Cyclin-Dependent Kinase Regulatory Subunit 2 Indicated Poor Prognosis and Facilitated Aggressive Phenotype of Hepatocellular Carcinoma

Cyclin-dependent kinase regulatory subunit 2 (CKS2) is a member of the cell cycle-dependent protein kinase subunit family, which is implicated as an oncogene in various malignancies. However, the clinical significance, oncogenic functions, and related mechanisms of CKS2 in hepatocellular carcinoma (HCC) remain largely unclear. In the present study, expression features and prognostic value of CKS2 were evaluated in the bioinformatic databases and HCC tissues. The effects of CKS2 on the malignant phenotypes of HCC cells were explored in vitro. According to the analyses of three bioinformatic databases, mRNA levels of CKS2 were elevated in HCC tissues compared with the normal tissues. Immunohistochemical assays found that high CKS2 expression was closely associated with liver cirrhosis (P = 0.019), poor differentiation (P = 0.02), portal vein invasion (P < 0.001), TNM stage (P = 0.019), tumor metastasis (P = 0.008), and recurrence (P = 0.003). The multivariate regression analyses suggested that CKS2 was an independent prognostic factor for overall survival (HR = 2.088, P = 0.014) and disease-free survival (HR = 2.511, P = 0.002) of HCC patients. Moreover, the bioinformatic analyses indicated that CKS2 might be associated with the malignant phenotypes in HCC progression. In addition, in vitro assays showed that CKS2 expression was higher in HCC cell lines than in normal liver cells. Knockdown of CKS2 remarkably repressed the proliferation, colony formation (P = 0.0003), chemoresistance, migration (P = 0.0047), and invasion (P = 0.0012) of HCC cells. Taken together, overexpression of CKS2 was significantly correlated with poor prognosis of HCC patients and the malignant phenotypes of HCC cells, suggesting that it was a novel prognostic biomarker and potential target of HCC.


Introduction
Hepatocellular carcinoma (HCC), accounting for 85-90% of all primary liver cancers, is the sixth most common type of cancer as well as the third most frequent cause of cancerrelated deaths [1,2]. Due to the infection of hepatitis B virus (HBV) or hepatitis C virus (HCV), HCC occurs more frequently in developing countries compared with developed countries [3]. Liver transplantation and radiofrequency (thermal) ablation (RF(T)A) are commonly applied in HCC patients at early and intermediate stages [4][5][6]. Despite the great efforts on pathology and physiology of HCC, it remains unclear for the molecular mechanisms underlying aggressive behaviors of HCC. Sorafenib, a multiple tyrosine kinase inhibitor, is the only systemic agent approved by the FDA for the first-line treatment of unresectable HCC patients [7]. While various targeted drugs (regorafenib, lenvatinib, and nivolumab) have been adopted in the treatment paradigm, the long-term survival of patients with HCC remains poor [8][9][10]. Therefore, it is of great importance to find novel prognostic biomarkers and a potential target for HCC.
Cdc kinase subunit (CKS) proteins are small (9 kDa) highly conserved cyclin-dependent kinase (CDK) binding proteins, which are essential components for cell cycle regulation [11,12]. The CKS family consists of two members, CKS1 and CKS2. CKS1, a well-known cell cycle-related protein, has been implicated in various tumors, including breast, lung, liver, and prostate cancers [13][14][15][16]. In addition, CKS2 is also observed in the transition of the cell cycle in multiple biological activities. Specifically, CKS2 could promote the early embryonic development and the somatic cell division [17]. However, accumulating evidence indicated that CKS2 might contribute to tumor progression [18]. Overexpression of CKS2 is determined in several cancer types and indicated a high risk of metastasis and recurrence. Though a recent study suggested the positive roles of CKS2 in biological behaviors of HCC cells [19], the potential clinical value and underlying functions of CKS2 remained largely unexplored. Based on the clinical samples and in vitro investigations, this study proposed CKS2 as a promising prognostic biomarker and therapeutic target for HCC.

Materials and Methods
2.1. Patient Information. HCC tissue samples and selfmatched adjacent nontumor tissues were obtained from 156 HCC patients (19 females and 137 males; age range, 35-74 years; mean age, 50.27) who underwent hepatectomy at the Affiliated Hospital of Nantong University (Jiangsu, China) between 2008 and 2012. Of them, 133 patients (85.3%) were diagnosed as HBsAg positive, 118 patients (75.6%) with liver cirrhosis, and 54 cases (34.6%) with an advanced stage (III/IV). The stages of all the enrolled patients were classified according to the 8th tumor node metastasis (TNM) classification system of the International Union Against Cancer. None of the patients received radiotherapy or preoperative chemotherapy before surgery. All patients were followed up until December 2017. The diagnosis of HCC was confirmed histologically. This study was approved by the Ethic Committees of the Affiliated Hospital of Nantong University.

Gene Set Enrichment
Analysis. RNA-seq of HCC samples from TCGA data was divided into two groups according to the median values of the expression of CKS2 (high vs. low expression). GSEA 3.0 software (Broad Institute, Cambridge, MA, USA; http://www.broad.mit.edu/gsea) was performed with reference from the Molecular Signatures Database (MSigDB, http://software.broadinstitute.org/gsea/msigdb). Thresholds were set following permutation tests for 1000 times. P value and normalized enrichment score (NES) were used to sort the possible pathways enriched in each group. Functional enrichment in the KEGG pathways and GO biological processes was assessed by hypergeometric test, which was used to identify a priori-defined gene sets that showed statistically significant differences between two groups. The test was performed by the R package clusterProfiler [20].
2.4. Immunohistochemical Staining. HCC and self-matched paracancerous tissues from 156 cases were fixed in formalin and embedded in paraffin for immunohistochemistry (IHC). In brief, following deparaffinization with xylene and rehydration with gradient ethanol, antigen retrieval was conducted by using sodium citrate buffer solution in a microwave. After that, sections were incubated with 3% hydrogen peroxidase to block endogenous peroxidase activity. Then, sections were incubated with primary anti-CKS2 rabbit monoclonal antibody (1 : 50, Abcam, USA) overnight at 4°C. After washing with phosphate buffer saline (PBS), samples were incubated with secondary HRP antibody (Dako, Denmark) for 2 h at room temperature. Following visualization by diaminobenzidine tetrachloride (DAB) and counterstaining by haematoxylin, sections were covered by coverslips with mounting media (Dako, Denmark).
2.5. Immunostaining Scores. Sections were independently assessed by two experienced histopathologists. Once it came to a conflicting result, the cases were evaluated again by a third histopathologist. The scores were semiquantitatively evaluated via two scoring parts: staining intensity and positive cell ratio. The immunostaining intensity was scored as follows: 0, no staining; 1, slightly yellow; 2, yellow brown; and 3, brown. The positive cell score was determined as follows: 0 (<5%), 1 (5~40%), 2 (40~75%), and 3 (>75%). The final score was calculated as the staining intensity score plus positive cell score. A score of 0-2 was considered negative, and a score of 3-6 was positive.
2.6. Cell Culture and Transfection. Hep3B, SMMC7721, Huh7, HepG2, SMMC7721, MHCC97H, and LO2 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640, Gibco, USA) or Dulbecco's modified Eagle medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA) and penicillin/streptomycin in a CO 2 incubator. For the transfection, cells were cultured in the 6-well plate and prewashed twice with 2 ml shRNA Transfection Medium (Santa Cruz, USA). KD-CKS2 Plasmid DNA solution (Santa Cruz, USA) was gently mixed with shRNA Plasmid Transfection Reagent (Santa Cruz, USA). Then, the suspension was added into indicated wells and incubated for 8 h, followed by incubation in the complete medium for 24 h. Finally, the transfection efficacy was verified by western blotting and RT-qPCR.

Cell
Counting Kit-8 Assay. The proliferation and viability of HCC cells was conducted using Cell Counting Kit-8 (CCK-8, Sigma, USA) according to the manufacturer's instructions. For the detection of proliferation, 1000 cells transfected with NC or KD-CKS2 were seeded in the 96-well plates. Then, the wells were exposed to CCK-8 solution at indicated time points and detected at 450 nm by using a plate reader (MD, USA). For the viability assay, the stock solution of sorafenib or regorafenib (Selleck, USA) was diluted in dimethyl sulfoxide (DMSO, Sigma, USA). Cells were treated with sorafenib at indicated concentrations for 48 h. Control group was treated with DMSO. Then, the plates were incubated with CCK-8 solution for 2 h at 37°C, followed by reading at 450 nm by a plate reader (MD, USA).

Colony Formation Assay. MHCC97H cells transfected
with KD-CKS2 and NC were seeded in six-well plates at a density of 500 cells/well. The cells were cultured for 12 days, followed by washing in PBS, fixing in 4% paraformaldehyde, and staining in 0.5% gentian violet. Then, the colony number was calculated and presented as mean ± SD of three independent experiments.
2.9. Transwell Assay. MHCC97H cells transfected with KD-CKS2 and NC were harvested and resuspended in serum-free medium. For the invasion assay, 8 μm Transwell chambers (Corning, USA) were precoated with Matrigel (BD Biosciences, USA) at the ratio of 1 : 5. For the migration assay, the chambers should not be pretreated with Matrigel. Then, the cells (1 × 10 5 cells/mL) were plated in the upper chambers, while the lower chamber was added with 600 μL DMEM medium supplemented with 10% FBS. Following incubation at 37°C for 24 h, cells were rinsed in PBS, fixed Semiquantitative analysis was conducted to assess the CKS2 protein levels between HCC and paracancerous tissues or among HCC cases at different stages. HCC: hepatocellular carcinoma; para: paracancerous tissues; CKS2: cyclin-dependent kinase regulatory subunit 2; TNM: tumor node metastasis. * * P < 0:01. and GraphPad 7.0 software. The χ 2 test was used to analyze the correlations between CKS2 expression and various clinicopathological features. Cox regression and Kaplan-Meier methods were used to analyze overall and disease-free survival of HCC patients. P < 0:05 was considered statistically significant.

Results
3.1. CKS2 mRNA Was Upregulated in Human HCC Tissues. CKS2 mRNA levels in HCC tissues extracted from several bioinformatic databases are shown in Figure 1. According to the TCGA database, CKS2 mRNA in HCC tissues (371 cases) were significantly higher (Figure 1(a); fold change, 3.14; P < 0:001) than that in normal liver tissues (50 cases). Similarly, higher CKS2 mRNA levels were also observed in HCC tissues compared with normal tissues in databases including the GSE14520 (Figure 1 Consistently, as shown in Figure 1(f), Oncomine databases demonstrated that the CKS2 expression gradually increased from normal livers (fold change, 4.12; P < 0:001), cirrhotic livers (fold change, 2.50; P < 0:001), and dysplasia livers (fold change, 1.94; P < 0:001) to HCC. Thus, these results indicated that CKS2 was highly expressed in HCC tissues and might be involved in the progression of HCC.

High CKS2 Expression Associated with Poor Survival in
Patients with HCC. The overall survival (OS) and diseasefree survival (DFS) of HCC patients analyzed by using Kaplan-Meier survival curves are elucidated in Figure 3. As shown in Figures 3(a) and 3(b), patients with high CKS2 expression had poorer OS (P = 0:01) and DFS (P = 0:002) than those cases with low CKS2 expression. Consistently, the survival analyses of TCGA databases also revealed that high CKS2 expression level was significantly correlated with lower OS (P = 0:014) and DFS (P < 0:001) in HCC patients (Figures 3(c) and 3(d)). These results demonstrated that high expression of CKS2 indicated poor survival of HCC patients. factors. Subsequent multivariate Cox analysis indicated that CKS2 (P = 0:002), together with portal vein invasion (P = 0:038), and TNM stage (P < 0:001) were independent prognostic factors for disease-free survival of 156 HCC patients (Table 3). Based on the analyses above, we proposed that CKS2 was an independent prognostic factor for HCC patients.
3.6. Potential Roles of CKS2 in HCC Progression. We further investigated the possible roles of CKS2 in HCC progression ( Figure 4). Cancer Hallmarks Analytics Tool showed that CKS2 might be associated with proliferation, resisting cell death, angiogenesis, invasion, and metastasis (Figure 4(a)). Then, the protein interaction analysis indicated that CKS2 might interact with some cell cyclin-related proteins, including CCNB1, CCNA2, CDK1, and CDK2 (Figure 4(b)). To explore the molecular functions related to CKS2, we identified the differentially expressed genes between CKS2-high and CKS2-low groups. Then, we performed the GO and KEGG analysis based on the top 116 genes that were positively or negatively associated with CKS2. GO enrichment found that the top involved biological processes included the chromosome segregation, DNA replication, catabolic process, and fatty acid metabolic process (Figure 4(c)). Pathway analyses suggested the top significant pathways consisting of the cell cycle, DNA replication, complement and coagulation cascade, and fatty acid metabolism pathways (Figure 4(d)). Furthermore, GSEA elucidated that CKS2 was implicated in cell cycle and DNA replication pathways (Figure 4(e)). As shown in Figure 4(f), CKS2 had significantly positive correlation with proliferative markers CCNB1, PCNA, and Ki-67 in HCC tissues, suggesting that CKS2 might contribute to HCC progression by regulating cell proliferation.

Discussion
HCC is one of the most common and malignant primary liver tumors with resistance to chemotherapy, especially for the cases at advanced stages. While patients benefit from some targeted drugs, HCC frequently develops tolerance to the currently available drug administration and subsequently leads to a poor prognosis [21]. Thus, it is imperative to find a prognostic marker and even a novel target for HCC. There is accumulating evidence that CKS2 is elevated in various cancer types, including colorectal, prostate, and bladder cancers [22][23][24]. However, its expression features and clinical significance in HCC has not been studied thoroughly. According to our analyses in multiple datasets, HCC tissues had higher CKS2 mRNA levels in comparison to normal liver tissues. Interestingly, CKS2 also gradually increased in the order from normal, inflammation, cirrhosis, dysplasia, and early stage HCC to advanced stage, suggesting that CKS2 might contribute to the progression of HCC.
Consistently, we also identified the overexpression of CKS2 protein in 156 HCC cases by the immunochemical staining analysis. Moreover, elevated CKS2 levels were significantly associated with various clinicopathological features, including liver cirrhosis, differentiation, portal vein invasion, TNM stage, and metastasis. CKS2 was previously recommended as a prognostic marker in colorectal, breast, gastric, and esophageal cancers [22,[25][26][27]. Thus, this study further discovered its roles in predicting the survival of HCC patients. As expected, CKS2 overexpression was obviously correlated with poor OS and DFS in the current HCC cohort. For HCC cases in TCGA cohort, similarly, high expression of CKS2 also led to a poorer outcome in contrast to low CKS2 expression. Taken together, CKS2 might be a prognostic marker to predict survival and recurrence of HCC patients.
CKS2 has been implicated in promoting the aggressive behaviors of cancer cells. Overexpression of CKS2 contributed to the tumor proliferation, migration, and invasion in thyroid cancer [28]. It could also accelerate tumorigenesis of squamous cell carcinoma in vivo [26]. While CKS2 was linked to HCC growth by recent studies [19,29], the possible mechanism remained partially unclear. In our study, we initially explored the underlying roles of CKS2 in HCC. The bioinformatic analyses identified that CKS2 was implicated in proliferation, angiogenesis, invasion, and metastasis. Additionally, enrichment analysis in KEGG pathways and GO terms revealed that CKS2-correlated genes were enriched in the cell cycle and DNA replication pathways, which are known as important factors in the proliferation of tumor cells. Besides, CKS2 was positively correlated with proliferative markers Ki-67 and PCNA, suggesting the potential role of CKS2 in HCC progression.
To validate the bioinformatic analysis, we further explored the roles of CKS2 in HCC cell lines. CKS2 overexpression was observed in HCC cell lines compared with normal hepatocytes. Consistent with the bioinformatic prediction, knockdown of CKS2 obviously downregulated the proliferation, colony formation, and invasion of MHCC97H cells. Moreover, silencing CKS2 also enhanced the efficacy of sorafenib against HCC cells. It was in line with the previous study that CKS2 promoted chemoresistance of cervical cancer [30]. For the mechanisms predicted by bioinformatic  was the first to evaluate CKS2 as a novel biomarker for OS and DFS of HCC patients. While the results are promising and attractive, further investigations are required to validate its clinical significance in a lager cohort and explore the underlying mechanisms regarding aggressive phenotypes of HCC.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declared no conflicts of interest.