 patients with stage II/III breast cancer randomized (1:1) to standard adjuvant therapy alone (control) or standard therapy with zoledronic acid (zoledronate) (19 doses of 4 mg in 5 years) (1) After a median of 84 months follow up, for the whole trial population, although there was no statistical difference in diseasefree survival, zoledronate reduced bone metastases (adjusted hazard ratio [HR] 0.78, 95% confidence interval [CI] 0.63 to 0.96, P .020) Moreover, in a preplanned analysis, zoledronate improved disease outcomes for women (n 1041) who were more than five years postmenopausal at diagnosis (adjusted HR for invasive disease free survival 0.77, 95% CI 0.63 to 0.96) (4) Furthermore, a meta analysis of individual patient data in 17 791 women from 22 randomized trials confirmed that in postmenopausal women adjuvant bisphosphonates reduced bone recurrences and breast cancer death by 34% (P .001) and 17% (P .004) respectively (5) These studies are likely to be practice changing but also highlight the unmet need for biomarkers to identify patients at risk of bone metastasis to guide selection for adjuvant bisphosphonate treatment. In the past decade, multiple gene expression datasets from analysis of breast cancer metastasis have identified key pathways underlying determinants of metastasis and provided information on which genes drive metastasis to specific organs, including the skeleton (6 11) Proteomic approaches also have high potential for the development of biomarkers for prediction of metastasis development (12) In this study, we have identified novel bone metastasis associated biomarkers from proteomics studies in cell lines, verified the increased expression of these proteins in bone homing cells, and carried out clinical validation in large training and independent validation sets on tissue microarrays (TMAs) from patients in the AZURE study, leading to a clinically validated composite biomarker with both prognostic and predictive utility. Methods Proteomic Analysis and Identification of Candidate Biomarkers Metastatic variants of the human breast cancer cell line MDA MB 231 home to bone (BM1, BM2) or lung (LM) when administered intravenously to nude mice, whereas the 'parental' MDA MB 231 cells (PCC) do not (8,13) We explored differences in the proteomes of BM1, BM2, LM, and PCC cells to identify differentially regulated proteins specifically associated with development of bone metastases in human breast cancer. Figure 1 indicates the key steps in our approach for the proteomic discovery of novel biomarkers. Differential protein expression between multiple independent cultures of these cell lines was quantified using two dimensional difference gel electrophoresis (2D DIGE) (14) Following analysis of the 2D DIGE gels, gel spots of interest were excised manually from silver stained DIGE gels, and tryptic peptides were generated for mass spectrometric analysis using an ingel digestion method. Proteins were identified using nano liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis on a QSTAR XL quadrupole time of flight hybrid mass spectrometer (AB Sciex, Warrington, UK) coupled online with an Agilent 1100 Series nano LC System (Agilent Technologies, Berkshire, UK) through electrospray ionization. The MS/MS raw data files from the LC/MS/MS analysis were processed by Analyst v2.0 and a script plug in Mascot.dll 1.6b24 (AB Sciex, Warrington, UK) then sent to the local Mascot database search engine (v2.3, Matrix Science, Boston, MA) Proteins with statistically significantly higher expression relative to PCC cells and greatest fold change in the bone metastatic cell lines (fold changes 2, P .029 by Wilcoxon MannWhitney test) but not with higher expression in the LM cells, were assessed for relevance in cancer and/or bone metastasis using published literature and verified by western blotting. Figure 1. Simplified flow diagram showing key proteomic steps used for discovery of novel biomarkers for risk of bone metastasis development. Proteins extracted from multiple independent cultures of cell lines were labeled using Cy5 fluorescent dye while an internal standard was labeled with Cy3. Separation of proteins (by isoelectric point and molecular weight) and image capture (fluoresence densitometry) creates protein array images that can be compared so as to detect differential protein expression between cell line types and replicates (intensity of fluoresence) Proteins of higher expression in bone homed cell lines were excised from silverstained two dimensional difference gel electrophoresis gels, reduced to peptides, and analyzed using tandem mass spectrometry. Identified proteins were assessed for known/reported relevance to breast cancer and/or bone metastasis prior to selection for validation of expression on breast cancer tissue microarrays. 2D DIGE twodimensional difference gel electrophoresis; IHC immunohistochemistry; LC/MS/MS liquid chromatography/mass spectrometry/mass spectrometry; TMA tissue microarray. D ow naded rom http/academ ic.p.com /jnci/article ab108/4/djv360/2412407 by gest on 10 April 2019 J. A. Westbrook et al. 3 of 10 a r t ic le Further details are available in the Supplementary Materials (available online) Clinical Validation of Potential Biomarkers of Bone Metastasis Risk Patients and Samples All analyses on patient samples were performed with Ethics approval and informed patient consent. Initially, protein expression of target molecules was characterized using a local TMA constructed from 364 breast cancer samples graded 1, 2, and 3 (Data Supplement, available online) The main patient based analyses were then performed on TMAs constructed from primary tumors from patients recruited into the AZURE trial (1) This provides an excellent resource for validation of protein biomarkers emerging from our proteomics studies because of the relatively high prevalence of bone metastatic outcomes and long follow up (median 84 months, interquartile range 66 93) Triplicate cores of breast tumor tissue were arrayed across replicate TMAs for immunohistochemistry. Immunohistochemistry Protein expression was assessed on TMAs using immunohistochemistry (15,16) Briefly, 5 mm serial sections of TMA were dewaxed in xylene and rehydrated through graded alcohols. Endogenous peroxidases were blocked (3% H202, 10 minutes) and antigens retrieved by microwaving slides. After cooling and washing, slides were blocked with goat serum (1:10; Zymed antibody diluent; 20 minutes) after which primary antibodies were applied (overnight, 4degC) Details of primary and secondary antibodies are presented in Supplementary Table 9 (available online) Following washing and incubation with HRP conjugated secondary antibodies, proteins were visualized using diaminobenzidine before counterstaining with haematoxylin, dehydration, and mounting.