Identification of Nucleobindin-2 as a Potential Biomarker for Breast Cancer Metastasis Using iTRAQ-based Quantitative Proteomic Analysis

Abstract

Metastasis is a lethal step in the progression of breast cancer. None of the metastasis-associated biomarkers identified up to now has a definite prognostic value in breast cancer patients. This study was designed to identify biomarkers for breast cancer metastasis and predictors of the prognosis of breast cancer patients. The differentially expressed proteins between 23 paired primary breast tumor and metastatic lymph nodes were identified by quantitative iTRAQ proteomic analysis. Immunohistochemistry was applied to locate and assess the expression of NUCB2 in paired primary breast tumor and metastatic lymph node tissues (n = 106). The relationship between NUCB2 expression and the clinicopathological characteristics of breast cancer patients (n = 189) were analyzed by χ² test. Kaplan-Meier analysis and Cox hazard regression analysis were utilized to investigate the relationship between its expression and prognosis of breast cancer patients. The iTRAQ proteomic results showed that 4,837 confidential proteins were identified, 643 of which were differentially expressed in the primary breast cancer tissues and the paired metastatic lymph nodes. NUCB2 protein was found decreased in paired metastatic lymph nodes (P = 0.000), with the positive expression rate being 82% in primary breast cancer tissues and 47% in paired metastatic lymph nodes, respectively. According to Kaplan-Meier analysis, the overall survival time of patients with positive expression of NUCB2 protein were shorter than those with negative NUCB2 expression (P = 0.004). Cox regression model suggested that NUCB2 was a risk factor of breast cancer patients (P = 0.045, RR = 1.854). We conclude that NUCB2 can be used as a potential biomarker for breast cancer metastasis and a prognostic predictor of breast cancer patients.

Keywords: Breast cancer; Metastasis; Nucleobindin-2.; Prognosis; iTRAQ.

Figure 1

Functional groups of differentially expressed proteins between the primary breast tumors and metastatic lymph nodes determined by GO analysis. The top ten functional groups of proteins up-regulated (A) or down-regulated (B) in metastatic lymph nodes compared with primary breast tumors are shown.

Figure 2

Expression of NUCB2 protein in primary breast tumors and metastatic lymph node tissues. The paraffin-embedded tissue sections were immunohistochemically stained for NUCB2 expression and statistically evaluated by the χ² test. (A) Representative immunohistochemical images showing the predominantly cytoplasmic staining of NUCB2 in breast tumor samples. Original magnification: 200×. (B) Summary of the positive expression rate of NUCB2 in breast tumor samples. PBT, primary breast tumor; MLN, metastatic lymph node.

Figure 3

Kaplan-Meier survival curves of patients with positive and negative NUCB2 expression. The overall survival rate of breast cancer patients that were positive or negative for NUCB2 expression was calculated with the Kaplan-Meier method and examined with log-rank test. There was significant difference between the two groups (log-rank P = 0.004).