Role of CENPL, DARS2, and PAICS in determining the prognosis of patients with lung adenocarcinoma

Abstract

Background: Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers, and is the leading cause of tumor-related death. Lung adenocarcinoma (LUAD) is the most prevalent subtype of NSCLC. Although significant progress of LUAD treatment has been made under multimodal strategies, the prognosis of advanced LUAD is still poor due to recurrence and metastasis. There is still a lack of reliable markers to evaluate the LUAD prognosis. This study aims to explore novel biomarkers and construct a prognostic model to predict the prognosis of LUAD patients.

Methods: The Genomic Data Commons-The Cancer Genome Atlas-Lung Adenocarcinoma (GDC-TCGA-LUAD) dataset was downloaded from the University of California, Santa Cruz (UCSC) Xena browser. The GSE72094 and GSE13213 datasets and corresponding clinical information were downloaded from the Gene Expression Omnibus (GEO) database. By analyzing these datasets using DESeq2 R package and Limma R package, differentially expressed genes (DEGs) were found. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to analyze possible enrichment pathways. A protein-protein interaction (PPI) network was constructed to explore possible relationship among DEGs by using the STRING database. A survival analysis was performed to identify reliable prognostic genes using the Kaplan-Meier method. A multi-omics analysis was performed using the Gene Set Cancer Analysis (GSCA). The Tumor Immune Estimation Score (TIMER) database was used to analyze the association between prognostic genes and immune infiltration. A Spearman correlation analysis was conducted to examine the correlation between prognostic genes and drug sensitivity. A multivariate Cox regression was used to identify independent prognostic factors. Next, a nomogram was constructed using the rms R package. Finally, the expressions of aspartyl-tRNA synthetase 2 (DARS2) and phosphoribosyl aminoimidazole carboxylase (PAICS) were detected using immunohistochemistry (IHC).

Results: We screened out 30 DEGs prior to functional enrichment and PPI network analysis revealing potential enrichment pathways and interactions of these DEGs. Then survival analysis revealed the CENPL, DARS2, and PAICS expression was negatively correlated with LUAD prognosis. Additionally, multi-omics analysis showed CENPL, DARS2, and PAICS expressions were significantly higher in LUAD tissues than normal tissues. CENPL, DARS2, and PAICS were all up-regulated in late stage and M1 stage. Correlation analysis indicated CENPL, DARS2, and PAICS may not be associated with activation or suppression of immune cells. Drug sensitivity analysis revealed many potentially effective drugs and small molecule compounds. Moreover, we successfully constructed a robust and stable nomogram by combining the DARS2 and PAICS expression with other clinicopathological variables. Finally, IHC results showed DARS2 and PAICS were significantly up-regulated in LUAD.

Conclusions: The CENPL, DARS2, and PAICS expression was negatively correlated with LUAD prognosis. A prognostic model, which integrated DARS2, PAICS, and other clinicopathological variables, was able to effectively predict LUAD patients prognosis.

Keywords: CENPL; DARS2; PAICS; lung adenocarcinoma (LUAD); prognosis.

Figure 1

Identification of differentially expressed genes. (A) PCA of normal and tumor samples from TCGA-LUAD. (B) PCA of early and late stage group from TCGA-LUAD. (C) Venn diagram of intersecting DEGs from normal and tumor samples from TCGA-LUAD, early and late stage groups from TCGA-LUAD, early and late stage groups from GSE13213 and GSE72094. (D) Heatmap of 30 DEGs in TCGA-LUAD dataset. (E) Heatmap of 30 DEGs in the GSE13213 dataset. (F) Heatmap of 30 DEGs in the GSE72094 dataset. TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma; PCA, principal component analysis; DEGs, differentially expressed genes.

Figure 2

Functional enrichment analysis and PPI network construction. (A) GO enrichment analysis of 30 DEGs. (B) KEGG enrichment analysis of 30 DEGs. (C) PPI network of DEGs. The cyan line stands for “from curated databases”. The magenta line stands for “experimentally determined”. The green line stands for “gene neighborhood”. The red line stands for “gene fusions”. The blue line stands for “gene co-occurrence”. The yellow-green line stands for “textmining”. The black line stands for “co-expression”. The purple line stands for “protein homology”. PPI, protein-protein interaction; GO, Gene Ontology; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3

Identification of key genes significantly associated with prognosis. (A) Venn diagram of DEGs associated with prognosis in TCGA-LUAD, GSE72094, and GSE13213 datasets, among which three intersecting genes were identified; CENPL, DARS2 and PAICS; (B,D,F) Kaplan-Meier OS curves based on CENPL, DARS2, and PAICS expression for LUAD patients in the GSE13213 dataset. (C,E,G) Kaplan-Meier OS curve based on CENPL, DARS2, and PAICS expression for LUAD patients in the GSE72094 dataset. CENPL, centromere protein L; DARS2, aspartyl-tRNA synthetase 2; PAICS, phosphoribosylaminoimidazole carboxylase; TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma; DEGs, differentially expressed genes; OS, overall survival.

Figure 4

Survival analysis of CENPL, DARS2, and PAICS expression in TCGA-LUAD dataset. (A-C) Kaplan-Meier OS curves based on CENPL, DARS2, and PAICS expression; (D-F) Kaplan-Meier PFS curves based on CENPL, DARS2, and PAICS expression; (G-I) Kaplan-Meier DSS curves based on CENPL, DARS2, and PAICS expression. OS, overall survival; CENPL, centromere protein L; DARS2, aspartyl-tRNA synthetase 2; PAICS, phosphoribosylaminoimidazole carboxylase; exp, expression; TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma; PFS, progression-free survival; DSS, disease-specific survival.

Figure 5

Expression of CENPL, DARS2, and PAICS in pan-cancer transcriptome and early and advanced stage LUAD patient data. (A-C) CENPL, DARS2, and PAICS expression of pan-cancer transcriptome data in TCGA-LUAD dataset. The red box stands for the primary tumor. The purple box stands for the metastatic tumor. The blue box stands for the normal tumor. (D-F) CENPL, DARS2, and PAICS expression of early and advanced stage LUAD patients in TCGA-LUAD, GSE13213, and GSE72094 datasets. (G) CENPL, DARS2, and PAICS expression of M0 and M1 patients in TCGA-LUAD dataset. *, P<0.05; **, P<0.01; ***, P<0.001. TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma; CENPL, centromere protein L; DARS2, aspartyl-tRNA synthetase 2; PAICS, phosphoribosylaminoimidazole carboxylase.

Figure 6

SNP and CNV analysis of CENPL, DARS2, and PAICS. (A-C) Mutation sites of CENPL, DARS2, and PAICS in TCGA-LUAD dataset; (D-F) CNV analysis of CENPL, DARS2, and PAICS in TCGA-LUAD dataset; (G-I) Spearman correlation between CENPL, DARS2, and PAICS CNVs and mRNA expression in TCGA-LUAD dataset. CENPL, centromere protein L; DARS2, aspartyl-tRNA synthetase 2; PAICS, phosphoribosylaminoimidazole carboxylase; SNP, single nucleotide polymorphism; CNV, copy number variation; FDR, false discovery rate; RSEM, RNA-Seq by Expectation-Maximization; LUAD, lung adenocarcinoma; TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma.

Figure 7

Immune cell infiltration analysis of CENPL, DARS2, and PAICS based on the TIMER database. (A) Correlation between CENPL and tumor purity, B cells, CD4⁺ T cells, CD8⁺ T cells, macrophages, neutrophil, and dendritic cells. (B) Correlation between DARS2 and tumor purity, B cells, CD4⁺ T cells, CD8⁺ T cells, macrophages, neutrophil, and dendritic cells. (C) Correlation between PAICS and tumor purity, B cells, CD4⁺ T cells, CD8⁺ T cells, macrophages, neutrophil, and dendritic cells. CENPL, centromere protein L; DARS2, aspartyl-tRNA synthetase 2; PAICS, phosphoribosylaminoimidazole carboxylase; TPM, transcripts per million.

Figure 8

Relationship between key prognostic genes and drug sensitivity. (A-C) Drug sensitivity analysis of CENPL, DARS2, and PAICS in GCP dataset. (D) Drug sensitivity analysis of CENPL, DARS2, and PAICS in the GDSC dataset. (E) Drug sensitivity analysis of CENPL, DARS2, and PAICS in the CTRP dataset. CENPL, centromere protein L; DARS2, aspartyl-tRNA synthetase 2; PAICS, phosphoribosylaminoimidazole carboxylase; GDSC, Genomics of Drug Sensitivity in Cancer; CTRP, Cancer Therapeutics Response Portal; FDR, false discovery rate.

Figure 9

Construction and validation of a prognostic model for LUAD patients. (A) Univariable and multivariable Cox analyses were performed by combining expression of CENPL, DARS2, and PAICS with other clinical parameters (age, gender, and tumor stage). (B) Nomogram for predicting the 1-, 3-, and 5-year OS probabilities of LUAD patients. (C) Time-dependent ROC curve of the training set in TCGA-LUAD dataset and the test set in the GSE13213 dataset. (D) Calibration plot of the established nomogram for predicting probabilities of 1-, 3-, and 5-year OS. HR, hazard ratio; CI, confidence interval; CENPL, centromere protein L; DARS2, aspartyl-tRNA synthetase 2; PAICS, phosphoribosylaminoimidazole carboxylase; TCGA-LUAD, The Cancer Genome Atlas-Lung Adenocarcinoma; TPR, true positive rate; FPR, false positive rate; AUC, area under the curve; OS, overall survival.

Figure 10

IHC for PAICS and DARS2 protein expression in resected LUAD specimens and normal tissues. (A) Representative images for PAICS protein expression in normal lung tissue. (B) Representative images for PAICS protein expression in LUAD specimen. (C) Analysis results of PAICS protein expression between normal lung tissues and LUAD specimens, the data represent the mean ± SD of three independent experiments. *, P<0.05. (D) Representative images for DARS2 protein expression in normal lung tissue. (E) Representative images for DARS2 protein expression in LUAD specimen. (F) Analysis results of DARS2 protein expression between normal lung tissues and LUAD specimens, the data represent the mean ± SD of three independent experiments. *, P<0.05. LUAD, lung adenocarcinoma; PAICS, phosphoribosylaminoimidazole carboxylase; DARS2, aspartyl-tRNA synthetase 2; IHC, immunohistochemistry; SD, standard deviation.