Comprehensive Expression Profiling and Molecular Basis of CDC28 Protein Kinase Regulatory Subunit 2 in Cervical Cancer

Abstract

More and more evidence suggests the oncogenic function of overexpressed CDC28 protein kinase regulatory subunit 2 (CKS2) in various human cancers. However, CKS2 has rarely been studied in cervical cancer. Herein, taking advantage of massive genetics data from multicenter RNA-seq and microarrays, we were the first group to perform tissue microarrays for CKS2 in cervical cancer. We were also the first to evaluate the clinical significance of CKS2 with large samples (980 cervical cancer cases and 422 noncancer cases). We further excavated the mechanism of the tumor-promoting activities of CKS2 in cervical cancer through analysis of genetic mutation profiles, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) significant enrichment of genes coexpressed with CKS2. According to the results, expression data from multilevels unanimously supported the overexpression of CKS2 in cervical cancer. Patients with cervical cancer in stage II from inhouse microarrays had significantly higher expression of CKS2, and CKS2 overexpression had an adverse impact on the disease-free survival status of cervical cancer patients in GSE44001. Both mutation types of mRNA high and mRNA low appeared in cervical cancer cases from the TCGA Firehose project. Gene coexpressed with CKS2 participated in pathways including the cell cycle, estrogen signaling pathway, and DNA replication. In summary, upregulated CKS2 is closely associated with the malignant clinical development of cervical cancer and might serve as a valuable therapeutic target in cervical cancer.

Figure 1

CKS2 expression in cervical cancer and noncancer samples from inhouse tissue microarrays, external microarrays, and RNA-seq datasets. (a) Violin plots for GPL570. (b) Violin plots for GPL571. (c) Violin plots for GPL6244. (d) Violin plots for GPL96. (e) Violin plots for GSE138080. (f) Violin plots for GSE26342. (g) Violin plots for GSE39001 (GPL201). (h) Violin plots for GSE4482 (GPL3515). (i) Violin plots for GSE4482 (GPL4926). (j) Violin plots for GSE46857. (k) Violin plots for GSE55940. (l) Violin plots for GSE7410. (m) Violin plots for TCGA-GTEx datasets (n). Violin plots for inhouse microarray. The expression shown in blue is for individuals without cancer, and the expression shown in red is for individuals with cancer.

Figure 2

The discriminatory ability of CKS2 expression in distinguishing cervical cancer from noncancer tissues in each microarray and RNA-seq dataset. (a) ROC curves for GPL570. (b) ROC curves for GPL571. (c) ROC curves for GPL6244. (d) ROC curves for GPL96. (e) ROC curves for GSE138080. (f) ROC curves for GSE26342. (g) ROC curves for GSE39001 (GPL201). (h) ROC curves for GSE4482 (GPL3515). (i) ROC curves for GSE4482 (GPL4926). (j) ROC curves for GSE46857. (k) ROC curves for GSE55940. (l) ROC curves for GSE7410. (m) ROC curves for TCGA-GTEx datasets. (n) ROC curves for inhouse microarray.

Figure 3

IHC staining of CKS2 in cervical cancer and noncancer tissues from tissue microarrays. (a) Moderate staining of CKS2 in cervical squamous cell carcinoma tissues (100x). (b) Moderate staining of CKS2 in cervical squamous cell carcinoma tissues (200x). (c) Moderate staining of CKS2 in cervical squamous cell carcinoma tissues (400x). (d) Strong staining of CKS2 in cervical squamous cell carcinoma tissues (100x). (e) Strong staining of CKS2 in cervical squamous cell carcinoma tissues (200x). (f) Strong staining of CKS2 in cervical squamous tissues (400x). (g) Negative staining of CKS2 in noncancer squamous epithelium tissues (100x). (h) Negative staining of CKS2 in noncancer squamous epithelium tissues (200x). (i) Negative staining of CKS2 in noncancer squamous epithelium tissues (400x). (j) Negative staining of CKS2 in noncancer squamous epithelium (100x). (k) Negative staining of CKS2 in noncancer squamous epithelium tissues (200x). (l) Negative staining of CKS2 in noncancer squamous epithelium tissues (400x).

Figure 4

CKS2 expression in cervical cancer patients at different stages from tissue microarray. (a) Violin plot showing differential CKS2 expression between stage I and stage II groups. (b) ROC curves of CKS2 expression discriminate stage I cervical cancer patients from stage II cervical cancer patients.

Figure 5

Flowchart of the inclusion of eligible microarrays and RNA-seq datasets for expression analysis.

Figure 6

Pooled SMD forest plot and SROC curves of CKS2 expression in cervical cancer and noncancer tissues for the inhouse tissue microarray, external microarrays, and RNA-seq datasets. (a) SMD forest. (b) SROC curves.

Figure 7

Survival analysis of CKS2 expression in cervical cancer from the TCGA database and GSE44001. (a) Kaplan-Meier survival curves for overall survival of cervical cancer patients from the TCGA database. (b) Kaplan-Meier survival curves for disease-free survival of cervical cancer patients from the TCGA database. (c) Kaplan-Meier survival curves for disease-free survival of cervical cancer patients from GSE44001. TPM: transcripts per kilobase million; HR: hazard ratio.

Figure 8

Genetic alteration of CKS2 in cervical cancer. The bar chart demonstrates the genetic alteration status of CKS2 in 310 cervical cancer samples profiled in mRNA expression and protein expression.

Figure 9

Functional enrichment analysis for genes positively correlated with CKS2 in cervical cancer. (a) Dot plot for biological process terms. (b) Dot plot for cellular component terms. (c) Dot plot for molecular function terms. (d) Dot plot for pathway terms.

Figure 10

Functional enrichment analysis for genes negatively correlated with CKS2 in cervical cancer. (a) Dot plot for biological process terms. (b) Dot plot for cellular component terms. (c) Dot plot for molecular function terms. (d) Dot plot for pathway terms.