Overexpression of CIP2A is an independent prognostic indicator in nasopharyngeal carcinoma and its depletion suppresses cell proliferation and tumor growth

Abstract

Background: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that acts as a prognostic marker for several human malignancies. In this study, we investigated the clinical significance of CIP2A and its function in nasopharyngeal carcinoma (NPC).

Methods: Quantitative RT-PCR, western blot, and immunohistochemistry analyses were used to quantify CIP2A expression in NPC cell lines and clinical samples. Kaplan-Meier curves were used to estimate the association between CIP2A expression and patient survival. The functional role of CIP2A in NPC cell lines was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation and xenograft growth.

Results: CIP2A levels were upregulated in NPC cell lines and clinical samples at both the mRNA and protein levels (P < 0.01). Patients with high CIP2A expression had poorer overall survival (HR, 1.98; 95% CI, 1.16-3.34; P = 0.01) and poorer disease-free survival (HR, 1.68; 95% CI, 1.07-2.62; P = 0.02) rates than patients with low CIP2A expression. In addition, CIP2A expression status was an independent prognostic indicator for NPC patients. The depletion of CIP2A expression inhibited c-Myc protein expression in NPC cell lines, suppressed cell viability, colony formation, and anchorage-independent growth in vitro, and inhibited xenograft tumor growth in vivo.

Conclusions: Our data demonstrate that high CIP2A expression in patients was associated with poor survival in NPC, and depletion of CIP2A expression inhibited NPC cell proliferation and tumor growth. Thus, these results warrant further investigation of CIP2A as a novel therapeutic target for the treatment of NPC.

Figure 1

Expression levels of CIP2A in NPC cell lines and clinical samples. (A-B) Expression levels of CIP2A mRNA (A) and protein (B) in NP69 and NPC cell lines. (C-D) Expression levels of CIP2A mRNA (C) and protein (D) in NPC tissues and normal nasopharyngeal epithelial tissues. GAPDH was used as the endogenous control. Data are presented as the mean ± SD, and P values were calculated with Student’s t-test.

Figure 2

Expression levels of CIP2A and survival of NPC patients. (A-H) Dicer1 protein expression is mainly localized to the cytoplasm. (A, E) Negative staining (A: 200×; E: 400×); (B, F) Weak staining: light yellow (B: 200×; F: 400×); (C, G) Moderate staining: yellow brown (C: 200×; G: 400×). (D, H) Strong staining: brown (D: 200×; H: 400×). (I-J) Patients with high CIP2A expression had poorer overall survival (I) and poorer disease-free survival (J) rates than patients with low CIP2A expression.

Figure 3

Effects of CIP2A depletion on MYC expression and NPC cell proliferation in vitro. (A-B) Effects of CIP2A siRNA on CIP2A and MYC protein expression in CNE-2 and SUNE-1 cells detected by western blot analysis (A) and immunofluorescence staining (B); (C-E) Effects of CIP2A siRNA on the cell viability (C), proliferation (D), and anchorage-independent growth (E) of CNE-2 and SUNE-1 cells. Data are presented as the mean ± SD. *P < 0.05 compared to the control using Student’s t-test.

Figure 4

Effects of CIP2A depletion on NPC xenograft tumor growth in vivo. (A) SUNE-1 cells stably expressing shCIP2A or scrambled control siRNA were subcutaneously injected into nude mice. SUNE-1 cells stably expressing shCIP2A had smaller tumors than scrambled controls after four weeks. (B) The growth curves of tumor volumes. (C) Representative picture of xenograft tumors. (D) Tumor weight. Data are presented as the mean ± SD. *P < 0.05 compared to the control using Student’s t-test.