Nucleobindin 2 in human breast carcinoma as a potent prognostic factor

Abstract

It is well-known that estrogens immensely contribute to the progression of human breast carcinoma, but their detailed molecular mechanisms remain largely unclear. In this study, we identified nucleobindin 2 (NUCB2) as a gene associated with recurrence based on microarray data of estrogen receptor (ER)-positive breast carcinoma cases (n = 10), and subsequent in vitro study showed that NUCB2 expression was upregulated by estradiol in ER-positive MCF-7 cells. However, NUCB2 has not yet been examined in breast carcinoma, and its significance remains unknown. Therefore, we further examined the biological functions of NUCB2 in breast carcinoma using immunohistochemistry and in vitro studies. NUCB2 immunoreactivity was detected in carcinoma cells in 77 of 161 (48%) breast cancer cases, and positively associated with lymph node metastasis and ER status of the patients. In addition, NUCB2 status was significantly associated with an increased risk of recurrence and adverse clinical outcome of the patients using both univariate and multivariate analyses. Results of siRNA transfection experiments showed that NUCB2 significantly increased cell proliferation, and migration and invasion properties in both MCF-7 and ER-negative SK-BR-3 cells. These results suggest that NUCB2 is upregulated by estrogens and plays an important role, especially in the process of metastasis, in breast carcinomas. NUCB2 status is considered a potent prognostic factor in human breast cancer.

Figure 1

Nucleobindin 2 (NUCB2) as an estrogen‐induced gene associated with breast carcinoma. (A) Scatter plot analysis of microarray data for 519 genes containing functional estrogen‐responsive element in breast carcinomas comparing the recurrence and non‐recurrence group (n = 5 in each group). Genes with an expression ratio, recurrence group to non‐recurrence group, of more than 2.0 or <0.5 are located outside the diagonal line, and classified as group A or group B, respectively. Genes with a ratio between 2.0 and 0.5 were classified as group C. NUCB2 showed the highest ratio in these genes (arrow). The right panel summarizes the gene list of group A. (B,C) Effects of estradiol on NUCB2 mRNA expression. MCF‐7 cells were treated with indicated concentrations of estradiol with or without (−) ICI 182780 or tamoxifen for 3 days (B) or treated with estradiol (10 nM) for the indicated period (C). The relative NUCB2 mRNA level summarized as a ratio (%) compared with the basal level (non‐treatment). Data are presented as the mean ± SD (n = 3). *P < 0.05 and ***P < 0.001 versus non‐treatment (left bar) (B) or 0 h (left plot) (C).

Figure 2

Immunohistochemistry for nucleobindin 2 (NUCB2) in breast carcinoma. (A) Immunoblotting for NUCB2 in MCF‐7 cells. MW, molecular weight. (B) NUCB2 immunoreactivity was detected in the carcinoma cells of invasive ductal carcinoma. (C) NUCB2 immunoreactivity was weakly and focally detected in morphologically normal mammary glands. (D) Positive control section of NUCB2 immunohistochemistry (gastric mucosa). (E) Negative control section of NUCB2 immunohistochemistry (same area as Fig. 2D). (F) NUCB2 immunoreactivity was detected in the carcinoma cells of ductal carcinoma in situ. Bar = 100 μm.

Figure 3

Disease‐free and breast cancer‐specific survival of 141 breast carcinoma patients according to nucleobindin 2 (NUCB2) status. (A,B) NUCB2 status was significantly associated with an increased risk of recurrence (P = 0.003) (A) and worse prognosis (P = 0.0002) (B). Solid line, positive for NUCB2 (n = 66); dashed line, negative for NUCB2 (n = 75). (C) Disease‐free survival curve according to NUCB2/Ki‐67 status. Solid line, positive for NUCB2/Ki‐67 labeling index (LI) ≥ 10% (n = 54); dashed line, positive for NUCB2/Ki‐67 LI < 10% (n = 12); dotted line, negative for NUCB2/Ki‐67 LI ≥ 10% (n = 50); dot‐dashed line, negative for NUCB2/Ki‐67 LI < 10% (n = 25). (D) Disease‐free survival curve according to NUCB2 immunointensity. Solid line, strongly positive (n = 16); dashed line, modestly positive (n = 50); dot‐dashed line, negative (n = 75). (E) NUCB2 status was associated with recurrence in 40 patients who received tamoxifen therapy. P‐value not available as there were no patients with recurrent disease in the NUCB2‐negative group. Solid line, positive for NUCB2 (n = 16); dashed line, negative for NUCB2 (n = 24). (F) NUCB2 status was significantly (P = 0.001) associated with recurrence in a group with estrogen receptor labeling index < 1% (n = 24). Solid line, positive for NUCB2 (n = 7); dashed line, negative for NUCB2 (n = 17).

Figure 4

Effects of nucleobindin 2 (NUCB2) on proliferation (A), and migration (B) and invasion (C,D) properties in breast carcinoma cells. (A–C) MCF‐7 (gray bar) and SK‐BR‐3 (open bar) were transfected with NUCB2‐specific siRNA (si1, si2) or control siRNA (siC). The relative cell proliferation was evaluated as a ratio (%) compared to that at 0 day after treatment (A). Migration ability was evaluated as an average number of cells in five middle power fields (MPF) (×200) on the lower surface of the membrane (B). Invasion ability was evaluated as the total number of cells (C). Data are presented as the mean ± SD (n = 6 [A]; n = 3 [B,C]). *P < 0.05; **P < 0.01; ***P < 0.001 versus control cells (left bar). (D) Representative microphotographs of results of invasion assay. Invaded carcinoma cells (arrows) were observed with 8 μm‐sized pore. Nucleii stained with hematoxylin. When MCF‐7 (upper panel) and SK‐BR‐3 (lower panel) cells were transfected with NUCB2‐specific siRNA (si2) (right panel), the number of invading cells was decreased compared to the corresponding control (transfection with control siRNA [siC], left panel). Bar = 100 μm.