FOXA2 suppresses endometrial carcinogenesis and epithelial-mesenchymal transition by regulating enhancer activity
Subhransu S. Sahoo, … , Ram S. Mani, Diego H. Castrillon
Subhransu S. Sahoo, … , Ram S. Mani, Diego H. Castrillon
Published June 15, 2022
Citation Information: J Clin Invest. 2022;132(12):e157574. https://doi.org/10.1172/JCI157574.
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Research Article Oncology

FOXA2 suppresses endometrial carcinogenesis and epithelial-mesenchymal transition by regulating enhancer activity

Abstract

FOXA2 encodes a transcription factor mutated in 10% of endometrial cancers (ECs), with a higher mutation rate in aggressive variants. FOXA2 has essential roles in embryonic and uterine development. However, FOXA2’s role in EC is incompletely understood. Functional investigations using human and mouse EC cell lines revealed that FOXA2 controls endometrial epithelial gene expression programs regulating cell proliferation, adhesion, and endometrial-epithelial transition. In live animals, conditional inactivation of Foxa2 or Pten alone in endometrial epithelium did not result in ECs, but simultaneous inactivation of both genes resulted in lethal ECs with complete penetrance, establishing potent synergism between Foxa2 and PI3K signaling. Studies in tumor-derived cell lines and organoids highlighted additional invasion and cell growth phenotypes associated with malignant transformation and identified key mediators, including Myc and Cdh1. Transcriptome and cistrome analyses revealed that FOXA2 broadly controls gene expression programs through modification of enhancer activity in addition to regulating specific target genes, rationalizing its tumor suppressor functions. By integrating results from our cell lines, organoids, animal models, and patient data, our findings demonstrated that FOXA2 is an endometrial tumor suppressor associated with aggressive disease and with shared commonalities among its roles in endometrial function and carcinogenesis.

Authors

Subhransu S. Sahoo, Susmita G. Ramanand, Yunpeng Gao, Ahmed Abbas, Ashwani Kumar, Ileana C. Cuevas, Hao-Dong Li, Mitzi Aguilar, Chao Xing, Ram S. Mani, Diego H. Castrillon

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Figure 1

FOXA2 is the endometrium-specific FOXA and is downregulated in ECs.

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FOXA2 is the endometrium-specific FOXA and is downregulated in ECs. (A) ...
(A) Tissue sections of normal human endometrium (secretory/HSecE, proliferative/HProE) and prostate (HProstate) immunostained for FOXA1 or FOXA2. FOXA1 is expressed in the prostate (luminal/secretory and basal layers) but not endometrium, whereas FOXA2 is expressed in endometrial epithelial cells and only weakly and heterogeneously in the basal layer of prostate glands. The 6 immunostains were performed under identical conditions, antibody concentrations, and chromogen incubation times and are thus directly comparable to one another. Insets for HProstate are higher magnifications of smaller boxed areas. Scale bars: 100 μm. (B) Western blot of immortalized endometrium (HEuEC) and prostate cancer (LNCaP) cell lines confirmed mutually exclusive patterns of FOXA1 and FOXA2 expression. (C) mRNA expression levels by qRT-PCR (n = 3) and correlation to protein expression by Western blot in 1 immortalized normal endometrial cell line (HEuEC) and 13 EC cell lines. FOXA2 was undetectable in 7/13 and significantly downregulated in 4/13 of the EC cell lines. Data shown as mean ± SEM. (D) Western blot analysis of estrogen receptor α (ERα) and progesterone receptor-A/B (PR-A/B) in EC cell lines. (E) Expression of FOXA2, ERα, and PR in normal human endometrium and grade 1–3 human endometrial carcinomas; representative images. Scale bars: 100 μm. (F) FOXA2 expression levels per H-scores in normal endometrium (n = 5) and ECs (grade 1, n = 16; grade 2, n = 23; grade 3, n = 21). Box-and-whisker plot represents medians with minimum and maximum values. P value was determined by 2-tailed Mann-Whitney U tests compared with normal cases. Data are shown as mean ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) Scatter plot shows correlation analysis of FOXA2 with ERα and PR in normal endometrium and ECs. FOXA2, ERα, and PR expression levels were determined by H-score. Pearson’s r determined correlation between data groups.