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. 2017 Oct 9;6(10):e389.
doi: 10.1038/oncsis.2017.87.

ESRP1 is overexpressed in ovarian cancer and promotes switching from mesenchymal to epithelial phenotype in ovarian cancer cells

H M Jeong  1 , J Han  2 , S H Lee  3 , H-J Park  4 , H J Lee  3 , J-S Choi  4 , Y M Lee  5 , Y-L Choi  6   7   8 , Y K Shin  1   4   9 , M J Kwon  3   10
Affiliations

ESRP1 is overexpressed in ovarian cancer and promotes switching from mesenchymal to epithelial phenotype in ovarian cancer cells

H M Jeong et al. Oncogenesis. .

Erratum in

Abstract

Epithelial splicing regulatory protein 1 (ESRP1) and 2 (ESRP2), epithelial cell-specific regulators of alternative splicing, are downregulated during the epithelial-mesenchymal transition (EMT). These factors have roles in tumor progression and metastasis in some cancers; however, their expression and function in ovarian cancer (OC) remain unclear. We found that ESRP1 and ESRP2 mRNAs were expressed at higher levels in OC cells than in immortalized ovarian surface epithelial (IOSE) cells, and confirmed their overexpression in OC tissues at the protein level. The Cancer Genome Atlas (TCGA) data analysis revealed frequent gene amplification of ESRP1 in OC tissues; however, we detected no significant correlation between ESRP1 gene copy number and gene expression in OC cells. Importantly, expression of ESRP1 and ESRP2 was inversely correlated with DNA methylation in OC cells, and ESRP2 overexpression in OC tissues was significantly associated with DNA hypomethylation. Notably, survival analysis using TCGA data from 541 OC tissues revealed that high ESRP1 expression was significantly associated with shorter 5-year survival of patients. Ectopic ESRP1 expression in mesenchymal OC cells promoted cell proliferation but suppressed cell migration. Furthermore, we found that ESRP1 drives a switch from mesenchymal to epithelial phenotype characterized by reduced cell migration in association with induction of epithelial cell-specific variant of CD44 and ENAH. Taken together, our findings suggest that an epigenetic mechanism is involved in ESRP1 overexpression, and that ESRP1 has a role in OC progression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ESRP1 and ESRP2 gene expression in human ovarian cancer cell lines, and protein expression in ovarian serous adenocarcinoma. (a) Box plot comparing the gene expression of ESRP1 and ESRP2 between normal and ovarian cancer tissues using TCGA data. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentile and the whiskers indicate the highest and lowest values of the results. Statistical differences between the two groups were evaluated using the Mann–Whitney test. (b) ESRP1 and ESRP2 gene expression in ovarian cell lines, determined by qRT–PCR. Data are presented as the mean±s.d. of two or three experiments. (c) Representative immunohistochemical staining of human ovarian tissues with anti-ESRP1 or anti-ESRP2 antibodies. ESRP1 (left) and ESRP2 (right) in normal ovarian surface epithelium and ovarian serous adenocarcinoma tissues. Magnification, × 100 or × 400. CA, carcinoma; NL, normal.
Figure 2
Figure 2
Genetic alterations of ESRP1 or ESRP2 in ovarian cancer tissues. (a) OncoPrint of gene copy number alterations of ESRP1 or ESRP2 in 557 ovarian cancer samples from TCGA data (top), and box plot showing the association between their mRNA levels and gene amplification or deletion (bottom). OncoPrint was generated using cBioPortal (http://www.cbioportal.org/). Statistical differences between the two groups were evaluated using the Mann–Whitney test. (b) ESRP1 gene copy number analysis in ovarian cancer cells. Gene copy numbers for two regions (Chr8 95653201–95653400 and 95686401–95686600, GRCh37) within the genomic region encoding ESRP1 were determined using qPCR. Data are presented as the mean±s.d. of two experiments. HBD_female, Human female blood gDNA. (c) Box plot comparing the ESRP1 gene copy number for region (Chr8 95653201–95653400) between the ESRP1-low and -high groups of ovarian cancer FFPE tissues. Statistical differences between the two groups were evaluated using the Mann–Whitney test. (d) ESRP1 genetic alterations in various cancer types using cBioPortal based on TCGA data. ACC, adrenocortical carcinoma; AML, acute myeloid leukemia; chRCC, chromophobe renal cell carcinoma; DLBC, diffuse large B-cell lymphoma; GBM, glioblastoma multiforme; LGG-GBM, lower-grade glioma-GBM; NSCLC, non-small cell lung cancer; PCPG, pheochromocytoma and paraganglioma; pRCC, papillary renal cell carcinoma; Uterine CS, uterine carcinosarcoma.
Figure 3
Figure 3
DNA methylation status in the promoter region of ESRP1 in ovarian cancer cell lines and tissues. (a) ESRP1 gene expression in ovarian cancer cells following treatment with epigenetic drugs. Data for qRT–PCR are presented as the mean±s.d. of two or three experiments. ESRP1 expression following treatment with epigenetic drugs was compared with that with no treatment (NT). Student’s t-test; *P<0.05, **P<0.01. (b) DNA methylation analysis of IOSE and ovarian cancer cells using bisulfite sequencing and (c) qMSP. DNA methylation level is expressed as percentage of methylated reference (PMR, %) values. Data for qMSP are presented as the mean±s.d. of two experiments. (d) Box plot showing the association between DNA methylation level (PMR) and ESRP1 expression in ovarian cancer FFPE tissues. Statistical differences between the two groups were evaluated using the Mann–Whitney test.
Figure 4
Figure 4
DNA methylation status in the promoter region of ESRP2 in ovarian cancer cell lines and tissues. (a) ESRP2 gene expression following treatment with epigenetic drugs. Data for qRT–PCR are presented as the mean±s.d. of two or three experiments. ESRP2 expression following treatment with epigenetic drugs was compared with that with no treatment (NT). Student’s t-test, *P<0.05. (b) DNA methylation analysis of IOSE and ovarian cancer cells using bisulfite sequencing and (c) qMSP. Data for qMSP are presented as the mean±s.d. of two experiments. (d) Box plot showing the association between DNA methylation and ESRP2 gene expression in ovarian cancer FFPE tissues. Statistical differences between the two groups were evaluated using the Mann–Whitney test.
Figure 5
Figure 5
Association between ESRP1 gene expression and OS or PFS in patients with ovarian serous adenocarcinoma. (a) Kaplan–Meier plot of 5-year OS, and PFS in total primary tumors and (b) stage III primary tumors. (a, b) Kaplan–Meier plot of 5-year OS, and PFS in total primary tumors and (c, d) stage III primary tumors.
Figure 6
Figure 6
Effect of ESRP1 on cell proliferation and migration in ovarian cancer cells. (a, b) Effect of enforced ESRP1 expression on cell proliferation in SK-OV3 and A2780 cells. (c) Soft agar formation assay in SK-OV3 stable cell lines. (d, e) Effect of ESRP1 knockdown on cell proliferation in OVCAR3 and Caov3 cells. ESRP1 overexpression or ESRP1 knockdown by siRNA treatment was determined using qRT–PCR and western blot. For in vitro cell proliferation assay, viable cell numbers were counted at each time point. (f) Effect of enforced ESRP1 expression or (g) ESRP1 knockdown on cell migration. Data for cell proliferation and migration are presented as the mean±s.d. of three or four experiments. Student’s t-test, *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.
Figure 7
Figure 7
Regulation of EMT markers and EMT-associated transcription factors by ectopic ESRP1 expression. (a) Association between ESRP1 and EMT markers' (CDH and VIM) gene expression in ovarian cell lines. Data are presented as the mean±s.d. of two experiments. (b) CDH1 or VIM gene expression upon ectopic ESRP1 expression in SK-OV3 and A2780 cells or (c) ESRP1 knockdown in OVCAR3 and Caov3 cells. (d) The mRNA levels of SNAI1, SNAI2, ZEB1, ZEB2 and TWIST1 in SK-OV3-Vec and SK-OV3-ESRP1 cells. qRT–PCR data (except for Figure 7a) are presented as the mean±s.d. of three or four experiments. Student’s t-test, *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.
Figure 8
Figure 8
Alternative splicing of CD44, ENAH and FGFR2 upon ectopic ESRP1 expression in mesenchymal ovarian cancer cells. (a) RT–PCR analysis of splice variant CD44 (left) and qRT–PCR analysis of CD44s (right) in SK-OV3-Vec and SK-OV3-ESRP1 cells. (b) RT–PCR analysis of splice variant ENAH in SK-OV3-Vec and SK-OV3-ESRP1 cells. M, marker. (c) qRT–PCR analysis of FGFR2IIIb and FGFR2IIIc in SK-OV3-Vec and SK-OV3-ESRP1 cells. Data for qRT–PCR are presented as the mean±s.d. of four experiments. Student’s t-test, ***P<0.001, n.s., not significant. (d) Schematic diagram for a switch from mesenchymal to epithelial phenotype upon ectopic ESRP1 expression in mesenchymal ovarian cancer cells.
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