High Skp2/Low p57(Kip2) Expression is Associated with Poor Prognosis in Human Breast Carcinoma

Abstract

Downregulation of p57(Kip2) is involved in tumor progression, and S-phase kinase-associated protein 2 (Skp2) is an E3 ligase that regulates a variety of cell cycle proteins. However, the prognostic value of p57(Kip2) and its correlation with Skp2 in breast cancer have not been fully elucidated. Here we report our study on the expression of p57(Kip2) and Skp2 in 102 breast cancer patients by immunohistochemistry, and analysis of clinicopathologic parameters in relation to patient prognosis. The expression of p57(Kip2) was negatively associated with Skp2 expression in breast cancer (r = -0.26, P = 0.009). Kaplan-Meier analysis indicated that both high Skp2 and low p57(Kip2) correlated with poor disease-free survival (DFS) (P = 0.05), and a group with the combination of high Skp2/low p57(Kip2) demonstrated even worse DFS (log-rank = 21.118, P < 0.001). In addition, univariate analysis showed that Skp2, p57(Kip2), histological grade, lymph node metastasis, and estrogen and progesterone receptors (ER and PR) were all associated with DFS, and multivariate analysis revealed that lymph node metastasis and Skp2 were independent prognostic biomarkers. The correlation between p57 and Skp2 was further demonstrated in multiple breast cancer cell lines and cell cycle phases. Half-life and immunoprecipitation (IP) experiments indicated that Skp2 directly interacts with p57(Kip2) and promotes its degradation, rather than its mutant p57(Kip2) (T310A). Overall, our findings demonstrate that Skp2 directly degrades p57(Kip2), and an inverse correlation between these proteins (high skp2/low p57(Kip2)) is associated with poor prognosis in breast cancer. Thus, our results indicate a combined prognostic value of these markers in breast cancer diagnosis and treatment.

Keywords: Skp2; breast cancer; degradation; p57Kip2; prognosis.

Figure 1

Representative slides demonstrating expression of p57^Kip2 and Skp2 in breast cancer tissues. Tumor cells exhibit (A) high (×200) and (C) low (×200) Skp2 nuclear/cytoplasmic staining and (B) high (×200) and (D) low (×200) p57^Kip2 stains in breast cancer tissues.

Figure 2

Association between Skp2 or p57^Kip2 expression with overall survival. Disease-free survival was plotted as a function of (A) Skp2 expression, (B) p57^Kip2 expression, and (C) Skp2 high/p57 low group. Analysis was done by Kaplan–Meier method.

Figure 3

Reverse expression of Skp2 and p57^Kip2 in breast cancer cell lines. (A) Expression of p57^Kip2 and Skp2 in six breast cancer cell lines. β-actin was used as a loading control. (B) The results were quantified in the graphs in the right. (C) T47D cells were released from 18 h nocodazole (0.4 μM/mL)-induced prometaphase arrest and collected at the indicated times. Skp2 and p57^Kip2 expression showed reverse expression in different time points. The results were quantified to Skp2/p57^Kip2 ratio in the graph as (D).

Figure 4

Association of Skp2 with p57^Kip2 degradation in breast cancer cell lines. (A) After transfection with Skp2 siRNA or control for 48 hours, T47D cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then subjected to immunoblot analysis with antibodys to p57^Kip2 or Skp2. (B) Cells were transfected with HA-Skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours and then incubated with 10 μM MG132 for 4 hours. After that, cell extracts were immunoprecipitated with an antibody against Flag and analyzed by immunoblotting. (C) After transfection with HA-skp2 and Flag-p57 (WT) or Flag-p57 (T310A) for 48 hours, cells were incubated for the indicated times with CHX (20 μg/mL). Cell lysates were then used for immunoblot analysis with antibodies to Flag or HA. β-Actin was used as a loading control.