Proteomic analyses reveal high expression of decorin and endoplasmin (HSP90B1) are associated with breast cancer metastasis and decreased survival

Abstract

Background: Breast cancer is the most common malignancy among women worldwide in terms of incidence and mortality. About 10% of North American women will be diagnosed with breast cancer during their lifetime and 20% of those will die of the disease. Breast cancer is a heterogeneous disease and biomarkers able to correctly classify patients into prognostic groups are needed to better tailor treatment options and improve outcomes. One powerful method used for biomarker discovery is sample screening with mass spectrometry, as it allows direct comparison of protein expression between normal and pathological states. The purpose of this study was to use a systematic and objective method to identify biomarkers with possible prognostic value in breast cancer patients, particularly in identifying cases most likely to have lymph node metastasis and to validate their prognostic ability using breast cancer tissue microarrays.

Methods and findings: Differential proteomic analyses were employed to identify candidate biomarkers in primary breast cancer patients. These analyses identified decorin (DCN) and endoplasmin (HSP90B1) which play important roles regulating the tumour microenvironment and in pathways related to tumorigenesis. This study indicates that high expression of Decorin is associated with lymph node metastasis (p<0.001), higher number of positive lymph nodes (p<0.0001) and worse overall survival (p = 0.01). High expression of HSP90B1 is associated with distant metastasis (p<0.0001) and decreased overall survival (p<0.0001) these patients also appear to benefit significantly from hormonal treatment.

Conclusions: Using quantitative proteomic profiling of primary breast cancers, two new promising prognostic and predictive markers were found to identify patients with worse survival. In addition HSP90B1 appears to identify a group of patients with distant metastasis with otherwise good prognostic features.

Figure 1. Scoring system used for DCN and HSP90B1 immunohistochemistry.

A, Strong DCN positivity in stroma (3+) and negative in carcinoma (0) (magnification 200×). B, Strong DCN positivity in carcinoma (3+), weak stromal positivity (1+) (200×). C, Moderate HSP90B1 positivity in carcinoma (1+) (200×). D, Strong HSP90B1 positivity in carcinoma (3+) (200×). Decorin antibody (Sigma-Aldrich, St. Louis, MO) used at a dilution of 1∶400. HSP90B1 antibody (Sigma-Aldrich, St. Louis, MO) used at a dilution of 1∶4000.

Figure 2. Venn diagram of Differentially expressed proteins.

Number of proteins differentially expressed for each comparison group as well as overlapping proteins. Node-negative breast cancer tissue (group A), node positive BC tissue (group B), and normal breast tissue (group N).

Figure 3. Overall and Disease-free survival based on high and low DCN and HSP90B1 staining.

A, OS curve for DE. B, DFS curve for DE. C, OS curve for HE. D, DFS curve for HE. DE: Decorin staining in malignant epithelial tissue. HE: HSP90B1 staining in malignant epithelial tissue. OS: Time from diagnosis to death from any cause. DFS: Time from diagnosis to any recurrence or death from any cause.

Figure 4. Overall survival curves using combinations of DE and HE expression levels.

Univariate Cox regression used to determine HR; logrank p-values reported; Bonferroni multiple testing adjustment for pairwise comparisons p = 0.05/5 = 0.01. DE: Decorin staining in malignant epithelial tissue. HE: HSP90B1 staining in malignant epithelial tissue. HR: Hazard ratio.

Figure 5. Overall survival curves for high and low DE staining based on tumour molecular subtype.

Univariate Cox regression used to determine HR; logrank p-values reported. Molecular subtypes were defined by IHC expression of ER, HER2 and Ki-67 as suggested by Cheang et al. (2009) and Hugh et al. (2009). DE: Decorin staining in malignant epithelial tissue. HR: Hazard ratio.

Figure 6. Overall survival curves for high and low HE staining based on tumour molecular subtype.

Univariate Cox regression used to determine HR; logrank p-values reported. Molecular subtypes were defined by IHC expression of ER, HER2 and Ki-67 as suggested by Cheang et al. (2009) and Hugh et al. (2009). HE: HSP90B1 staining in malignant epithelial tissue. HR: Hazard ratio.

Figure 7. Overall survival curves based on DE/HE expression and hormone treatment.

A, Survival curves for cases with high and low DE that did not receive hormone treatment. B, Survival curves for cases with high and low DE that received hormone treatment. C, Survival curves for cases with high and low HE that did not receive hormone treatment. D, Survival curves for cases with high and low HE that received hormone treatment. Univariate Cox regression used to determine HR and 95% CI. DE: Decorin staining in malignant epithelial tissue. HE: HSP90B1 staining in malignant epithelial tissue.