Rel Family Transcription Factor NFAT5 Upregulates COX2 via HIF-1α Activity in Ishikawa and HEC1a Cells

Abstract

Nuclear factor of activated T cells 5 (NFAT5) and cyclooxygenase 2 (COX2; PTGS2) both participate in diverse pathologies including cancer progression. However, the biological role of the NFAT5-COX2 signaling pathway in human endometrial cancer has remained elusive. The present study explored whether NFAT5 is expressed in endometrial tumors and if NFAT5 participates in cancer progression. To gain insights into the underlying mechanisms, NFAT5 protein abundance in endometrial cancer tissue was visualized by immunohistochemistry and endometrial cancer cells (Ishikawa and HEC1a) were transfected with NFAT5 or with an empty plasmid. As a result, NFAT5 expression is more abundant in high-grade than in low-grade endometrial cancer tissue. RNA sequencing analysis of NFAT5 overexpression in Ishikawa cells upregulated 37 genes and downregulated 20 genes. Genes affected included cyclooxygenase 2 and hypoxia inducible factor 1α (HIF1A). NFAT5 transfection and/or treatment with HIF-1α stabilizer exerted a strong stimulating effect on HIF-1α promoter activity as well as COX2 expression level and prostaglandin E2 receptor (PGE2) levels. Our findings suggest that activation of NFAT5-HIF-1α-COX2 axis could promote endometrial cancer progression.

Keywords: cyclooxygenase 2; endometrial cancer; hypoxia inducible factor 1α.

Figure 1

NFAT5 expression in Grades 1 and 3 endometrial cancer tissues. (a). Representative images of NFAT5 immunohistochemistry expression analysis in Grade 1 (n = 15) and Grade 3 (n = 11) endometrial cancer tissue. Staining shows excessive NFAT5 expression in Grade 3 tumor samples compared to Grade 1. Scale bar—100 µm. (b). In parallel, mRNA expression level of NFAT5 from FFPE tissue samples was quantified by qRT-PCR, *, p < 0.05 based on unpaired t-test. Data were normalized to ribosomal housekeeping gene, L19.

Figure 2

Gene expression alteration in Ishikawa cells with NFAT5 overexpression. (a). Heat-map shows gene expression alteration by NFAT5 overexpression in Ishikawa cells (FDR < 0.05 and log2FC ≥ 0.3). Upregulation and downregulation of genes are shown by red and blue color coding, respectively. (b). Box and whisker plots of log2 fold change of genes of interest in control and NFAT5 overexpressed Ishikawa cells.

Figure 3

Effect of NFAT5 on COX2 transcript and protein levels in Ishikawa cells. (a). Ishikawa cells were treated with 0.5 mM DMOG for 24 h. mRNA expression level of NFAT5 was quantified by qRT-PCR. Data were normalized to L19 and presented as mean ± SEM. (n = 6; *, p < 0.05). (b). NFAT5 protein abundance was investigated by SDS-PAGE and western blot analysis. Ishikawa cells were treated with 0.5 mM DMOG for 24 h. Data were normalized to each corresponding level of pan-actin and shown as mean ± SEM. (n = 6; ****, p < 0.0001, a.u: arbitrary unit). (c). mRNA expression level of NFAT5, HIF1A, and PTGS2 were quantified by qRT-PCR. Ishikawa cells were transfected with NFAT5 overexpression plasmid for 24 h (n = 5; **, p < 0.01, ****, p < 0.0001). (d). NFAT5 and PTGS2 protein abundance were investigated by SDS-PAGE and western blot analysis using the indicated antibodies. Ishikawa cells were transfected with NFAT5 overexpression plasmid for 24 h (n = 6; *, p < 0.05; **, p < 0.01). (e). Luciferase activity of HIF-1α that was normalized to renilla post Ishikawa cells transfected with NFAT5 overexpression plasmid for 24 h (n = 6; ****, p < 0.0001). (f). mRNA expression level of PTGS2 was quantified by qRT-PCR. Ishikawa cells were treated with 0.5 mM DMOG for 24 h (n = 6; **, p < 0.01). (g). COX2 protein abundance was investigated by SDS-PAGE and western blot analysis using the indicated antibodies. Ishikawa cells were treated with 0.5 mM DMOG for 24 h (n = 6; **, p < 0.01).

Figure 4

Effect of NFAT5 overexpression in Ishikawa biological activity. (a). FACS assisted cell cycle analysis on control and NFAT5 overexpressed Ishikawa cells (n = 7; *, p < 0.05). (b). Cell proliferation analysis on control and NFAT5 overexpressed Ishikawa cells with BrdU ELISA assay (n = 4; **, p < 0.01). Data were normalized to each control and shown as mean ± SEM. (c). Representative bright field images of wound healing scratch assay on the control and NFAT5-overexpressed Ishikawa cells. (d). Wound closure rate on the control and NFAT5 overexpressed Ishikawa cells 24 h post scratch (n = 4; **, p < 0.01), scale bar—200 µm.

Figure 5

Synergistic effect of NFAT5 overexpression and DMOG on COX2 transcript and protein levels in Ishikawa cells. (a). mRNA expression level of NFAT5 and PTGS2 quantified by qRT-PCR. Ishikawa cells were treated with 0.5 mM DMOG for 24 h post with or without 24 h transfection with NFAT5 overexpression plasmid. Data were normalized to L19 and presented as mean ± SEM. (NFAT5, n = 6; *, p < 0.05; **, p < 0.01). (b–d). NFAT5 and COX2 protein abundance were investigated by SDS-PAGE and western blot analysis using the indicated antibodies. Ishikawa cells were subjected to with or without NFAT5 transfection followed by treatment with or without DMOG. Data were normalized to each corresponding level of pan-actin and shown as mean ± SEM. (n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001, a.u: arbitrary unit). (e). Effect of hypoxia on HIF-1α induction with or without NFAT5 transfection using Luciferase promoter assay. Data shown as mean ± SEM. (n = 6; **, p < 0.01). (f). Effect on PGE2 levels with or without NFAT5 transfection, followed by treatment with or without DMOG (0.5 mM, 24 h). Data shown as mean ± SEM. (n = 4; *, p < 0.05).