INK4 cyclin-dependent kinase inhibitors as potential prognostic biomarkers and therapeutic targets in hepatocellular carcinoma

Abstract

The INK4 family is an important family of cyclin-dependent kinase inhibitors (CDKIs) and consists of CDKN2A, CDKN2B, CDKN2, and CDKN2D. Abnormal expression of CDKN2A has been reported in hepatocellular carcinoma (HCC) and is associated with the prognosis of patients and infiltration of immune cells. However, there is a lack of systematic research on the roles of the other INK4 family members in the diagnosis, prognosis, and immune regulation of HCC. Using online public databases and clinical samples, we comprehensively analyzed the INK4 family in HCC. All four INK4 proteins were overexpressed in HCC and correlated with advanced cancer stage and poor prognosis. INK4 expression accurately distinguished tumor from normal tissue, particularly CDKN2A and CDKN2C. The INK4 family participated in cell-cycle regulation and the DNA damage repair pathway, which inhibited genotoxic-induced apoptosis in tumorigenesis. INK4 proteins were positively correlated with the infiltration of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) and immune checkpoints (CTLA-4, PD1, and PD-L1). CDKN2D had the highest correlation (correlation coefficient >0.3) with all the above-mentioned infiltrating immune cells and immune checkpoints, indicating that it may be useful as an immunotherapy target. The INK4 family was valuable for diagnosis and predicting the prognosis of HCC and participated in the occurrence, progression, and immune regulation of HCC, demonstrating its potential as a diagnostic and prognostic biomarker and therapeutic target in HCC.

Keywords: Hepatocellular carcinoma; INK4; biomarkers; immune; prognosis.

Figure 1. Transcription levels of INK4 family members in 20 different types of cancers in the ONCOMINE database

Panels show the numbers of datasets with either significant up-regulation (red) or down-regulation (blue) of mRNA expression of the target genes. The threshold was designed using the following parameters: P-value of 0.01 and fold-change of 1.5.

Figure 2. Overexpression of INK4 family in HCC

(A) The expression levels of INK4 family in 374 HCC tissues and 50 normal liver tissues based on TCGA. (B) The expression levels of INK4 family in 50 HCC tissues and their paired adjacent normal liver tissues based on TCGA. (C) The ROC curve of INK4 family based on TCGA. (D–G) Quantitative PCR analysis of INK4 family members in clinical HCC samples. (H) Immunohistochemical-staining results of INK4 family members in clinical HCC samples. ***P<0.001.

Figure 3. Relationship between mRNA expression of INK4 family and cancer stages and prognosis

(A) The correlation analysis of mRNA expression of all INK4 family members and T stages of HCC. (B) The correlation analysis of mRNA expression of all INK4 family members and pathologic stage. The Kaplan–Meier survival curves for OS (C), PFS (D), and DSS (E). Comparing patients with high (red) and low (black) expression of INK4 family members in HCC.

Figure 4. Biological process and KEGG pathway enrichment analysis

(A–D) Biological process and KEGG analysis of all INK4 family members, including CDKN2A (A), CDKN2B (B), CDKN2C (C), and CDKN2D (D). (E) Venn diagram showing the intersection of coexpressed genes of INK4 family members. (F) Biological process and KEGG pathway enrichment analysis of intersected coexpressed genes of INK4 family members. (G) Ten hub genes selected using CytoHubba.

Figure 5. Associations between INK4 family members and immune cell infiltration in HCC

(A) The correlation analysis of CDKN2A and the infiltration levels of six types of immune cells (macrophages, neutrophils, dendritic cells, CD4+ T cells, CD8+ T cells, and B cells). (B) The correlation analysis of CDKN2B and the infiltration levels of immune cells. (C) The correlation analysis of CDKN2C and the infiltration levels of immune cells. (D) The correlation analysis of CDKN2D and the infiltration levels of immune cells.

Figure 6. Correlation of the expression of INK4 family members with immune checkpoints in HCC

(A) The correlation analysis of PD1 (PDCD1) and the expression of all INK4 family members. (B) The correlation analysis of PD-L1 (CD274) and the expression of INK4 family members. (C) The correlation analysis of CTLA-4 and the expression of INK4 family members.