A Comprehensive Pan-Cancer Analysis Reveals Cyclin-Dependent Kinase Inhibitor 2A Gene as a Potential Diagnostic and Prognostic Biomarker in Colon Adenocarcinoma

Abstract

Introduction Cyclin-dependent kinase inhibitor 2A (CDKN2A) is a suppressor carcinogenic gene that is upregulated across various types of cancer including breast, liver, thyroid, and bile duct cancer due to its crucial role in cell cycle regulation and cell division. Nevertheless, it is mostly investigated at the genetic level, but it is still poorly studied on pan-cancer analysis as a biomarker and this study shows its significant potential diagnostic and prognostic characteristics. However, this study aims to investigate the role of CDKN2A as a diagnostic and prognostic biomarker across various types of cancer focusing primarily on colon adenocarcinoma (COAD). Methods We investigated CDKN2A gene expression in a pan-cancer analysis across different types of cancer to show its diagnostic potential characteristics by using various bioinformatic tools, including Tumor Immune Estimation Resource (TIMER) 2.0, Gene Expression Profiling Interactive Analysis (GEPIA), and University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) database. TIMER was used to profile gene expression across 32 types of cancer composed of 10,000 RNA-seq samples obtained from the Cancer Genome Atlas (TCGA) and to analyze the tumor-infiltrating immune cells. In addition, GEPIA and UALCAN were further used to analyze gene expression, in terms of gene regulation, pathological stages, and clinical parameters, including gender, age, and race. Therefore, we used GEPIA, UALCAN, and Kaplan-Meier plotter particularly across adenocarcinoma to investigate CDKN2A prognosis by studying its high expression association with the patient's overall survival rate to show the tumor progression. Then, we looked into the genetic alteration of CDKN2A by using the cBio Cancer Genomics Portal (cBioPortal), including 10 pan-cancer studies. We concluded the analysis with gene validation by using a public cohort in Gene Expression Omnibus (GEO). Results CDKN2A showed a trend of upregulation in most cancers and it was significantly upregulated in five cancers, which were commonly identifiable in three databases, including breast invasive carcinoma (p < 0.001), kidney chromophobe (p < 0.001), kidney renal clear cell carcinoma (p < 0.001), kidney renal papillary cell carcinoma (p < 0.001), and COAD (p < 0.001). The upregulation was significantly different in association with pathogenic stages II and III (pr(>F) = 0.00234) which was identifiable significantly in COAD more than in other cancers. The gene showed a high upregulation in association with poor prognosis of patient survival in three cancers, including COAD (log-rank p = 0.011), mesothelioma (log-rank p = 5.9e-07), and liver hepatocellular carcinoma (log-rank p = 0.0045). Therefore, COAD was the only comprehensively analyzed tumor to show a diagnostic and prognostic potential characteristic during high upregulation of CDKN2A. Furthermore, CDKN2A displayed a rare mutation in the form of deep deletion (9%) and revealed an upregulation associated with CD4+ T cells (p = 0.0108), macrophage (p = 0.0073), and neutrophils (p = 0.0272) as immune cells infiltrating COAD. Conclusion Our study demonstrates the pan-cancer relevance of CDKN2A and revealed a novelty in showing CDKN2A underscores its potential as a diagnostic prognostic biomarker in COAD since CDKN2A is mostly studied at a genetic level across COAD.

Keywords: cancer biomarker; cancer genomic profiling; cdkn2a; cyclin-dependent kinase inhibitor 2a; diagnostic marker; overexpression; prognostic markers.

Figure 1. The differential gene expression analysis across various types of cancer by TIMER 2.0

The red columns represent the tumor tissues and the blue ones represent the normal tissues while the stars indicate the differential significance between the tumor and normal samples. ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05. BRCA: Breast invasive carcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; HNSC: head and neck squamous cell carcinoma; HNSC-HPV: head and neck squamous cell carcinoma associated with human papillomavirus; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; STAD: stomach adenocarcinoma; THCA: thyroid carcinoma; UCEC: uterine corpus endometrial carcinoma; BLCA: bladder urothelial carcinoma; ESCA: esophageal carcinoma; ACC: adrenocortical carcinoma; BRCA-Basal, BRCA-Her2, BRCA-luminal; CSCC: cervical squamous cell carcinoma; CESC: endocervical adenocarcinoma; BLBC: basal-like breast cancer; GBM: glioblastoma multiforme; LAML: acute myeloid leukemia; LGG: low-grade gliomas, MESO: mesothelioma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; SARC: sarcoma; TGCT: testicular germ cell tumors; THYM: thymoma; UCS: uterine carcinosarcoma; SKCM: skin cutaneous melanoma; UVM: uveal melanoma.

Figure 2. Gene expression profiling of CDKN2A for the common tumors

A-E: Gene expression by TIMER, F-J gene expression by UALCAN.  *p ≤ 0.001,  ***p ≤ 0.001. Red boxes represent tumor tissues, and the grey and blue boxes represent normal tissues. BRCA: Breast invasive carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; COAD: colon adenocarcinoma; num: number; T: tumor; N: normal.

Figure 3. Gene expression analysis across the pathological stages for the common diagnostic tumors

A-E: Pathological stages by TIMER, F-J pathological stages by UALCAN.  Pr (>F) < 0.05, ***p ≤ 0.001. BRCA: Breast invasive carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; COAD: colon adenocarcinoma.

Figure 4. Gene clinical parameters across COAD by UALCAN

A. The expression of the gene in COAD across s races, B. The gene expression in COAD vs based on normal and gender including males and females, C. Expression of the CDKN2A gene in COAD based on age, and F. The gene expression in COAD based on the P53 mutation status including mutant and non-mutant. COAD: Colon adenocarcinoma; UALCAN: University of Alabama at Birmingham Cancer Data Analysis Portal.

Figure 5. The CDKN2A gene correlation with other genes across COAD

A. CKDN2A vs ZFAND2A, B. CDKN2A vs GAS5, and C. CDKN2A vs C7ORF40. The scattered red points represent the tumor correlation. ZFAND2A: Zinc finger AN1-type containing 2A; GAS5: growth arrest-specific 5; 7ORF40: small nucleolar RNA host gene 15.

Figure 6. Data representation of overall survival for COAD against the CDKN2A expression

A. Overall survival generated by GEPIA, B. Overall survival generated by UALCAN, and C. Overall survival generated by the KM plotter. p < 0.05. The red line represents high gene expression, and the black line represents low gene expression. It shows poor prognosis in COAD during CDKN2A high expression. COAD: Colon adenocarcinoma, HR: hazard ratio, CDKN2A: cyclin-dependent kinase inhibitor 2A; UALCAN: University of Alabama at Birmingham Cancer Data Analysis Portal; KM: Kaplan-Meier.

Figure 7. Gene alternation across various types of cancers in a pan-cancer analysis visualized by cBioPortal

A. Gene alternation across various types of cancers, B. The CDKN2A gene copy number alternations highlighting a rare deep deletion, and C. The copy number alternation based on age raging from light red of 0 years old till dark red indicating max-age parameter of 98 years old. cBioPortal: cBio Cancer Genomics Portal.

Figure 8. The purity and the immune cell infiltration in the COAD microenvironment upon CDKN2A upregulated expression

A. The upregulation of CD4+ T Cell, macrophage, neutrophil, and dendritic cells across the downregulation purity of CDKN2A and B. The regulation of B cell and CD8+ T cell. p-value (P) and partial correlation (Partial cor.). CD4+ T Cell: T helper lymphocyte cells, B Cell: B lymphocyte cells; CD8+ T Cell: cytotoxic T lymphocyte cells; COAD: colon adenocarcinoma.

Figure 9. The corrected volcano plot after obtaining the data from GEO of cohort case GSE84984

CDKN2A is marked in bold black color on the red upregulated side of the volcano plot in COAD patients comparing control samples and tumor samples while the blue side of the plot represents the downregulated genes in this cohort case. The grey marked zone represents the genes that have no significance according to the logFC > 0.5 and p < 0.05. CDKN2A: Cyclin-dependent kinase inhibitor 2A; GEO: Gene Expression Omnibus.

Figure 10. The gene expression across various types of cancer in GEPIA

A. ACC, B. BLCA, C. BRCA, D. CESC, E. CHOL, F. COAD, G. DLBC, H. ESCA, I. HNSC, J. KICH, K. KIRC, and L. KIRP. *p ≤ 0.001. Red boxes represent tumor tissues, and the grey and blue boxes represent normal tissues. GEPIA: Gene Expression Profiling Interactive Analysis; ACC: adrenocortical carcinoma; BLCA: urothelial bladder carcinoma; BRCA: breast invasive carcinoma; CESC: endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: diffuse large B-cell lymphoma; ESCA: esophageal carcinoma; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma.

Figure 11. The gene expression across various types of cancer in GEPIA

A. LAML, B. LGG, C. LIHC, D. LUAD, E. LUSC, F. OV, G. THYM, H. PAAD, I. READ, J. SARC, K. SKCM, L. STAD, M. TGCT, and N. UCEC. *p ≤ 0.001. Red boxes represent tumor tissues, and the grey and blue boxes represent normal tissues. GEPIA: Gene expression profiling interactive analysis; LAML: acute myeloid leukemia; LGG: low-grade gliomas; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; OV: ovarian serous cystadenocarcinoma; THYM: thymoma; PAAD: pancreatic adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: tenosynovial giant cell tumors; UCEC: uterine corpus endometrial carcinoma.

Figure 12. The upregulated gene expression across various types of cancer in UALCAN

A. BLCA, B. BRCA, C. CESC, D. CHOL, E. COAD, F. ESCA, G. GBM, H. HNSC, I. KICH, J. KIRC. The red box represents the tumor samples, and the blue box represents normal samples. UALCAN: University of Alabama at Birmingham Cancer Data Analysis Portal; BLCA: urothelial bladder carcinoma; BRCA: breast invasive carcinoma; CESC: endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; ESCA: esophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma.

Figure 13. The upregulated gene expression across various types of cancer in UALCAN

A. KIRP, B. LUAD, C. LUSC, D. PAAD, E. PRAD, F. READ, G. SARC, H. STAD, I. THCA, J. LIHC, and K. UCEC. The red box represents the tumor samples, and the blue box represents normal samples. UALCAN: University of Alabama at Birmingham Cancer Data Analysis Portal; KIRP: kidney renal papillary cell carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; PAAD: pancreatic adenocarcinoma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; STAD: stomach adenocarcinoma; THCA: thyroid carcinoma; LIHC: liver hepatocellular carcinoma; UCEC: uterine corpus endometrial carcinoma.

Figure 14. The significant overall survival in high CDKN2A expression based on overall survival analysis in GEPIA

A. COAD, B. MESO, C. PCPG, D. LIHC. Logrank p < 0.05. The red line shows the high gene expression correlated with overall survival prognosis, and the black line shows the low gene expression correlated with overall survival. GEPIA: Gene Expression Profiling Interactive Analysis; COAD: colon adenocarcinoma; MESO: mesothelioma; PCPG: pheochromocytoma and paraganglioma; LIHC: liver hepatocellular carcinoma; CDKN2A: cyclin-dependent kinase inhibitor 2A.

Figure 15. The significant overall survival in high CDKN2A expression based on overall survival analysis in UALCAN

A. COAD, B. LIHC, C. MESO, D. PCPG. p-value < 0.05. The red line shows the high gene expression correlated with overall survival prognosis, and the blue line shows the low gene expression correlated with overall survival. UALCAN: University of Alabama at Birmingham Cancer Data Analysis Portal; COAD: colon adenocarcinoma; LIHC: liver hepatocellular carcinoma; MESO: mesothelioma; PCPG: pheochromocytoma and paraganglioma; CDKN2A: cyclin-dependent kinase inhibitor 2A.