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. 2023 Jan 4;15(1):92-107.
doi: 10.18632/aging.204455. Epub 2023 Jan 4.

Comprehensive bioinformatics analysis reveals the significance of forkhead box family members in pancreatic adenocarcinoma

Wei Hu  1 , Mingxu Li  1 , Yan Wang  2 , Chengcheng Zhong  1 , Xinxin Si  3 , Xiao Shi  3 , Zhong Wang  1
Affiliations

Comprehensive bioinformatics analysis reveals the significance of forkhead box family members in pancreatic adenocarcinoma

Wei Hu et al. Aging (Albany NY). .

Abstract

Background: Forkhead box proteins (FOXs) play important roles in multiple biological processes; while little is known regarding the role of FOX members in pancreatic adenocarcinoma (PAAD). This study aimed to comprehensively investigate the function of FOX family members in PAAD.

Methods: Expression and prognostic value of FOXs were analyzed by R language and GEPIA. Genetic alteration and promoter methylation level were analyzed using CBioPortal and UALCAN. Protein-protein interactions and gene functions were analyzed using STRING and DAVID. TIMER and SENESCopedia were utilized to analyze the correlation of FOXs with immune cell infiltration or tumor senescence. Protein levels of FOXs were detected by immunohistochemistry.

Results: Expression of 15 of 50 FOXs were significantly elevated in PAAD. Among these 15 differentially expressed FOXs (DE-FOXs), 4 were significantly associated with the clinical cancer stage and 4 were negatively associated with overall survival. Functions of DE-FOXs were related to epithelial tube morphogenesis, nuclear chromatin, and DNA-binding. Promoter methylation and genomic alterations were not major causes of FOX dysregulation. Most DE-FOX was correlated with diverse immune infiltration cells. Seven of the DE-FOXs were positively related to tumor senescence. The protein levels of FOXM1, FOXP1, and FOXN3 were negatively correlated with OS in the collected PAAD patients.

Conclusions: FOXM1, FOXP1, and FOXN3 have prognostic value. Seven FOXs were related senescence, whereas most DE-FOXs were related to immune infiltration in PAAD. Our findings are instructive for future research on FOX family and provide novel insights into the selection of FOXs with potential prognostic or therapeutic target value.

Keywords: biomarker; forkhead box family; pancreatic adenocarcinoma; prognosis; senescence.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Differential expression and correlation with tumor stages of FOXs in PAAD patients. (A) Differentially expressed genes were selected with P < 0.01 and |Log2FC| Cutoff ≥1 among the datasets GSE15471, GSE16515, and TCGA. The Venn diagram shows an overlap of 15 genes that had higher expression in cancer tissues than in adjacent tissues. (B) Expression of representative FOXs in PAAD (GSE15471, GSE16515 and GEPIA). GSE15471: number(normal)=39, number(tumor)=39; GSE16515: number(normal)=36, number(tumor)=16. (C) Correlations between FOX expression and tumor stage in PAAD patients (TCGA). The expression of FOXK1, FOXQ1, and FOXP1 was correlated with the pathological stage of PAAD patients (p < 0.05). PAAD: pancreatic adenocarcinoma.
Figure 2
Figure 2
Survival analysis of 15 differentially expressed FOXs in PAAD patients (GSE21501, GSE28735, and TCGA). (A) High expression of FOXM1 was correlated with short OS in all three datasets. (B) High expression of FOXC1, FOXK1, and FOXN2 was negatively correlated with OS in two of three data sets. (C) High levels of FOXP1 were related to short OS in the TCGA and GSE21051 databases, but long disease-free survival (DFS) in the GSE28735 database. (D) Expression of FOXN3 was positively correlated with OS in the GSE28735 and GSE21051 databases, but showed no significant relationship in the TCGA database. (E) Expression of FOXF2 was positively correlated with OS in the GSE28735 and GSE21051 databases but was negatively related to OS in the TCGA database.
Figure 3
Figure 3
Genetic alterations and promoter methylation levels of the 15 differentially expressed FOXs in PAAD patients. (A) Summary of genetic alterations of different expressed FOXs in PAAD. All FOXs were altered in 19 samples of 175 patients with PAAD, accounting for 11% of the queried patients/samples. (B) Results from UALCAN showed downregulation of the promoter methylation level of FOXD1, FOXJ1, and FOXK1 in tumor tissues. (C) The methylation levels of FOXC2, FOXF1, FOXF2, FOXM1, and FOXN3 were upregulated in tumor tissues.
Figure 4
Figure 4
Protein-protein interaction (PPI) and gene function analyses of the 15 differentially expressed FOXs and related interactors in PAAD patients. (A) STRING analysis of 15 differentially expressed FOXs and 50 interaction proteins. Network nodes represent proteins. Edges represent protein–protein associations, which include known interactions, predicted interactions, and others. PPI enrichment p< 0.001. (B) All KEGG pathway enrichment results for the 15 FOXs and 50 interaction proteins. (C) All GO enrichment analysis results for the 15 FOXs and 50 interaction proteins. MF: Molecular Function; CC: Cellular Component; BP: Biological Process.
Figure 5
Figure 5
Correlations between FOX expression and immune cell infiltration. (A–C) The expression correlation between distinct FOXs and immune cells was analyzed by TIMER. p value is shown in the figures. Partial.cor, purity-corrected partial Spearman’s rho value.
Figure 6
Figure 6
Correlation between differentially expressed FOXs and tumor senescence. (A) Positive correlation between FOXs and IL-1α in pancreatic adenocarcinoma (PAAD), n=179. (B) Positive correlation between FOXs and IL-6 in PAAD, n=179. (C) Negative correlation between FOXs and IL-6 in PAAD, n=179. (D) Senescence score of FOXM1 and FOXA1 calculated using SENESCopedia. *, p < 0.01.
Figure 7
Figure 7
Validation of the prognostic role of FOXs in PAAD patient tissues. (A) Representative immunohistochemistry (IHC) images (200X) in PAAD tissues with low or high FOXM1, FOXP1, FOXN3, FOXF2, and FOXK1 protein levels. (B) Expression of FOXM1, FOXP1, and FOXN3 was negatively correlated with OS in PAAD patient pathology samples. Expression of FOXF2 and FOXK1 had no significant correlation with OS.
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