Carboxypeptidase N2 as a Novel Diagnostic and Prognostic Biomarker for Lung Adenocarcinoma

Abstract

Carboxypeptidase N2 (CPN2) is a plasma metallo-protease that cleaves basic amino acids from the C-terminal of peptides and proteins. Emerging evidence showed that carboxypeptidases perform many diverse functions in the body and play key roles in tumorigenesis. However, the clinical significance and biological functions of CPN2 in lung adenocarcinoma remain unclear. Our study aimed to explore the potential role and functions of CPN2 in lung adenocarcinoma. The results showed that the transcription level of CPN2 was significantly increased in the tumor tissues of lung adenocarcinoma patients compared to the adjacent normal tissues in The Cancer Genome Atlas cohort (P < 0.05). The survival plots showed that the overall survival of patients with a high expression of CPN2 was significantly lower than that of patients with a low expression of CPN2, both in the Kaplan-Meier database and the clinical sample cohort (P < 0.05). The tissue microarray analysis found that CPN2 protein expression was significantly positively correlated with node status and tumor stage as well as tumor malignancy (P < 0.05). Further univariate and multivariate Cox regression analyses showed that CPN2 may act as an independent prognostic factor in patients with lung adenocarcinoma (P < 0.05). In addition, the analysis of co-expression genes from LinkedOmics showed that CPN2 was positively associated with many genes of fibrillar collagen family members and the PI3K-Akt pathway. The gene set enrichment analysis showed that a higher expression of CPN2 may participate in mTOR, TGF-BETA, NOTCH, TOLL-like-receptor, WNT, and MAPK signaling pathway in lung adenocarcinoma. Notably, the knockdown of CPN2 significantly inhibited the ability of cell proliferation, clone formation, invasion, and migration. Our findings suggested that the upregulation of CPN2 is associated with a worse clinical outcome in lung adenocarcinoma and cancer-related pathways, which laid the foundation for further research on CPN2 during carcinogenesis.

Keywords: CPN2; biomarker; diagnosis; lung adenocarcinoma; prognosis.

Figure 1

The CPN2 transcription level was upregulated in lung cancer patients from The Cancer Genome Atlas cohort. (A) CPN2 expression was significantly upregulated in lung cancer tissues compared with the adjacent normal samples according to the total samples (P < 0.01). Two-tailed Wilcoxon test. (B, C) CPN2 expression was significantly increased in lung adenocarcinoma (B) and squamous cell carcinoma (C) tissues compared with the adjacent normal samples (P < 0.01). Two-tailed Wilcoxon test. (D) The receiver operating characteristic curve showed that the CPN2 gene expression level was a promising biomarker with high sensitivity and specificity for the clinical diagnosis of lung adenocarcinoma, but not squamous cell carcinoma.

Figure 2

CPN2 was associated with survival outcome in lung adenocarcinoma cancer from the Kaplan–Meier plotter. (A–C) Overall survival of CPN2 in the lung cancer (A), lung adenocarcinoma (B), and lung squamous cell carcinoma (C) cohorts, respectively. (D–F) Overall survival plot of CPN2 in stage I (D), stage II (E), and stage III (F) in the lung adenocarcinoma cohort, respectively. Each group was divided according to the median expression cutoff value of CPN2 which equal to 24, and P <0.05 was considered statistically significant.

Figure 3

CPN2 expression was significantly upregulated in clinical lung adenocarcinoma tissues. (A) Immunohistochemistry analysis of CPN2 expression levels in adjacent and lung adenocarcinoma tissues, with the scale bar equal to 100 μm. CPN2 was highly expressed in tumor tissues. Immunohistochemistry staining assays were performed with an anti-CPN2 antibody (magnification, ×200). (B) CPN2 expression was significantly upregulated in lung adenocarcinoma tissues according to the total samples with a staining score. Two-tailed Wilcoxon test. (C) Receiver operating characteristic curve of CPN2 protein expression in clinical samples for the diagnosis of lung adenocarcinoma.

Figure 4

A high CPN2 protein expression was significantly associated with poor survival in clinical lung adenocarcinoma patients from our cohort by Kaplan–Meier survival curves. (A) The Kaplan–Meier survival curves based on a log-rank test showed that lung cancer patients with a high CPN2 expression had poorer survival than those with a low CPN2 expression (P < 0.0001). (B) Kaplan–Meier curves of lung adenocarcinoma patients with TNM stages I and II (P < 0.0001). (C) Kaplan–Meier curves of lung adenocarcinoma patients with TNM stages III and IV (P > 0.05).

Figure 5

Co-expression genes of CPN2 using Linkedomics analysis. (A) Protein–protein interaction of its co-expression genes. Co-expression genes with Pearson correlation coefficient >0.3 were gathered to draft this network, and the top 10 hub genes are shown in yellow color. (B) KEGG enrichment analysis of these co-expression genes. (C) Validation of 10 hub genes in The Cancer Genome Atlas lung adenocarcinoma database.

Figure 6

Protein–protein interaction network and functional enrichment analysis of genes interacted with CPN2 through Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis. (A) Ten genes interacted with CPN2 confirmed by STRING database. (B) Gene ontology biological process enrichment analyzed by these interaction genes. (C) Kyoto Encyclopedia of Genes and Genomes enrichment analyzed by these interaction genes. False discovery rate <0.5 was considered statistically significant.

Figure 7

Gene set enrichment analysis of CPN2 in lung adenocarcinoma. The high expression of CPN2 was significantly enriched in the mTOR signaling pathway (A), TGF-BETA signaling pathway (B), NOTCH signaling pathway (C), TOLL-like-receptor signaling pathway (D), WNT signaling pathway (E), and MAPK signaling pathway (F). False discovery rate <0.25 was considered statistically significant.

Figure 8

CPN2 knockdown significantly inhibited lung cancer cell proliferation, migration, and invasion. (A) The knockdown expression of CPN2 was confirmed by qRT-PCR in lung cancer cells. (B) CCK-8 assay was used to examine the effect of CPN2 knockdown on proliferation in lung cancer cells. (C) Colony formation assays were used to examine the effect of CPN2 knockdown on growth in lung cancer cells. (D) Wound healing assay was used to determine the motility of CPN2 knockdown in lung cancer cells. The quantification of migrated cells is shown in the right panel. (E) Transwell assay was used to examine the effect of CPN2 knockdown on migration and invasion in lung cancer cells. The quantification of migrated cells is shown in the right panel. (F) CCK-8 assay was used to detect the effect of CPN2 overexpression on proliferation in lung cancer cells with CPN2 knockdown. (G) Transwell assay was used to detect the effect of CPN2 overexpression on migration and invasion in lung cancer cells with CPN2 knockdown. All data represent the mean ± SD of three independent experiments. **P < 0.01.