Annexin A1 Expression Is Associated with Epithelial-Mesenchymal Transition (EMT), Cell Proliferation, Prognosis, and Drug Response in Pancreatic Cancer

Abstract

Annexin A1 (ANXA1) is a calcium-dependent phospholipid-binding protein overexpressed in pancreatic cancer (PC). ANXA1 expression has been shown to take part in a wide variety of cancer biology, including carcinogenesis, cell proliferation, invasion, apoptosis, and metastasis, in addition to the initially identified anti-inflammatory effect in experimental settings. We hypothesized that ANXA1 expression is associated with cell proliferation and survival in PC patients. To test this hypothesis, we analyzed 239 PC patients in The Cancer Genome Atlas (TCGA) and GSE57495 cohorts. ANXA1 expression correlated with epithelial-mesenchymal transition (EMT) but weakly with angiogenesis in PC patients. ANXA1-high PC was significantly associated with a high fraction of fibroblasts and keratinocytes in the tumor microenvironment. ANXA1 high PC enriched multiple malignant gene sets, including hypoxia, tumor necrosis factor (TNF)-α signaling via nuclear factor-kappa B (NF-kB), and MTORC1, as well as apoptosis, protein secretion, glycolysis, and the androgen response gene sets consistently in both cohorts. ANXA1 expression was associated with TP53 mutation alone but associated with all KRAS, p53, E2F, and transforming growth factor (TGF)-β signaling pathways and also associated with homologous recombination deficiency in the TCGA cohort. ANXA1 high PC was associated with a high infiltration of T-helper type 2 cells in the TME, with advanced histological grade and MKI67 expression, as well as with a worse prognosis regardless of the grade. ANXA1 expression correlated with a sensitivity to gemcitabine, doxorubicin, and 5-fluorouracil in PC cell lines. In conclusion, ANXA1 expression is associated with EMT, cell proliferation, survival, and the drug response in PC.

Keywords: ANXA1; GSEA; biomarker; gene set; metastasis; pancreatic cancer; survival; treatment response; tumor microenvironment.

Figure 1

Association of Annexin A1 (ANXA1) expression with epithelial–mesenchymal transition (EMT), angiogenesis, and stromal cells in The Cancer Genome Atlas (TCGA) and GSE57495 cohorts. (A) Scatter plots of ANXA1 expression with the EMT score. (B) Boxplots of EMT-associated gene expression: cadherin1 (CDH1) and CDH2, snail family transcriptional repressor 1 (SNAI1) and SNAI2, and twist-related protein 1 (TWIST1) by ANXA1 low and ALXA1 high pancreatic cancer (PC). (C) Scatter plots of ANXA1 expression with the angiogenesis score. (D) Boxplots of infiltrating fraction of endothelial cells, microvascular endothelial (mvE) cells, and lymphatic endothelial (lyE) cells by ANXA1 low and ANXA1 high PC. (E) Boxplots of angiogenesis-associated genes expression: platelet and endothelial cell adhesion molecule 1 (PECAM1) and sphingosine-1-phosphate receptor 1 (S1PR1) by ANXA1 low and ANXA1 high PC. (F) Boxplots of the infiltrating fraction of fibroblasts and keratinocytes by ANXA1 low and ANXA1 high PC. Median cut-off within each cohort was used to divide them into ANXA1 low and ANXA1 high groups (n = 88, respectively, in the TCGA and n = 31 and 32, respectively, in the GSE57495 cohort). Spearman’s rank correlation was used for the correlation analysis. For group comparison, p-values were calculated by the Mann–Whitney U test.

Figure 2

Gene set enrichment analysis of pancreatic cancer with high annexin A1 (ANXA1) expression in the TCGA and GSE57495 cohorts. Enrichment plots of hypoxia, transforming growth factor (TGF)-β signaling, tumor necrosis factor (TNF)-α signaling, MTORC1, apoptosis, protein secretion, glycolysis, and the androgen response of hallmark gene sets with NES and FDR. Median cut-off within each cohort was used to divide into ANXA1 low and ANXA1 high groups (n = 88, respectively, in the TCGA and n = 31 and 32, respectively, in the GSE57495 cohort). NES, normalized enrichment score and FDR, false discovery rate.

Figure 3

The association of ANXA1 with gene mutation and homologous recombination deficiency (HRD) in the TCGA pancreatic cancer cohort. (A) Bar plots of the mutation rate of KRAS, TP53, CDK2A, and SMAD4 by ANXA1 low and ANXA1 high groups. p-values were calculated with Fisher’s exact test. (B) Boxplots of the mutation-related scores, including altered fraction, single nucleotide variant (SNV) and indel neoantigens, and silent and non-silent mutations, and (C) HRD scores by ANXA1 low (blue) and ANXA1 high (red) groups. Median cut-off was used to divide into ANXA1 low and ANXA1 high groups (n = 88, respectively). p-values were calculated by the Mann–Whitney U test.

Figure 4

Association of the ANXA1 expression and infiltrating immune cells in pancreatic cancer in the TCGA and GSE57495 cohorts. Boxplots of the infiltrating fraction of CD8⁺ T cells, CD4⁺ T cells, T-helper types 1 and 2 (Th1 and Th2), regulatory T cells (Tregs), M1 and M2 macrophages, and Mast cells by ANXA1 low and ANXA1 high groups. Median cut-off within each cohort was used to divide into ANXA1 low and ANXA1 high groups (n = 88, respectively, in the TCGA and n = 31 and 32, respectively, in the GSE57495 cohort). p-values were calculated by the Mann–Whitney U test.

Figure 5

Association of the ANXA1 expression and tumor aggressiveness in PC. Boxplots of (A) the clinical factors: AJCC stage, N-category, and histological grade and (B) proliferation-related factors: proliferation score and MKI67 expression in the TCGA cohort. p-values were calculated by the Kruskal–Wallis and Mann–Whitney U tests. (C) Correlation plots between the ANXA1 expression and cell proliferation-related score: E2F targets, G2M checkpoint, and Mitotic spindle score in the TCGA and GSE57495 cohorts. The median cut-off was used to divide them into the ANXA1 low and ANXA1 high groups (n = 88, respectively). Spearman’s rank correlation was used for the analysis. AJCC: American Joint Committee on Cancer.

Figure 6

Association of the ANXA1 expression with patient survival in the TCGA and GSE57495 cohorts. (A) Kaplan–Meier curve between low (blue line) and high (red line) with disease-free survival (DFS), disease-specific survival (DSS), progression-free survival (PFS), and overall survival (OS). The median cut-off within each cohort was used to divide them into the ANXA1 low and ANXA1 high groups (n = 88, respectively, in the TCGA and n = 31 and 32, respectively, in the GSE57495 cohort). (B) Kaplan–Meier curve between low and high with PFS in the pathological grades 1/2 (n = 62, respectively) and grade 3 groups (n = 24, respectively) in the TCGA cohort. p-values were calculated by log-rank test.

Figure 7

Correlation of the ANXA1 expression with the drug sensitivity of several drugs in primary and metastasis PC cell lines. The correlation plots of the ANXA1 expression with the level of drug sensitivity area under the curve (AUC) of gemcitabine, paclitaxel, doxorubicin, and 5-fluorouracil. Spearman’s rank correlation coefficient was used for the analysis.