Correlation of Skp2 overexpression to prognosis of patients with nasopharyngeal carcinoma from South China

Abstract

S-phase kinase-associated protein 2 (Skp2), which plays a role in cell cycle regulation, is commonly overexpressed in a variety of human cancers and associated with poor prognosis. However, its role in nasopharyngeal carcinoma (NPC) is not well understood. In this study, we examined the clinical significance of Skp2, with a particular emphasis on overall survival (OS) and disease-free survival (DFS), in NPC cases in South China, where NPC is an epidemic. Additionally, we explored the function of Skp2 in maintaining a cancer stem cell-like phenotype in NPC cell lines. Skp2 expression was assessed for 127 NPC patients using tissue microarrays and immunohistochemistry and analyzed together with clinicopathologic features, OS, and DFS. Skp2 expression was detectable, or positive, in 75.6% of patients. Although there was no correlation between Skp2 and any clinicopathologic factor, Skp2 expression significantly portended inferior OS (P = 0.013) and DFS (P = 0.012). In the multivariate model, Skp2 expression remained significantly predictive of poor OS [P = 0.009, risk ratio (RR) = 4.06] and DFS (P = 0.008, RR = 3.56), and this was also true for clinical stage (P = 0.012 and RR=3.201 for OS; P = 0.002 and RR=1.94 for DFS) and sex (P = 0.016 and RR=0.31 for OS; P = 0.006 and RR = 0.27 for DFS). After Skp2 knockdown, a colony formation assay was used to evaluate the self-renewal property of stem-like cells in the NPC cell lines CNE-1 and CNE-2. The colony formation efficiency in CNE-1 and CNE-2 cells was decreased. In Skp2-transfected CNE-1 and CNE-2 cells, side population (SP) proportion was increased as detected by flow cytometry. Skp2 is an independent prognostic marker for OS and DFS in NPC. Skp2 may play a role in maintaining the cancer stem cell-like phenotype of NPC cell lines.

Figure 1.. Immunohistochemical staining of Skp2 in tumor samples from 150 patients with nasopharyngeal carcinoma (NPC). A, the NPC tissue microarray shows that 23 samples dropped off the chip and 127 were assessable. B, representative image of a Skp2-positive sample shows that many tumor cells are stained in brown-yellow (×200). C, magnification of panel B shows brown-yellow granules in the nuclei of positive tumor cells (×400). D, representative image of a Skp2-negative sample shows that no tumor cells are stained in brown-yellow (×200). E, magnification of panel D shows no brown-yellow granules in the nuclei of tumor cells (×400).

Figure 2.. Kaplan-Meier survival curves of 96 Skp2-positive and 31 Skp2-negative patients with NPC. Skp2 expression is associated with poor overall survival (A, P = 0.013) and disease-free survival (B, P = 0.012) of patients with NPC.

Figure 3.. Knockdown of Skp2 significantly decreases the colony formation efficiency in human NPC cells. CNE-1 and CNE-2 cells were transfected with Skp2 siRNA (siSkp2) or scrambled siRNA (NC) for 48 h. A, Western blotting shows no Skp2 expression in Skp2 siRNA-transfected cells, and no changes of Skp2 expression in scrambled siRNA-transfected cells and untransfected cells. B, the colony formation assay shows that the number of colonies is significantly lower in Skp2 siRNA-transfected cells than in scrambled siRNA-transfected cells (P < 0.01).

Figure 4.. Overexpression of Skp2 increases the percentage of side population (SP) cells in human NPC cells. CNE-1 and CNE-2 cells were transiently transfected with pcDNA3.1-Skp2 plasmids or empty vector for 48 h. Hoechst 33342 staining was performed with or without 50 µmol/L verapamil. The percentage of SP cells significantly increased after Skp2 transfection; verapamil blocked ABCG2 activity and decreased SP cells.