Abstract
Background/Aim: To characterize the potential roles of CEP55 in colorectal cancer development and assess its eligibility as a prognostic diagnosis tool for colorectal cancer. Patients and Methods: Immunohistochemical (IHC) analysis of CEP55 immunoreactivity in 166 cancer specimens from colorectal cancer patients. Results: CEP55 was not found to statistically significantly affect different patient clinical parameters. Multivariate analysis illustrated that patients with N stage (1+2) colorectal cancer and high CEP55 expression had a significantly lower five-year survival rate than patients with N stage (1+2) colorectal cancer and low CEP55 expression. Conclusion: There is a correlation between CEP55 and advanced N-stage colorectal cancer. Thus, CEP55 may be a potential diagnostic biomarker for colorectal cancer patients.
Colorectal cancer holds the fourth highest incidence rate of all cancers in the USA and was estimated to account for 8.4%, or the second highest majority, of deaths due to cancers in 2019 (1). Though cancer screening policy contributes to early diagnosis and favorable clinical outcomes, cancer treatment remains a critical clinical challenge, especially for those with advanced stage cancers. When comparing the localized, regional, and distant stages of colon cancer, the five-year survival rate drops significantly between the prognostic and diagnostic stages of the disease, from 90% to 71%, and even as low as 14% (1). Therefore, using cancer staging or molecular prognostic markers to identify disease progression can provide important information for therapeutic decision making.
Some molecular markers are already viewed as early indicators of colorectal carcinogenesis (2). By using these discoveries, the present research sought to identify possible tumor markers that can be used to identify patients whose cancer is likely to relapse or metastasize. This would enable practitioners to schedule more intense follow-ups or treatments and thus, improve tumor outcomes. Cancer occurs under special conditions, and many potential tumor markers are based on the special characteristics of tumor growth and abnormal immunity. Abnormal cell cycles are important signifiers of these unique characteristics (3).
As part of a cell’s life cycle, cell abscission involves parallel pathways, including centriolin and midbody assembly (4). Many structural and regulatory proteins are required for midbody assembly, such as Aurora B, MKLP2, PRC1, and ECT2 (4). These proteins are targeted by centrosomal protein 55 (CEP55), which is normally strongly restricted to the testis, thymus, and placenta (5-7). These proteins are either absent or mislocalized from the midbody microtubules in CEP55-knockdown cells (4). During abscission, CEP55 at the midbody, recruits proteins that are directly involved in membrane fission, such as TSG101 and ALIX (8). This feature can make both the up-regulation and down-regulation of CEP55 result in unsuccessful cytokinesis, and even aneuploidy, which is a common characteristic of tumor cells (9). In cells with depleted CEP55, an increased proportion of multinucleation is observed (8). The up-regulation of CEP55 has also been reported in many types of cancer, such as bladder cancer, head and neck squamous cell carcinoma, gastric cancer, and colon cancer (6, 10-13).
Although CEP55 expression is a potential diagnostic biomarker for colorectal cancer, its prognostic role remains unclear (2, 6, 10). As such, this study aimed to investigate the potential association between CEP55 expression and the clinical outcomes of colorectal cancer patients.
Patients and Methods
Study subjects and ethics statement. This study included a total of 166 patients who received pathologically confirmed CRC diagnoses at Changhua Christian Hospital between 1997 and 2000. Patients with histories of other malignancies or missing clinical data were excluded. Cancers were staged according to the American Joint Committee on Cancer (AJCC) Colon Cancer Staging, 7th edition. Clinical data, including gender, age, stage, T, N, and M stages, and follow-up information, were obtained from an established database (14). This study was approved, and consent was waived by the Institutional Review Board and the Ethics Committee of the Changhua Christian Hospital, Changhua, Taiwan (IRB No. 121008).
Immunohistochemistry staining of CEP55. Immunohistochemistry (IHC) staining was performed at the Department of Surgical Pathology, Changhua Christian Hospital, Changhua, Taiwan, as previously described (15, 16). IHC staining were performed on tissue microarray sections (4 μm) of formalin-fixed, paraffin-embedded, pre-chemotherapy primary colorectal tumors. The antibody used was anti-human CEP55 (CEP55 antibody, sc-374051, 1:50 dilution, Santa Cruz Biotechnology, Dallas, TX, USA).
Evaluation of CEP55 immunoreactivity. Immunoreactivity was analyzed by pathologists using a previously described scoring system (14, 15). In brief, Immunoreactivity scores were defined as cell staining intensity (0=nil; 1=weak; 2=moderate; and 3=strong) multiplied by the percentage of stained cells (0-100%), resulting in scores ranging from 0 to 300 (15).
Statistical analysis. The Student t-test, and the χ2 test were applied for continuous or discrete data analysis. The associations between the CEP55 expression and patient survival outcome were estimated using the Kaplan–Meier method and assessed using the log-rank test. Potential confounders were adjusted by Cox regression models, with the CEP55 expression fitted as indicator variables. Overall survival time was defined as the interval between the date of surgery and the date of last follow-up or death. All statistical analyses were conducted using the SPSS statistical software program (version 15.0) (SPSS, Inc., Chicago, IL, USA). All statistical tests were two-sided, and p<0.05 was considered statistically significant.
Results
CEP55 expression is not significantly associated with clinical parameters. This study analyzed a total of 166 patients with colorectal cancer, including 71 females and 95 males. Representative IHC staining is shown in Figure 1. The average age of the patients was 64 years (SD: 13.4). At least 19% of the patients were diagnosed in the M1 stage, and 13% of the patients were diagnosed in stage 1. We stratified cases into different groups according to clinical parameters, such as age, gender, stage, T value, N value, and M value. There are no significant differences in CEP55 expression levels related to these parameters (Table I).
Representative immunostaining of (A) low and (B) high expression of CEP55 in colorectal cancer specimens.
Relationships of CEP55 expression with clinical parameters in colorectal cancer patients.
CEP55 is an independent prognostic marker for patients with advanced N-stage cancer. We used univariate analysis to determine the prognostic role of CEP55. As illustrated in Table II, patients with stage II, III, or IV cancer had lower overall five-year survival rates than patients with stage I cancer, as expected (stage II, III, and IV: 40%, stage I: 73%, p=0.05). However, CEP55 expression was not statistically significant as a prognostic marker (Table II and Figure 2A). The result did not change under multivariate analysis (HR=1.317, 95%CI=0.893-1.943, p=0.165, Table III). We further verified the prognostic role of CEP55 according to the subgroup of clinical parameters. As shown in Table III, CEP55 played no significant role under most of the subgroups. Nevertheless, in patients with advanced N stage cancer, lower CEP55 levels were associated with significantly poorer clinical outcomes (HR=1.896, 95%CI=1.034-3.475, p=0.039, Table III and Figure 2B).
Univariate analysis of the influence of various parameters on overall survival in colorectal cancer patients.
Kaplan–Meier actuarial analysis of CEP55 expression with respect to overall survival among (A) all patients and (B) patients with N=1+2.
Multivariate analysis of the influence of CEP55 expression according to clinical parameters on overall survival in colorectal cancer patients.
Discussion
As a centrosome-associated protein, CEP55 is capable of homodimerization (7). It transfers from the centrosome to the midbody when the cell cycle enters the cytokinesis stage (4). The midbody is regulated by proteins, such as Aurora B, MKLP2, PRC1, and ECT2. In addition, it has been reported that CEP55 plays an important role in the membrane fission event of cytokinesis by regulating these proteins (7). The up-regulation of CEP55 can result in aneuploidy and thus, an increase in the number of multinucleated cells, which may lead to tumorigenesis (13). Over-expression of CEP55 has been found in hepatocarcinoma, oral squamous cell carcinoma, lung cancer, ovarian carcinoma, and colon carcinoma (10, 17-20). Furthermore, CEP55 expression levels vary significantly in size and degree (13, 17, 21). This variation can also be found in patients with different stages of lymph node metastases (17, 20, 21). Since research has already identified correlations between lymph node metastases, CEP55, and many types of cancers, the present study sought to determine if there are any similar correlations between these factors and colorectal cancer.
To our knowledge, this is the first study to uncover the relationship between CEP55 expression and advanced N stage colorectal cancer by using multi-variate analysis of 166 patients, as previous research has only indicated that CEP55 is over-expressed in patients with colorectal cancer (22). This discovery may prove fundamental to the application of CEP55 expression as a prognostic tool for N-stage colorectal cancer. Furthermore, because we determined that the presence of CEP55 expression can affect the survival rate of patients with lymph node metastases, future studies should aim to establish the mechanisms behind this phenomenon. No relationships were determined in the comparisons of the collected data. With CEP55 expression, the five-year survival rate dropped significantly for colorectal cancer patients with lymph node metastases (Table III). This finding is consistent with the results of previous studies of other types of cancers (17, 20, 21). However, due to the lack of statistical significance (p-value=0.372, Table II), the extent of CEP55 expression does not appear to impact survival rate.
On the other hand, our research identified no similar connections in patients with M0 or M1 stages of cancer. This may be because patients in the M0 stage of cancer can present with mild sickness or more serious symptoms. This diversity in disease presentation may decrease the correlation between CEP55 and the M0 stage of cancer. In addition, because M1 is classified as the worst prognostic status of cancer with the lowest survival rate, it may mask the impact of CEP55 expression on M1 patients’ clinical outcomes. Indeed, the present study’s results showed that CEP55 had no impact on this group.
This study had several limitations. First, the statistical results indicated that CEP55 expression had no impact on long-term survival when using the 12-year survival rate as an example (Figure 1). A potential cause of this factor may have been the limited number of patients included in this study. Follow-up research studies with more patients may provide the answer. Second, because this study only enrolled patients from Taiwan, which may have resulted in selection bias, subsequent research should consider selecting patients from various regions. Third, this study used a hazard ratio for analysis, which can only show instantaneous risks and may have resulted in selection bias due to the imbalanced number of patients with low and high CEP55 expression and the varying survival rates (23). Therefore, it may prove useful for future studies to establish other parameters, such as the relative risk and odds ratios.
In conclusion, our research shows that the expression of CEP55 in colorectal tumor patients is associated with lymph node metastases and, therefore, a poorer five-year survival rate. Based on this correlation, CEP55 should undergo further investigation as a potential prognostic marker.
Footnotes
Authors’ Contributions
RHH and CMY performed the experiments. RHH and WSS designed the study. WWS performed the statistical analyses. CMY and WWS analyzed and interpreted the data. WKY and CMW drafted the manuscript. RHH and WWS critically revised the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare that they have no competing interests relative to this study.
- Received December 10, 2020.
- Revision received December 20, 2020.
- Accepted December 21, 2020.
- Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
