Overexpressing PLOD Family Genes Predict Poor Prognosis in Pancreatic Cancer

Abstract

Background: Pancreatic cancer is a common malignant tumor. Multiple studies have shown that procollagen lysyl-hydroxylase (PLOD) family genes were closely related to tumor progression and metastasis in a variety of human cancers. This study aimed to explore the prognosis and biological role of PLOD family genes in pancreatic adenocarcinoma (PAAD).

Methods: GEPIA, GEO, HPA, CCLE, Kaplan-Meier plotter, cBioPortal, LinkedOmics, DAVID6.8, STRING, and TIMER were employed to determine the prognostic values and biological function of PLOD family members in PAAD.

Results: The mRNA and protein expression patterns of PLOD family members were noticeably up-regulated in PAAD compared with normal tissues. PLOD family gene expression was also up-regulated in pancreatic cancer cell lines. PLOD1 was correlated with histological and pathological grades of pancreatic cancer. PLOD2 was related to histological grade. The high expression of PLOD1-2 was correlated with the poor overall survival rate and relapse-free survival rate in patients with PAAD. Additionally, PLODs showed high sensitivity and specificity in distinguishing pancreatic cancer from normal tissues. Through the functional enrichment analysis of PLOD-related genes in PAAD, we found that PLODs were enriched in collagen fiber tissue structure, lysine degradation, and collagen biosynthesis. Pathway analysis confirmed that PLODs regulated the proliferation, migration, and metastasis of pancreatic cancer through the RalGEF-Ral signaling pathway. Furthermore, the level of expression of PLOD1-2 was positively correlated with the activity of tumor-infiltrating immune cells, including CD8+T cells, neutrophils, macrophages, and dendritic cells. The level of expression of PLOD3 was inversely correlated with the level of infiltration of CD8+T cells. PLOD1 and PLOD2 were highly expressed in pancreatic cancer tissues with TP53 and KRAS mutations, respectively. However, the level of expression of PLOD3 in SMAD4 wild-type pancreatic cancer was increased.

Conclusion: The findings showed that individual PLOD genes or PLOD family genes could be potential prognostic biomarkers for PAAD.

Keywords: PLOD; mutations; pancreatic cancer; prognosis; tumor-infiltrating immune cells.

Figure 1

Expression of PLOD family members in different tumor tissues. (A–C) depict the transcription levels of PLOD1, PLOD2, and PLOD3 in different types of cancer, respectively. Red, up-regulated; green, down-regulated.

Figure 2

(A) Boxplot results of the level of expressions of PLODs family members in PAAD analyzed using GEPIA. Red box, tumor samples; grey box, normal samples. T, tumor; N, normal. (B and C) Boxplot results of PLOD family member levels of expression in PAAD were analyzed using GSE16515 and GSE15471 respectively. *Denotes P < 0.05, **Denotes P < 0.01, ***Denotes P < 0.001.

Figure 3

CCLE analysis of the expression of members of the PLODs family in pancreatic cancer cell lines. Figure (A–C) show the expression of PLOD1/2/3 in pancreatic cancer cell lines, respectively. The horizontal axis in the figure represents the expression of the gene, the ordinate represents different cell lines, the size of the dot in the figure represents the level of expression, and the different colors also represent the level of expression.

Figure 4

IHC images of different members of the PLOD family were detected using the Human Protein Atlas (HPA). (A) shows PLOD1 in pancreatic cancer and not in normal pancreatic tissue. (B and C) show highly expressed PLOD2 and PLOD3 in pancreatic cancer but not expressed in normal pancreatic tissue.

Figure 5

Survival analysis of PLOD family members in PAAD. (A) Over-expressions of PLOD1 and PLOD2 were associated with shorter OS in PAAD. (B) Over-expressions of PLOD1 and PLOD2 were associated with shorter RFS in PAAD. (C) PLODs have high accuracy in predicting the outcome (normal or tumor).

Figure 6

Expression levels of PLOD family genes in pancreatic cancer with KRAS mutations (A-C). Expression levels of PLOD family genes in pancreatic cancer with TP53 mutations (D-F).Expression levels of PLOD family genes in pancreatic cancer with SMAD4 mutations (G-I).Expression levels of PLOD family genes in pancreatic cancer with CDKN2A mutations (J-L).

Figure 7

(A) Summary of alterations in PLOD family members in PAAD. (B) mRNA expression alterations (RNA Seq V2 RSEM) of the PLOD family genes in PAAD patients.

Figure 8

(A) The correlation between each type of TIICs (B-cells, CD4+ T-cells, CD8+ T-cells, neutrophils, macrophage, and dendritic cells) and PLOD1. (B) The correlation between each type of TIICs (B-cells, CD4+ T-cells, CD8+ T-cells, neutrophils, macrophages, and dendritic cells) andPLOD2. (C) The correlation between each type of TIICs (B-cells, CD4+ T-cells, CD8+ T-cells, neutrophils, macrophages, and dendritic cells) and PLOD3).

Figure 9

Signaling modules correlated with PLOD family genes in PAAD. (A–C) The volcano plot showed the co-expression genes correlated with PLOD1, 2, and 3 in the PAAD cohort from the TCGA database. (D and E) Venn diagram showing the positively and negatively co-expressed genes correlated with PLOD1, 2, and 3. (F–H) Scatter plot of PLOD1 to 3 correlations with RALA.

Figure 10

(A) Positively correlated gene enrichment. (B) Negatively correlated gene enrichment. Entry GO: BP, CC, and MF terms, KEGG pathway.

Figure 11

Protein interaction network of 30 functional partners with a confidence score above 0.9 based on the STRING database. PLOD1, 2, and 3 are the seed genes. Ten interacting partners with the highest confidence scores were colored and placed in the outer shell. The other twenty interacting partners were placed in the inner shell. The blue lines represent the correlation between proteins and the thickness of the lines indicates the strength of data support.