Kang et al (28) performed immunohistochemical staining analyses and identified that CKS2 was more strongly expressed in the less differentiated area compared to the well differentiated area, even in the same tumor tissue of each patient, occasionally. The study divided 60 cases of gastric cancer tissues into two groups; low and high CKS2 expression levels. Subsequently, the SPSS^® software (SPSS, Inc., Chicago, IL, USA) package was used to determine the association between the results of the immunohistochemical study and the clinicopathological parameters. The analyses showed that a high level of CKS2 strongly correlated with tumor size, histological tumor differentiation and pTNM stage. A total of 82.1% of the patients in the high CKS2 expression group had tumor diameters of >4 cm, whereas 73.3% were poorly differentiated and 83.3% were pTNM stage IV. Multivariate logistic analysis was used to assess the predictive value of the CKS2 expression status for clinicopathological significance. The results corroborated that histological tumor differentiation, pTNM stage and a high level of CKS2 were significant covariables. A high CKS2 expression level was also found to be closely associated with tumor size and pTNM stage in gastric cancer tissues (12).

Shen et al (9) applied RT-qPCR and immunohistochemical staining analyses to determine the CKS2 mRNA and protein expression levels in 48 hepatocellular carcinoma tissues. The SPSS^® statistical software package was used to study the associations between CKS2 expression and various clinicopathological features. The analyses showed that a high CKS2 expression level strongly correlated with poor differentiation. No association was found between CKS2 overexpression and tumor size. In another study by Shen et al (29), the same results were exhibited in cholangiocarcinoma, and CKS2 overexpression was associated with poor differentiation.

Jung et al (14) studied the correlation between the CKS2 gene expression level and clinical characteristics in CRC tissues. The analysis of variance statistical test was used and the results showed that a high level of CKS2 expression was associated with early tumor stage. The CKS2 mRNA levels of 16 patients in stage Ⅱ were higher compared to eight patients in stage Ⅲ. Yu et al (15) also reported results that the overexpression of CKS2 was correlated with the pathological stage in CRC tissues. Using western blot analyses, this study reported that tumor tissues from 15 stage III CRC patients demonstrated significantly higher CKS2 protein levels compared to those from 15 stage II patients. CKS2 expression was also correlated with tumor size and differentiation. The reason for the results differing from Jung et al (14) may be due to the smaller number of patients from stage Ⅲ in the latter.

In 2006, Kawakami et al (17) first reported that CKS2 expression is strongly correlated with the progression of human BC. This study subjected 21 BC samples to the RT-qPCR assay for CKS2, and identified a considerable difference between superficial and invasive BC. CKS2 had a significantly greater level of upregulation in invasive compared to superficial BC (P=0.04).

Chen et al (18) used the RT-qPCR analysis on 45 patient samples with a diagnosis of superficial BC (of these, seven patients developed muscle-invasive BC during the follow-up period) and 10 samples from normal bladders. Expression of the CKS2 gene in superficial BC tissues was >10-fold higher compared to normal bladder tissues, and was ∼4-fold higher following disease transition in patients developing muscle-invasive cancer. CKS2 had significantly increased expression levels following transition from superficial BC to muscle-invasive cancer.

Tanaka et al (12) reported that 31 cases exhibited serosal invasion among 40 cases of high CKS2 expression in gastric cancer tissues.

From these outcomes it can be inferred that CKS2 may function as a tumor oncogene. CKS2 may promote cancer invasion and it may be a useful biomarker for predicting disease outcome and the requirement for early preventive treatments.

Lymph node metastasis is extremely common for cancers, and almost all will develop to lymph node metastasis of tumors (30).

Kita et al (11) examined the mRNA level of CKS2 in esophageal squamous cell carcinoma tissues from 62 esophageal cancer patients. The patients with values below the median expression level in tumor tissues were assigned to the low expression group, whereas those with values above the median were assigned to the high expression group. Of the 31 patients assigned to the high expression group, 26 (83.9%) presented with lymphatic invasion. This result suggested that high expression of CKS2 was significantly associated with the incidence of lymphatic invasion. Wang et al (10) also observed the correlation between high CKS2 expression and lymph node metastasis in esophageal carcinoma tissues.

Tanaka et al (12) showed the results that 40 cases had high CKS2 expression and 69 cases had low CKS2 expression in 109 cases of gastric cancer tissues. Of the 40 cases with high CKS2 expression, 32 (80%) had lymph node metastasis, indicating that CKS2 promotes lymph node metastasis. Kang et al (28) also observed the correlation between high CKS2 expression and lymph node metastasis in gastric cancer tissues.

Lyng et al (31) identified the gene expression in 29 cervical cancer patients with and 19 patients without lymph node metastases using a genomic microarray technique. CKS2 was noted to be elevated in node-positive compared to node-negative tumors.

From these outcomes it can be suggested that CKS2 may function as a tumor oncogene and it promotes lymph node metastasis from carcinoma in situ.

The liver is the most common site of metastases from the digestive tract tumors. Tanaka et al (12) reported that five cases (12.5%) had an incidence of liver metastasis in 40 gastric cancer tissues with a high level of CKS2 mRNA expression. In 69 cases with a low level of CKS2 mRNA expression, just one case (1.4%) had the incidence of liver metastasis, indicating that high expression of CKS2 was associated with liver metastasis.

Lin et al (32) identified conflicting results to Tanaka et al (12). The mRNA profiling of CKS2 was undertaken by RT-qPCR to confirm the differential expression between liver metastases and primary tumors. CKS2 was noted to be downregulated in 28 metastases tissues relative to 40 primary colon tumors.

The prognosis for cancer patients is extremely important. Tanaka et al (12) used Kaplan-Meier survival curves to evaluate the survival rate in gastric cancer patients according to the levels of CKS2 mRNA expression. The overall 5-year survival rate was significantly higher in the CKS2 low expression group (59.9%) compared to the CKS2 high expression group (23.9%). Thus, CKS2 mRNA expression was associated with the prognosis.

CKS2 is a transcription target downregulated by p53 (7). Kang et al (28) used SUN638 and AGS gastric cancer cells that were transfected with GFP; GFP-CKS2 plasmids. The western blot results showed that the protein expression level of p53 in GFP-CKS2-overexpressing SUN638 and AGS cells was lower compared to the GFP control cells, suggesting that the increased CKS2 protein affected the protein level of p53. However, it requires further study to understand how CKS2 overexpression causes the downregulation of p53.

Cholangiocarcinoma cells, QBC939, were stably transfected with shCKS2 or shCtrl plasmids, and stable CKS2-knockdown cells were established. RT-qPCR and western blot analysis were used to determine the cell cycle associated genes. CKS2 downregulation resulted in a marked reduction in the protein and mRNA levels of cyclin A and cyclin B1 when compared with shCtrl cells. Flow cytometry showed that CKS2 downregulation increased the cells at the G₂/M phase and decreased the cells at the S and G₁ phases. In this study, CKS2-knockdown induced cholangiocarcinoma cell cycle arrest in G₂ phase by decreasing the expressions of cyclin A and cyclin B1. These results suggest that CKS2 may serve as a cell cycle checkpoint protein for S/G₂ transition and this is one of the mechanisms of how CKS2 promotes cholangiocarcinoma progression (29).

Tissues from 10 gastric cancer patients were prepared and RT-qPCR analysis was performed (28). Cell cycle regulators, cyclin A, cyclin B1 and CDK1, were evidently upregulated in tumor tissues compared to normal tissues. The study also used the small interfering RNA (siRNA)-mediated gene-silencing method to suppress the transcript level of CKS2 in AGS gastric cancer cells. When the transcript levels of CKS1 and CDK1 were compared with the CKS2 level, the genes were upregulated, possibly by a complicated mechanism in which the proteins that have high similarities, such as CKSs and CDKs, complement each other to maintain their basic role.

CDK1 and cyclin B1 are known to be important players in the cell cycle. Numerous studies have demonstrated that cyclin B1-CDK1 protein kinase, also known as mitosis promoting factor, is essential for mitosis and that in its absence, cells are unable to progress past the G₂ phase of the cell cycle (33,34). CKS2 binds to CDK1 via interaction with the catalytic subunit of CDK1, and subsequently affects cell cycle.

Tanaka et al (12) used the siRNA-mediated gene-silencing method to suppress the transcript level of CKS2 in MKN74 gastric cancer cells and examined caspase-3 expression by flow cytometry. CKS2-siRNA cells had increased caspase-3 activity (10.8%) compared to the siRNA-negative control (2%), indicating that the suppression of CKS2 expression increased caspase-3 activity by >5-fold. The knockdown of CKS2 expression increased Bax expression at the protein level, shown by western blot analyses. Caspase-3 and Bax proteins are involved in cell apoptosis, these results show that apoptosis is induced by inhibiting CKS2 expression.

Shen et al (29) observed the changes in the expression of Bcl-2 family proteins in cholangiocarcinoma cells by western blot analysis. Bax increased following CKS2 knockdown (shCKS2). The study also examined the activation of Bax by immunofluorescence and found that Bax activation increased in shCKS2 cells. In the nude mouse tumorigenesis experiment, the 32 kDa proenzyme caspase-3 was cleaved to obtain its active form of 17 kDa following CKS2 knockdown. These data suggest that CKS2 knockdown enhances the susceptibility of cholangiocarcinoma cells to a Bax-mediated mitochondrial caspase-dependent apoptosis. To further confirm CKS2 knockdown promoting cholangiocarcinoma cells under apoptosis, flow cytometry was used to evaluate the apoptotic susceptibility of QBC939-shCKS2 and shCtrl cells. Data indicated that the apoptosis rate of shCKS2 cells (42.5%) was significantly higher compared to shCtrl cells (12.3%) under hypoxia growth conditions. From these results it is shown that CKS2 downregulation can promote cholangiocarcinoma under apoptosis.