CDC20 overexpression predicts a poor prognosis for patients with colorectal cancer

The human colon epithelial cell line, NCM460, was obtained from Sun Yat-sen University Cancer Center; two other human colon epithelial cell lines, CCD841-coN and CCD112-coN, were purchased from ATCC. Human colon cancer cell lines DLD1, HT29, HCT116, Lovo, SW620 and THC8307 were obtained from Sun Yat-sen University Cancer Center. NCM460, CCD841-coN and CCD112-coN cells were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS). DLD1, HT29, HCT116, Lovo and THC8307 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. SW620 cells were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS. All cells were grown in 10-centimeter cell culture dishes (NEST Biotechnology, Wuxi, China) in 5% CO₂ in a humidified atmosphere at 37°C.

A total of 244 paraffin-embedded archived samples were used for immunohistochemistry, including 126 samples with adjacent non-tumorous (ANT) tissues and 20 samples with liver metastasis. All patients underwent their initial surgery between 2001 and 2009 at Sun Yat-sen University Cancer Center after providing informed consent. None of these patients received preoperative therapy. Informed consent from patients and approval from the Institute Research Ethics Committee were obtained before the use of the clinical materials. Clinical and pathologic classification and staging were determined according to the American Joint Committee on Cancer (AJCC) TNM staging system. Table 1 shows the clinical information related to the 244 CRC samples. Overall survival (OS) was defined as the interval between the date of surgery and the date of death or last known follow up.

CDC20 expression was compared between colon cancer cells and colon normal epithelial cells by Western blotting analysis, as described previously[14]. CDC20 protein expression was determined with anti-rabbit immunoglobulin G (1:2000; Bethyl) according to the manufacturer's suggested protocols. A rabbit anti-α-tubulin monoclonal antibody (1:20,000; Abcam) was used as the loading control.

Altered CDC20 protein expression was also studied in 244 human colorectal cancer tissues by immunohistochemistry, as described previously[15]. Briefly, the tissue sections were deparaffinized, rehydrated, endogenous-peroxide-blocked and antigen-retrieved sequentially and were then incubated with a rabbit anti-CDC20 antibody (1:150; Bethyl) overnight at 4°C. Then, the tissue sections were washed with PBS and treated with anti-rabbit secondary antibody for 20 minutes, followed by further incubation with the streptavidin horseradish peroxidase complex. The sections were developed with diaminobenzidine tetrahydrochloride (DAB) and further counterstained with hematoxylin. The degree of immunostaining was evaluated by two independent observers who were blind to the clinical data of the patients. The percent of positive cells was scored as ≤ 10% = 0, >10% to ≤ 25% = 1, >25% to ≤ 50% = 2, >50% to ≤ 75% = 3 or >75% = 4. The intensity of nuclear staining was scored as negative = 0, weak = 1, moderate = 2, or strong = 3. The two scores were then multiplied to calculate the final score. CDC20 expression was considered low if the final score was equal to or less than four; otherwise, CDC20 expression was considered high.

As shown in Figure 1, CDC20 expression was evaluated in colorectal cancer cell lines and tissues to investigate the role of CDC20 in tumorigenesis. Western blotting analysis revealed elevated CDC20 expression in colon cancer cell lines (DLD1, HT29, HCT116, Lovo, SW620 and THC8307) compared with normal colon epithelial cell lines (NCM460, CCD841-coN and CCD112-coN) (Figure 1A, upper row) (Figure 1A, lower row). CDC20 overexpression was further evaluated by IHC in 244 paraffin-embedded colon cancer tissues. As shown in Figure 1B, CDC20 was primarily localized in the cancer cell nucleus. Interestingly, a comparison of CDC20 expression in tumor tissues and ANT tissues in the paired 126 cases showed significant CDC20 overexpression in cancer cells (Figure 1B, right panel, P < 0.001, Wilcoxon signed rank test); the representative slides are shown in Figure 1B, left panel. Moreover, CDC20 expression was further increased in the matched metastatic liver tissues compared to the primary cancer tissues, and representative slides are shown in Figure 1C, left panel. The primary colorectal cancer tissues were compared to their respective metastatic liver tissues, and the increase was significant (Figure 1C, right panel, P < 0.001, Wilcoxon signed rank test). These results together suggested CDC20 overexpression in colorectal cancer.

We further analyzed the link between CDC20 expression and the clinical characteristics of colorectal cancer in 244 paraffin-embedded, archival primary colorectal cancer tissues by IHC. The samples included 42 cases of clinical stage I (17.2%), 53 cases of clinical stage II (21.7%), 72 cases of clinical stage III (29.5%) and 77 cases of clinical stage IV (31.6%) colorectal cancer. As shown in Table 1, 114 of the total 244 CRC cases (46.7%) demonstrated high CDC20 expression, whereas 130 cases (53.3%) had low CDC20 expression.

As shown in Table 2, a statistical analysis revealed a strong correlation between CDC20 expression and the clinicopathologic characteristics including clinical stage (P = 0.008), N classification (P = 0.020), M classification (P = 0.013), pathologic differentiation (P = 0.008) and vital status (P < 0.001). Representative IHC stained slides are presented to demonstrate the correlation between CDC20 expression and clinical stage (Figure 2A) and pathologic differentiation (Figure 2B), respectively.

A Kaplan-Meier analysis and the log-rank test were carried out to evaluate the effects of CDC20 expression and clinicopathological characteristics on patient survival. Interestingly, CDC20 expression in colorectal cancer showed a negative correlation with patient survival time (P < 0.001). High CDC20 expression indicated a shorter overall survival (OS) (median OS: 44 months) compared to lower CDC20 expression (median OS: not reached) (Figure 3A, P < 0.001). The overall two-, three- and five-year cumulative survival rates of patients with high CDC20 expression were 61.4%, 45.8% and 27.2%, respectively. For patients with low CDC20 expression, the rates were 81.5%, 72.3% and 53.8%, respectively (Figure 3A). Furthermore, CDC20 expression was significantly correlated with OS in the advanced clinical stages (stage III and IV) (Figure 3C-3D, P = 0.026 (C); P < 0.001(D)). However, no significant correlation between OS and CDC20 expression was found in the early clinical stages (stages I and II) (Figure 3B, P = 0.826). Moreover, multivariate analysis indicated that N classification, M classification, pathologic differentiation and CDC20 expression were independent prognostic factors for colorectal cancer (Table 3).