The roles of the cell division cycle-associated gene family in hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) places as the sixth most common cancer and the fourth leading cause of cancer-related deaths globally (1). In China, the incidence of HCC is 10–20 per 100,000, ranking the second of all malignant tumor mortality (2). Although different curative or palliative therapies exist for HCC, the long-term survival rate of patients with HCC is still extremely low (3). The mechanisms of the growth, progression, and metastasis of HCC have been investigated; however, the molecular features of the disease have not yet been identified. Therefore, novel prognostic markers and prospective drug targets need to be discovered to improve the prognosis and individualized treatments for patients with HCC.

The 8 members of the cell division cycle-associated (CDCA) gene family (CDCA1–8) are significant regulators of cell proliferation. Studies have demonstrated that abnormal expression of CDCAs can cause cancer (4,5). The protein encoded by CDCA1 (also referred to as NUF2) is crucial for the nuclear division and stability of microtubules (6). CDCA2 regulates the DNA damage response in the cell cycle by binding to protein phosphatase 1 γ (PP1γ) (7). CDCA3 forms a portion of the ubiquitin ligase (E3) complex SKP1-Cullin-RING-F-box (SCF), which can degrade the endogenous cell cycle inhibitor WEE1 to regulate the cell cycle (8). CDCA4 is a G1/S transition-related cell cycle regulator and also modulates p53 expression (9,10). CDCA5 is a key regulator of the cohesion and separation of sister chromatids during cell division (11). CDCA6 (also referred to as CBX2) encodes a polycomb protein complex that maintains the transcriptional repression of multiple genes throughout the growth cycle through chromatin remodeling and histone modification (12). CDCA7 is a cMyc target gene engaged in cMyc-mediated cell transformation (13). Finally, CDCA8 is an essential component of the vertebrate chromosome passenger complex, which has important regulatory involvement in mitosis and cell division (14).

In HCC, the roles of CDCAs are assumed to be complex and distinct. Previous studies of HCC have evidenced the overexpression of CDCA3 and CDCA4, which may participate in cell proliferation, migration, invasion, and apoptosis (15,16). A number of studies have also reported that CDCA5 and CDCA8 play important roles in the development of HCC (17,18). For instance, studies have found CDCA5 to be expressed at high levels in HCC, which has a significant correlation with tumor progression and a poor prognosis (18,19). However, previous studies only focused on several members of CDCA gene family and failed to investigate the expression of this gene family at multiple levels such as tissue and cell. Hence, it is necessary to study the expression of CDCA gene family from multiple levels to understand the individual roles of the whole gene family members including CDCA1-8 in HCC and their potential mechanisms of action.

In the present study, we used online bioinformatics analysis tools to analyze the relationships of CDCA family members with the pathogenesis and progression of HCC, in order to ascertain the expression patterns, underlying functions, and unique prognostic values of CDCAs in HCC. We present the following article in accordance with the MDAR reporting checklist (available at http://dx.doi.org/10.21037/jgo-21-110).

Methods

Ethics statement

This research was approved by the institutional ethics committee of Sun Yat-Sen University Cancer Center and was carried out in accordance with the principles of the Helsinki Declaration (as revised in 2013). All data used in this study were retrieved from publically available sources, so there was no requirement to obtain informed participant consent.

Oncomine database analysis

The Oncomine database (www.oncomine.org), an online cancer microarray database for DNA or RNA sequencing (seq) analysis, was used to investigate expression of CDCAs (20). In the current study, Student’s t-test was used to compare the transcriptional levels of CDCAs in tissues from diverse cancer types and their corresponding normal tissues. The cut-off values for the P value and fold change were defined as 0.01 and 1.5, respectively.

UALCAN

UALCAN (http://ualcan.path.uab.edu/) is an integrative and interactive network resource which can be used to analyze level 3 RNA-seq data and clinical data of 31 different cancers from The Cancer Genome Atlas (TCGA) database. This portal can be used to analyze differences in the expression levels of a query gene between tumor and normal samples, and to estimate the influence of a gene’s expression level and clinicopathological characteristics on patient survival (21). In our study, we used the portal to evaluate the messenger RNA (mRNA) expression of the 8 CDCA family members in HCC tissues as well as their connection with clinicopathological variables of patients with HCC. Differences in transcriptional expression were compared with Student’s t-test, and P<0.01 was deemed to be statistically significant.

Human Protein Atlas (HPA)

The HPA (https://www.proteinatlas.org) is an open accessed knowledge resource. All data in it can be retrieved freely to explore the human proteome (22). In this study, we obtained the immunohistochemical (IHC) data of CDCA gene family in HCC and normal tissues for protein level investigation.

Cancer Cell Line Encyclopedia (CCLE)

CCLE (https://portals.broadinstitute.org/ccle) is a free online database, which compiles chromosomal copy number, large-scale parallel sequencing, and gene expression data of human cancer cell lines (23). In this database, there are about one thousand cell lines data for genomic analysis and visualization. We verified the expression of CDCA gene family in HCC cell lines using datasets downloaded from the CCLE database. Differences in transcriptional expression were compared with Student’s t-test using GraphPad Prism 9, and P<0.01 was deemed to be statistically significant.

LinkedOmics

LinkedOmics (http://www.linkedomics.org/login.php) is an open portal website containing multi-omics datasets for all 32 cancer types in TCGA. This portal can be used by biologists and clinicians to access, analyze, and compare multi-omics data within as well as among tumor types (24). In this study, LinkedOmics was used to perform prognostic analyses of the CDCA gene family in patients with HCC.

TCGA database and cBioPortal

The TCGA database contains sequencing information as well as pathological information on 30 different cancers (25). The cBioPortal for Cancer Genomics (http://www.cbioportal.org/) is an open source resource that facilitates investigation of multidimensional datasets of cancer genomes (26). The liver HCC (TCGA, Provisional) dataset, which contains information from 371 patients with pathological results, was chosen for further exploration of CDCAs using cBioPortal. The genomic profiles of HCC patients, including the frequency of gene alterations, z-scores of mRNA expression (RNA Seq V2 RSEM), and co-expression and correlations of genes in the CDCA family, were analyzed with the cBioPortal online tool.

Functional enrichment and bioinformatics analysis

Metascape (http://metascape.org/gp/index.html#/main/step1) is an online portal integrating functional enrichment, interactome analysis, gene annotation, and membership search, which utilizes more than 40 bioinformatics knowledgebases (27). In our study, to identify the most frequently altered linked genes, a gene list comprising the CDCA family genes was analyzed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) tools in Metascape.

Statistical analysis

In the Oncomine database analysis, student’s t-test was used to compare the transcriptional levels of CDCAs in tissues from diverse cancer types and their corresponding normal tissues. The cut-off values for the P value and fold change were defined as 0.01 and 1.5, respectively. In the UALCAN database analysis, differences in transcriptional expression were compared with Student’s t-test, and P<0.01 was deemed to be statistically significant. In the CCLE database analysis, differences in transcriptional expression were compared with student’s t-test, and P<0.01 was deemed to be statistically significant. In this study, CCLE database analysis was performed using GraphPad Prism 9, and all other databases analyses were performed with database online tools.

Results

Transcriptional levels of CDCA genes in patients with HCC

First, we identified CDCA genes in the human genome. To investigate the different prognostic and potential therapeutic values of CDCA family members in HCC, the Oncomine database was used to compare the transcriptional levels of the 8 CDCA genes between tissue samples from 20 different cancers and samples from normal controls (Table 1 and Figure 1). The mRNA expression levels of CDCA1, CDCA2, CDCA3, CDCA4, CDCA5, CDCA7, and CDCA8 were upregulated in patients with HCC. In the Chen liver dataset, CDCA1 mRNA was overexpressed 5.752-fold in HCC tissues compared to normal tissues (P=1.90E−27) (28), while in the Wurmbach liver dataset, it was upregulated in HCC, with a fold change of 4.453 compared to normal tissues (P=2.16E−08) (29). The results of analysis of the Wurmbach liver dataset also showed that the mRNA expression of CDCA2 showed a fold increase of 1.813 in HCC compared to normal tissues (P=1.94E−04) (29). CDCA3 overexpression was also found in HCC: the fold change in the Wurmbach liver dataset was 3.241 (P=3.39E−08), while that in the Roessler liver 2 dataset was 1.633 (P=6.04E−42) (29,30). The transcriptional expression of CDCA4, CDCA5, CDCA7, and CDCA8 was also upregulated in patients with HCC. CDCA4 was identified to be expressed at a higher level in HCC tissues compared to normal tissues in the Wurmbach liver and Roessler liver 2 datasets, with fold changes of 1.832 and 1.545, respectively (P=1.55E−05 and 8.68E-38, respectively) (29,30). Furthermore, CDCA5 was significantly upregulated in HCC, with fold changes of 4.400 and 2.422 in the Chen liver dataset and Wurmbach liver dataset, respectively (P=4.55E−24 and 4.84E−06, respectively) (28,29). CDCA7 was also discovered to have a higher expression in HCC tissues than normal tissues in the Chen liver dataset, with a fold change of 1.955 (P=7.28E−08) (28). Additionally, overexpression of CDCA8 was also found in HCC, with a fold change of 5.159 in the Chen liver dataset (P=3.98E−24) (28). Meanwhile, Roessler reported 1.760-fold and 1.583-fold increases in CDCA8 mRNA expression in HCC (P=1.66E−06 and 1.99E−37, respectively), while Wurmbach described a 1.693-fold rise in CDCA8 mRNA expression in HCC tissues (P=2.19E−05) (29,30). In line with the Oncomine analyses, there was no significant difference in the expression levels of mRNA of CDCA6 between HCC and normal tissues.

Table 1 The significant changes of CDCA expression in transcription level between different types of HCC and normal samples
Full table

Figure 1 The transcriptional levels of CDCA family members in various cancers. CDCA, cell division cycle-associated.

Next, the UALCAN database was used to explore differences in the mRNA expression levels of CDCAs between HCC and normal tissues. As shown in Figure 2, we found that the expression levels of all CDCA family members in HCC were obviously higher than those in normal tissues (all P<0.05).

Figure 2 The expression levels of CDCA family members in HCC tissues and normal tissues. CDCA, cell division cycle-associated; HCC, hepatocellular carcinom.

To further verify the prognostic values of CDCA family members in HCC, we used the IHC data from HPA database to compare the protein expression of CDCA genes between HCC tissues and normal tissues. The data of CDCA2, CDCA5, CDCA6 and CDCA8 were available, which showed that these four proteins expressed more highly in HCC tissues than normal tissues (Figure 3).

Figure 3 The protein levels of CDCA family members in HCC tissues and normal tissues with IHC staining. CDCA, cell division cycle-associated. Original magnification: ×100.

We also verified the mRNA expression of CDCA genes from cell level with the CCLE database. The results were presented in Figure 4. We found that all CDCA gene family members were highly expressed in the HCC cell lines from CCLE database. We further compared the mRNA expression level of CDCA genes in HCC cell lines before and after knockdown. The mRNA expression data of all CDCA genes knockdown except CDCA3 were available in the CCLE database. The results showed that, in HCC cell lines, CDCA genes knockdown resulted in lower mRNA expression.

Figure 4 The expression levels of all CDCA family members in HCC cell lines and the comparison of expression levels of CDCA family members in HCC cell lines before and after knockdown. CDCA, cell division cycle-associated.

Relationship between the clinicopathological variables and CDCA mRNA expression of HCC patients

After discovering that the mRNAs of all CDCA genes were overexpressed in patients with HCC, we explored the relationships between CDCAs and HCC stage through UALCAN. The mRNA expression levels of CDCA genes were shown to be significantly positively correlated with the stage of HCC (Figure 5). The level of CDCA6 mRNA expression was highest in patients with stage 4 HCC, while the highest expression of other CDCA genes was observed in patients with stage 3 HCC.

Figure 5 The expression levels of CDCA family members in HCC based on individul cancer stage. CDCA, cell division cycle-associated; HCC, hepatocellular carcinoma.

The role of CDCAs in the survival of HCC patients

We also analyzed the prognostic values of CDCAs in HCC using LinkedOmics. As shown in Figure 6, the results revealed that elevated expression levels of CDCA1–8 mRNA were significantly correlated with short overall survival (OS) (P<0.05) in HCC patients. This result indicated that overexpression of CDCA1–8 may constitute a poor prognostic factor for HCC, and these genes may be used as biomarkers to predict the survival of patients with HCC.

Figure 6 The prognostic value of the CDCA gene expression in HCC patients. CDCA, cell division cycle-associated; HCC, hepatocellular carcinoma.

Genetic mutations and correlations of CDCA genes in HCC

We next analyzed the mutations, correlations, and networks of CDCA genes in HCC using the “TCGA Provisional” database as well as the cBioPortal online tool for HCC. CDCA genes were altered in 167 of 373 (45%) HCC samples. CDCA1, CDCA6, and CDCA2 were the 3 genes that showed the highest genetic variation, with mutation rates of 27%, 15%, and 12%, respectively. There were 3 main types of genetic alterations: deep deletion, mRNA overexpression, and amplification (Figure 7A). We further analyzed the mRNA expression of CDCA genes to examine their correlations with each other using cBioPortal [mRNA sequencing (RNA-seq) version V2 RSEM], together with Pearson’s correlation coefficient. We found that any 2 CDCA family members were significantly positively correlated with each other (Figure 7B). Then, we established the gene relation network to show CDCA genes and their the 50 most frequently altered adjacent genes. We found that cell cycle-related genes, such as AHCTF1, AKT1, BIRC5, CENPF, CENPL, and CENPQ, were closely associated with CDCA gene alterations (Figure 7C).

Figure 7 Genetic correlations and mutations of CDCA family members in HCC. (A) Genetic mutations of CDCA family members in HCC. (B) Genetic correlations of CDCA family members in HCC. (C) The network of CDCA genes and the 50 most frequently altered neighboring genes. CDCA, cell division cycle-associated; HCC, hepatocellular carcinoma.

Predicted functions and pathway enrichment of CDCA genes in HCC

Before using the GO tools in Metascape, we compiled a list of CDCA genes and 50 neighboring genes that exhibited alterations most frequently (Figure 8). The results of enrichment analyses revealed that CDCA gene alterations influenced the following pathways: R-HSA-68886: M Phase; R-HSA-69620: Cell Cycle Checkpoints; GO:1903827: regulation of cellular protein localization; GO:1902850: microtubule cytoskeleton organization involved in mitosis; M139: PID MYC PATHWAY; M14: PID AURORA B PATHWAY; R-HSA-2468052: Establishment of Sister Chromatid Cohesion; R-HSA-5689901: Metalloprotease DUBs; GO:0006997: nucleus organization; R-HSA-8866654: E3 ubiquitin ligases ubiquitinate target proteins; CORUM:1464: Mis12 centromere complex; R-HSA-432142: Platelet sensitization by LDL; R-HSA-3108232: SUMO E3 ligases SUMOylate target proteins; GO:0034508: centromere complex assembly; GO:0051301: cell division; R-HSA-3214858: RMTs methylate histone arginines; and R-HSA-68875: Mitotic Prophase.

Figure 8 The functions of CDCA genes and genes significantly related to CDCA mutations. CDCA, cell division cycle-associated.

Discussion

According to reports, CDCA gene abnormalities occur in many cancers (4,5). Despite the carcinogenetic and prognostic functions of CDCA family members in several cancers having been well documented (31-33), an in-depth bioinformatics analysis of their roles in HCC had yet to be performed. Therefore, this study analyzed the expressions and mutations as well as the prognostic values of the CDCA family genes in HCC.

CDCA1 is a crucial constituent of the NDC80 kinetochore complex, which is necessary for kinetochore-microtubule connection as well as chromosome separation (34). Previous research showed that CDCA1 promoted the growth and inhibited the apoptosis of HCC cells (35). Wang et al. further showed that high expression of CDCA1 is significantly related to the poor survival of patients with HCC, and CDCA1 therefore holds promise as a prognostic biomarker to aid in the accurate prediction of early recurrence of HCC after surgical treatment (34). In the present study, the expression of CDCA1 mRNA in HCC tissues was significantly higher than that in normal tissues and was significantly correlated with the individual cancer stage; this finding was consistent with the results of previous studies. Moreover, high CDCA1 mRNA expression was also significantly related to poor survival in HCC patients, indicating that CDCA1 participates in HCC tumorigenesis.

Up to now, researchers have gained little insight into the performance and function of CDCA2 in HCC. It has been reported that CDCA2 is a cell cycle-related protein, the expression of which is related to other members of the CDCA gene family (36). According to previous studies, CDCA2 plays a key role in regulating the expression of PP1γ-dependent essential DNA damage responses in the cell cycle as well as preserving the characteristic chromosome structure for transiting to interphase (7,37). Several studies have revealed CDCA2 to be highly expressed in tissue samples from patients with oral squamous cell carcinoma, neuroblastoma, and adenocarcinoma of the lung (38-40). Recent research indicated that CDCA2 might target CCND1, at least in part by activating the PI3K/AKT pathway to promote colorectal carcinoma cell proliferation and tumorigenesis (41). Our current study showed that CDCA2 was expressed more highly in HCC tissues than in normal tissues. Furthermore, we found that CDCA2 expression was related to HCC stage. Among all the HCC patients, a high expression of CDCA2 was significantly related to poor OS, which indicated that CDCA2 has carcinogenic effects in HCC.

CDCA3 plays a significant role in cell mitosis and control of the G1 phase (8). The involvement of CDCA3 has been reported in lung cancer, prostate cancer, and oral squamous cell carcinoma (42-44). Furthermore, in colorectal cancer, CDCA3 is upregulated, and its upregulation is correlated with the proliferation and apoptosis of cancer cells. This effect may be achieved in colorectal cancer through activation of the nuclear factor-kappa B (NF-κB) signaling pathway by CDCA3 via interaction with TRAF2 (8). Studies have also shown that CDCA3 expression is elevated in liver cancer and may be involved in cell proliferation, migration, invasion, and apoptosis (15,16). In our present study, CDCA3 expression was significantly higher in HCC tissues than in adjacent normal tissues, and the mRNA expression of CDCA3 was strongly related to cancer stage. Furthermore, a high expression level of CDCA3 was found to be significantly correlated with poor OS in patients with HCC.

CDCA4 was first discovered when mouse hematopoietic stem cells were screened against a reduced cDNA library. It was named hematopoietic progenitor protein (HEPP) on the basis of its preferred expression in adult bone marrow hematopoietic progenitor cells (45). Alderman et al. found CDCA4 to be highly expressed in melanoma and to be significantly associated with poor prognosis. Their study also found that microRNA-15a can directly regulate the expression of the CDCA4 gene, thereby regulating the proliferation of melanoma cells (46). In this study, similar tumorigenicity of CDCA4 was demonstrated in HCC. According to the results of our study, the mRNA expression of CDCA4 in HCC tissues was higher than that in adjacent normal tissues, and its expression level was significantly related to cancer stage and OS in HCC patients.

CDCA5 is also considered to be an oncogene due to its overexpression in multiple cancers (11,18). A previous study showed that the CDCA5 gene is extremely important for the genesis and progression of HCC, in which it is highly expressed and is significantly associated with tumor progression and poor prognosis (18). Similar to observations made in a previous study, the mRNA expression of CDCA5 was found to be higher in HCC tissues in our study and was significantly correlated with cancer stage. A higher expression of CDCA5 was also remarkably related to shorter OS in HCC patients.

As a vital component of polycomb repressive complexes 1 (PRC1), CDCA6 is involved in the gene expression and heterochromatin regulation (47). A previous study showed that, in breast cancer, CDCA6 expression is positively correlated to tumor size and TNM stage (48). CDCA6 has been reported as a potential drug target because its expression in association with adverse clinical outcomes in prostate cancer patients (49). In this study, our results were consistent with these previous founding. We found that CDCA6 was overexpressed in HCC tissues and high expression of it correlated with poor outcome in HCC patients, indicating that CDCA6 is an oncogene.

CDCA7 has been regarded as a cMyc target gene (13). A recent study found that the abnormal upregulation of CDCA7 in patients with breast cancer was related to a dismal prognosis and induced the progression of Enhancer of Zeste Homolog 2 (EZH2)-mediated triple-negative breast cancer (32). The results of our current study showed that CDCA7 was expressed at a higher level in HCC tissues compared with adjacent normal tissues, and the expression level was significantly related to cancer stage. It was also found that in HCC patients, high CDCA7 mRNA expression was related to poor OS.

As an important regulatory gene during mitosis, CDCA8 has been found to have enhanced transcriptional activity in embryos, embryonic stem cells, and cancer cells. Meanwhile, CDCA8 knockdown can effectively inhibit the proliferation of lung cancer cells, colon cancer cells, and human embryonic stem cells, and can promote and induce cell differentiation (50-52). A study by Jiao et al. revealed that the overexpression of CDCA8 in breast cancer reduced patient survival (53). In the present study, CDCA was expressed at significantly higher levels in HCC tissues, and its expression was correlated with disease stage. Accordingly, higher expression levels of CDCA8 were also associated with shorter OS in HCC patients.

In this study, GO and KEGG analyses were also carried out to identify the associations of CDCA genes and the most frequently altered linked genes with HCC initiation and prognosis. According to our research, closer attention should be paid to the following pathways: R-HSA-68886: M phase; R-HSA-69620: cell cycle checkpoints; R-HSA-68875: mitotic prophase; R-HSA-3214858: RMTs methylate histone arginines; GO:0051301: cell division; GO:0034508: centromere complex assembly; R-HSA-3108232: SUMO E3 ligases SUMOylate target proteins; and R-HSA-432142: Platelet sensitization by LDL.

Some limitations of the present study should be noted. First, we analyzed data retrieved from online databases, and further studies with larger sample sizes are needed to validate our findings. Also, we failed to investigate the underlying clinical roles and mechanisms of distinct CDCA genes in HCC, which demands further research.

Conclusions

In conclusion, we systematically investigated the prognostic value and expression levels of CDCA genes in HCC, which clarified the heterogeneity as well as complexity of the biological properties of HCC at the molecular level. According to our observations, overexpression of CDCA genes in HCC tissues likely plays a considerable role in HCC oncogenesis. Moreover, overexpression of CDCA genes may also serve as a molecular marker to improve prognostic accuracy and survival for patients with HCC.

Acknowledgments

Funding: This work was funded by grants from the Science and Technology Program of Guangzhou, China (No. 201803010071) and the Rural Science and Technology Commissioner Program of Guangdong Province, China (No. 202010130627).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at http://dx.doi.org/10.21037/jgo-21-110

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jgo-21-110). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This research was approved by the institutional ethics committee of Sun Yat-Sen University Cancer Center and was carried out in accordance with the principles of the Helsinki Declaration (as revised in 2013). All data used in this study were retrieved from publicly available sources, so there was no requirement to obtain informed participant consent.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Reynolds)