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. 2022 Nov 2:2022:9328972.
doi: 10.1155/2022/9328972. eCollection 2022.

Systematic Pan-Cancer Analysis and Experimental Verification Identify FOXA1 as an Immunological and Prognostic Biomarker in Epithelial Ovarian Cancer

Kai Wang  1 , Chenan Guan  2 , Junhui Yu  1 , Xing Chen  1 , Xianwen Shang  1 , Shuangshuang Mei  1 , Xingjun Feng  1 , Lingzhi Zheng  1   3
Affiliations

Systematic Pan-Cancer Analysis and Experimental Verification Identify FOXA1 as an Immunological and Prognostic Biomarker in Epithelial Ovarian Cancer

Kai Wang et al. Dis Markers. .

Abstract

Background: Epithelial ovarian cancer (EOC) has the lowest survival rate among female reproductive cancers present with symptoms of aggressive malignancies, poor prognosis, drug resistance and postoperative recurrence. The majority of patients with EOC are diagnosed at an advanced stage due to the therapeutic challenges including lack of early diagnosis and effective therapeutic targets for EOC.

Methods: Pan-cancer analyses were performed to explore the features of forkhead-box (FOX) A1 (FOXA1) using data from TCGA and GTEx databases. R package "clusterprofiler" was used to perform the enrichment analysis of FOXA1 in EOC. Data downloaded from Drug Sensitivity in Cancer (GDSC) database were used to evaluate the association between FOXA1 and antitumor drug sensitivity. In experimental verification, FOXA1 expression was detected using qRT-PCR and western blot assays. Western blot, immunofluorescence staining, and Transwell assays were used to assess the influence of FOXA1 silencing on epithelial-mesenchymal transition (EMT) of EOC cells.

Results: We found that FOXA1 was highly expressed in EOC and predicted poorer survival of EOC patients. We observed that FOXA1 expression was positively correlated EMT-related pathways. Through experimental verification, we found the underlying function of FOXA1 to promote EMT in ovarian cancers. The results from western blot, immunofluorescence staining, and Transwell assays showed that FOXA1 silencing impeded the progression of EMT and invasiveness of the cancer cells. Furthermore, CCK-8 and invasion assays suggested that siRNA-FOXA1 attenuated the ability of cancer cells to metastasize and proliferate. Dual-luciferase reporter assays confirmed the binding activity of FOXA1 to the promoter of connective tissue growth factor (CTGF). In addition, we found that FOXA1 was closely correlated immunosuppressive microenvironment of EOC. High FOXA1 expression may contribute to the resistance of many anticancer drugs.

Conclusions: Our results predict and validate the function of FOXA1 in promoting EMT and the progression of disease in EOC. Targeting FOXA1 may improve the sensitivity of EOC treatment.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Pan-cancer expression. (a) Pan-cancer expression of FOXA1. (b) The expression of FOXA1 in paired tumor and adjacent normal tissues in indicated tumor types from TCGA cohort. (c) The expression of FOXA1 in indicated cells.
Figure 2
Figure 2
FOXA1 expression in different tumor stages. (a–i) The FOXA1 expression in different tumor stages in indicated tumor stages from TCGA cohort.
Figure 3
Figure 3
Gene alteration of FOXA1. (a) The genetic alteration of FOXA1 in TCGA pan-cancer. (b) The correlation between FOXA1 expression and copy number in TCGA pan-cancer. (c) The correlation between FOXA1 expression and methylation level in TCGA pan-cancer.
Figure 4
Figure 4
Prognostic value of FOXA1. (a–l) The Kaplan-Meier analyses of FOXA1 in indicated tumor types. The optimum cutoff value of FOXA1 in each tumor type was set.
Figure 5
Figure 5
TME analysis. (a–c) The Kaplan-Meier analyses of FOXA1 in EOC datasets, including GSE26193 (a), GSE26712 (b), and GSE63885 (c). The optimum cutoff value of FOXA1 in each dataset was set. (d) The correlation between TME-related signature scores and FOXA1 expression.
Figure 6
Figure 6
FOXA1 expression was upregulated in ovarian cancer cells. (a–c) FOXA1 expression was assessed by qRT-PCR (a) and western blot (b) in IOSE80 (human ovarian epithelial cell line from normal tissues) and OVCAR-3/A2780/3AO/SKOV-3 cells (human ovarian cancer cell lines). Relative mRNA and protein levels of FOXA1 were quantified in (a) and (c); N = 3, two-way ANOVA. (d, e) FOXA1 knockdown via transient transfection with siRNA-2 in OVCAR-3 cell was confirmed by qRT-PCR (d) and western blot (e, f). siRNA-NC-transfected cells were set as the control. The experiments were repeated independently for 3 times.
Figure 7
Figure 7
Effects of FOXA1 silencing on OVCAR-3 cell proliferation and invasion. (a) The proliferation was measured by CCK8 assay at 24, 48, and 72 h, respectively. The results were obtained from the 6 replicates in each group and presented as average value ± SD. (b) The invasiveness of OVCAR-3 cells was detected by the Transwell migration assay. Representative images were selected (magnification ×100). (c) The number of invaded cells were quantified and obtained from three independent experiments. (d) Protein levels of E-cadherin, vimentin, and Snail were measured by western blot after 48 h following siRNA transfection. (e) Quantifications of representative blots were demonstrated in bar graphs. Protein levels of E-cadherin, vimentin, and Snail in the FOXA1-silenced group (siRNA-FOXA1) were compared to those in the control group (siRNA-NC) and presented in the form of average ± SD. (f, g) Representative immunofluorescence staining for E-cadherin (f) and vimentin (g) in the siRNA-NC- or siRNA-FOXA1-transfected OVCAR3 cells (original magnification ×630).
Figure 8
Figure 8
The suppressive effects of FOXA1 silencing on EMT through TGF-beta 1 signaling pathway. (a) Predicted binding site of FOXA1 at the wild-type CTGF promoter region and the mutant CTGF promoter (-399 to -390) was present. (b) Relative luciferase activity was presented as per the ratio of the intensity of firefly luciferase to that of Renilla. (c) Western blot analysis on protein expression levels of FOXA1, CTGF, MMP-2, E-cadherin, and Snail in the siRNA-NC-, or the siRNA-FOXA1-transfected cells treated with or without TGF-β1. Quantifications of protein expressions was represented as mean ± standard deviation (SD) of the results from six independent replicates in each group. (d) Expressions of FOXA1, CTGF, cleaved TGF-beta 1, and EMT-associated markers including E-cadherin, vimentin, and Snail were analyzed by western blot after 48 h after the transfected cells treated with or without lithium chloride. Quantification of the representative blots was represented as mean ± standard deviation (SD) of the results from three independent experiment in bar graphs.
Figure 9
Figure 9
GSEA or FOXA1. (a–c) The GSEA results of FOXA1 in TCGA-OV cohort, including GSEA-GO (a), GSEA-KEGG (b), and GSEA-Reactome (c).
Figure 10
Figure 10
The correlation analysis. (a) The correlation between MHC genes and FOXA1. (b) The correlation between immunosuppressive genes and FOXA1. (c) The correlation between immune activating genes and FOXA1. (d) The correlation between chemokine receptors and FOXA1.
Figure 11
Figure 11
Effect of FOXA1 on the efficacy of immunotherapy. (a) The FOXA1 expression in indicated groups in GSE135222. (b) The Kaplan-Meier curve of FOXA1 in GSE135222. (c) The FOXA1 expression in indicated groups in checkmate cohort. (d) The Kaplan-Meier curve of FOXA1 in checkmate cohort.
Figure 12
Figure 12
The correlation analysis between FOXA1 and IC50 values of indicated anticancer drugs.
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