Identification of CKS2 as a novel prognostic biomarker and potential therapeutic target for oral squamous cell carcinoma

Abstract

Background: The cyclin-dependent kinase subunit 2 (CKS2) is recognized to have a substantial impact on the pathogenesis and advancement of several malignant neoplasms. Nevertheless, its biological function and prognostic significance in oral squamous cell carcinoma (OSCC) have yet to be thoroughly investigated. Our primary objective was to clarify the contribution of CKS2 in the progression and prognosis of OSCC.

Methods: We first conducted a thorough examination of online databases to investigate the expression of CKS2, and subsequently corroborated our discoveries by analyzing clinical specimens that we collected. According to the clinicopathological data, we then explored the prognostic significance of CKS2. Furthermore, we predicted the role of CKS2 in OSCC progression by employing weighted gene co-expression network analysis (WGCNA) in conjunction with functional enrichment analysis. We conducted functional experiments in vitro to confirm our speculations. Additionally, we explored other potential functions of CKS2 in immune infiltration, tumor mutation burden (TMB), and drug sensitivity. Finally, we established and validated a nomogram that effectively integrated CKS2-related genes and other relevant clinical factors.

Results: Our findings indicated a significant upregulation of CKS2 expression in OSCC tissues compared to normal groups, which was positively associated with poor clinical outcomes. We also predicted and validated the role of CKS2 in promoting proliferation by regulating the cell cycle. Additionally, its upregulation was significantly correlated to enhanced immune cell infiltration, high TMB, and increased sensitivity of anti-tumor agents. Following verification, the nomogram was conducted to quantify an individual's survival probability.

Conclusions: In general, our study indicates that CKS2 is a novel prognostic biomarker and potential therapeutic target in OSCC.

Keywords: Cyclin-dependent kinase subunit 2 (CKS2); cell proliferation; immunity; oral squamous cell carcinoma (OSCC); prognosis.

Figure 1

CKS2 was overexpressed in OSCC tissues and associated with poor prognosis. (A) Differential expression of CKS2 in 33 different tumor tissues and paired normal tissues from TCGA and GTEx databases. Each dot represents the expression of samples. (B) The prognostic impact of CKS2 expression level based on the survival heatmap, showing significance in ACC, KIRP, LGG, LIHC, MESO, PAAD, PRAD, SKCM, STAD and UVM. (C) Differential analysis of CKS2 expression between OSCC and normal samples in OSCC-TCGA, GSE30784, and GSE42743 datasets. (D) Representative images of immunohistochemistry staining for CKS2 in ANCT and OSCC tissues. Magnification at 50× (up) and 200× (down). (E) Immunohistochemical staining score of CKS2 in ANCT (n=30) and OSCC (n=85) tissues. (F-J) The correlation between CKS2 expression and clinical features in TAGA-OSCC cohort, including the grade, gender, age, N, and lymph nodes positive (by hematoxylin-eosin staining). (K-L) Kaplan-Meier survival curves of overall survival (K) and progression free survival (L) based on TCGA-OSCC patients with high- and low-expression CKS2. (M) Kaplan-Meier survival curves of overall survival based on GSE41613 dataset. Differences between the two groups were compared using a log-rank test. *, P<0.05; **, P<0.01; ***, P<0.001. CKS2, cyclin-dependent kinase subunit 2; TCGA, The Cancer Genome Atlas; N, node; HE, hematoxylin and eosin; OSCC, oral squamous cell carcinoma; GTEx, Genotype-Tissue Expression; ACC, adrenocortical cancer; KIRP, kidney papillary cell carcinoma; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; MESO, mesothelioma; PAAD, pancreatic adenocarcinoma; PRAD, prostate adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach cancer; UVM, uveal melanoma; ANCT, adjacent non-cancerous tissues.

Figure 2

Identification of the key module relayed to CKS2 and functional enrichment analysis in TCGA-OSCC and GSE30784 datasets. (A,B) The correlation coefficients and P values of module-trait relationships Each row corresponds to a module eigengene. (C,D) GO analysis for CKS2-realated key module. (E,F) KEGG enrichment analysis for CKS2-realated key module. (G,H) GSEA results show the significant enriched pathway based on CKS2 expression. CKS2, cyclin-dependent kinase subunit 2; TCGA, The Cancer Genome Atlas; OSCC, oral squamous cell carcinoma; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, The Gene Set Enrichment Analysis.

Figure 3

CKS2 promotes proliferation of OSCC by regulating cell cycle. (A,B) Western blot and RT-qPCR were used to detect the CKS2 expression after transfection in OSCC cell lines. (C) CCK-8 assay showed that down regulation of CKS2 expression inhibited the proliferation of CAL33 and SCC25 cells, up-regulation of CKS2 expression promoted the proliferation of SCC1 and SCC15 cells. (D,E) Clone formation assay showed that down regulation of CKS2 expression inhibited the proliferation of CAL33 and SCC25 cells, up-regulation of CKS2 expression promoted the proliferation of SCC1 and SCC15 cells. Cells were stained with crystal violet. Magnification at 1×. **, P<0.01; ***, P<0.001. OSCC, oral squamous cell carcinoma; CKS2, cyclin-dependent kinase subunit 2; TCGA, The Cancer Genome Atlas; RT-qPCR, quantitative reverse transcription PCR; CCK-8, cell counting kit-8; NC, negative control.

Figure 4

Immunological significance of CKS2 in OSCC. (A) The distribution of infiltrating immune cells in 306 TCGA-OSCC samples based on CKS2 expression. (B) The differences in the infiltrating levels of 22 immune cells between the high- and low-CKS2 expression groups. (C-G) The correlations between the CKS2 expression and the enrichment of five core immune cells. (H) The correlations between the CKS2 expression and the tumor mutation burden. CKS2, cyclin-dependent kinase subunit 2; OSCC, oral squamous cell carcinoma; TCGA, The Cancer Genome Atlas.

Figure 5

Drug prediction of IC50 differences in CKS2 high and low group based on the GDSC database. (A-F) High level of CKS2 expression was significantly correlated with low IC50 of recommended therapeutic drugs in OSCC. (G-I) Other commonly used anti-tumor drugs most associated with the expression level of CKS2. IC50, half maximal inhibitory concentration; CKS2, cyclin-dependent kinase subunit 2; GDSC, Genomics of Drug Sensitivity in Cancer; OSCC, oral squamous cell carcinoma.

Figure 6

Construction and validation of a nomogram based on CKS2-related genes. (A) Forest plot of univariate Cox regression analysis of 6 hub genes associated with CKS2 expression and OS in TCGA-OSCC cohort. (B,C) LASSO regression analysis screened 5 CKS2-related genes. (D,E) Uni- and multivariate Cox regression models are conducted to uncover the association of clinical features and CKS2-derived risk score with OSCC survival outcome. (F) The ROC curves show the predictive power of CKS2-derived risk score and clinical features on the prognosis of OSCC patients. (G) Construction of prognostic nomogram for predicting OS of OSCC patients based on TCGA dataset. (H,I) The ROC curves and calibration curves for 1-, 3-, and 5-year in the TAGA-OSCC training set. (J,K) The ROC curves and calibration curves for 1- and 3-year in the GSE42743 testing set. *, P<0.05; **, P<0.01. T, tumor; N, node; AUC, area under the curve; CI, confidence interval; OS, overall survival; CKS2, cyclin-dependent kinase subunit 2; TCGA, The Cancer Genome Atlas; OSCC, oral squamous cell carcinoma; LASSO, least absolute shrinkage and selection operator; ROC, receiver operating characteristic.