CAPG and GIPC1: Breast Cancer Biomarkers for Bone Metastasis Development and Treatment

Abstract

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, lung-metastatic, 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.

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 silver-stained 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 = two-dimensional difference gel electrophoresis; IHC = immunohistochemistry; LC/MS/MS = liquid chromatography/mass spectrometry/mass spectrometry; TMA = tissue microarray.

Figure 2.

Macrophage-capping protein (CAPG) and PDZ domain–containing protein GIPC1 (GIPC1) protein expression in cells and patient breast cancer tissue. Left hand panel shows western blot verification of high CAPG and GIPC1 protein expression in BM1 and BM2 cell lysates compared with parental cells (expression changes >2-fold with P < .03, by Student’s t test); upper part shows blot expression with loading control. Data are means ± SD. Right hand panel illustrates examples of differential protein expression in AZURE tissue microarray breast tumor cores for CAPG and GIPC1. In each case, the antibody localization has been used in conjunction with diaminobenzidine (DAB; brown), and the scoring is based on the intensity of staining in the cytoplasmic compartment in the tumor cells only. Scale bar = 200 µm. CAPG = macrophage-capping protein; GIPC1 = PDZ domain–containing protein GIPC1; PCC = MDA-MB-231 cells.

Figure 3.

Control arm of the AZURE trial population. Kaplan-Meier estimates of the survival function showing association between protein expression and time to first distant recurrences (DRs). Upper left) Training set; first DR is skeletal only. Upper right) Training set; first DR includes skeletal as well as other distant site(s). Lower left) Validation set; first DR is skeletal only. Lower right) Validation set; first DR includes skeletal as well as other distant site(s). High indicates a tissue microarray (TMA) score of 3. Low indicates TMA score of 1 or 2. P values refer to the two-sided log-rank test. CAPG = macrophage-capping protein; GIPC1 = PDZ domain–containing protein GIPC1.

Figure 4.

Combined sets. Kaplan-Meier estimates of the survival function for time to distant recurrence and overall survival for control and zoledronate arms. High signifies both macrophage-capping protein (CAPG) and PDZ domain–containing protein GIPC1 (GIPC1) high; low signifies other combinations where not both CAPG and GIPC1 are high. A and B) Skeletal only; first distant recurrence only skeletal. C and D) Skeletal and other; first report of distant recurrence included skeletal and other site(s) of metastasis. E and F) Nonskeletal; first distant recurrence does not contain any skeletal component. G and H) First skeletal recurrence irrespective of timing and sites of local and nonskeletal recurrences. I and J) Overall survival. P values refer to the two-sided log-rank test. DR = distant recurrence; OS = overall survival.

Figure 5.

Forest plot showing effect of zoledronate in subgroups defined by the bivariate score composed of macrophage-capping protein and PDZ domain–containing protein GIPC1 in relationship to time to distant recurrence events and overall survival. Skeletal only (first distant recurrence event only skeletal); skeletal and other (first distant recurrence event reported includes skeletal and other site[s] of metastasis); nonskeletal (first distant recurrence event does not include skeletal recurrence); first skeletal (first skeletal event irrespective of whether other distant events have occurred first). All hazard ratios are adjusted for systemic therapy plan, estrogen receptor status, and lymph node involvement. Hazards ratios of less than 1 indicate improvement with zoledronate. CI = confidence interval; HR = hazard ratio.