CIP2A influences survival in colon cancer and is critical for maintaining Myc expression

Abstract

The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor that stabilises the c-Myc protein. CIP2A is overexpressed in several tumours, and expression levels are an independent marker for long-term outcome. To determine whether CIP2A expression is elevated in colon cancer and whether it might serve as a prognostic marker for survival, we analysed CIP2A mRNA expression by real-time PCR in 104 colon cancer samples. CIP2A mRNA was overexpressed in colon cancer samples and CIP2A expression levels correlated significantly with tumour stage. We found that CIP2A serves as an independent prognostic marker for disease-free and overall survival. Further, we investigated CIP2A-dependent effects on levels of c-Myc, Akt and on cell proliferation in three colon cancer cell lines by silencing CIP2A using small interfering (si) and short hairpin (sh) RNAs. Depletion of CIP2A substantially inhibited growth of colon cell lines and reduced c-Myc levels without affecting expression or function of the upstream regulatory kinase, Akt. Expression of CIP2A was found to be dependent on MAPK activity, linking elevated c-Myc expression to deregulated signal transduction in colon cancer.

Figure 1. CIP2A mRNA expression is significantly correlated with advanced tumour stage.

The panels show box and whisker plots documenting relative CIP2A mRNA levels in tumors stratified according to UICC stage (A) (UICC I vs. II p<.0001; II vs. III p = .0046; III vs. IV p = .0002), according to lymph node metastasis (B; N- vs. N+ p<.0001), according to distant metastasis (C; M0 vs. M1 p<.0001), and according to histological grading (D: G2 vs. G3 p = .0084).

Figure 2. Patients with CIP2A high mRNA expression have an overall lower survival rate than patients with CIP2A low mRNA expression.

The graphs show Kaplan–Meier curves of OS according to CIP2A mRNA expression. (red: CIP2A mRNA expression below median fold expression value of 10,5 above normal tissue), green: CIP2A mRNA expression above median fold expression value of 10,5 above normal tissue) (A & B) All patients with respect to CIP2A mRNA expression normalized to housekeeping gene (n = 75) (A: b2MG; B: GAPDH) (C) Patients in Stage UICC II with respect to CIP2A mRNA expression normalized to housekeeping gene GAPDH (n = 29) (D) Patients in Stage UICC III with respect to CIP2A mRNA expression normalized to housekeeping gene GAPDH (n = 21).

Figure 3. CIP2A protein levels in colon cancer correlate with CIP2A mRNA expression.

The panels show representative examples of immunofluorescence staining, showing CIP2A protein expression in cancer cells of patients with low (A+B) or high CIP2A (C+D) mRNA expression (A+C x100, B+D x200 magnification).

Figure 4. Depletion of CIP2A downregulates c-Myc protein expression in colon cancer cells.

(A) Immunoblot analysis of CIP2A and c-Myc protein expression in Caco2, HCT116 and SW620 cells transfected with siRNA targeting CIP2A or control siRNA. Cells were harvested 72 h after transfection (n = 3 for each cell line). (B) Real-time PCR analysis of CIP2A and c-Myc mRNA expression in HCT116 cells transfected with siRNA targeting CIP2A or control siRNA (n = 3). (C) Depletion of CIP2A does not change activation status of AKT or its downstream targets. The panels show immunoblots of the indicated proteins and phosphorylated proteins (p) in Caco2, HCT116 and SW620 cells transfected with siRNA targeting CIP2A or control siRNA as before (n = 3 for each cell line).

Figure 5. CIP2A is required for growth of HCT116 cells.

(A) Immunoblot analysis of CIP2A and c-Myc protein expression in HCT116 cells infected lentiviral with shRNA targeting CIP2A or a ctr. shRNA. Numbers below lines indicate the c-myc protein expression relative to c-myc levels in control cells (n = 2). (B) Colony formation of HCT116 cells after 7 days. (Top), density of colonies stained with crystal violet; (bottom), representative of the indicated cultures (n = 3).

Figure 6. CIP2A expression is regulated by MAPK signalling.

Caco2, HCT116 and SW620 cells were treated with DMSO or the MEK inhibitor UO126 for 24(n = 3 for each cell line). (A) Immunoblot analysis of CIP2A and p-ERK protein expression in Caco2, HCT116 and SW620. (B) Real-time PCR analysis of CIP2A mRNA expression (*<0.05; **<0.005).