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 6

Foxa2 reconstitution in FP cells suppresses cell growth and tumor phenotypes.

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Foxa2 reconstitution in FP cells suppresses cell growth and tumor pheno...
(A) Brightfield and indirect immunofluorescence images of FP cell line derived from Foxa2/Pten mouse uterine tumor. Scale bars: 50 μm. (B) Western blot shows enforced Foxa2 expression after lentiviral transduction in FP cells (FP-Foxa2). For controls, transduction was performed with empty vector (FP-EV). (C) Immunofluorescence of FOXA2 (red) validates nuclear localization in the FP-Foxa2 cells. Scale bar: 50 μm. (D) Growth comparison of FP-EV and FP-Foxa2 cells by real-time live cell imaging (n = 3). Data shown as mean ± SEM; ****P < 0.0001, 2-tailed t test. (E) Comparison of cell cycle analysis in FP-EV and FP-Foxa2 cells by flow cytometry (n = 3). The peaks in blue and yellow show percentage of cells in G0/G1 and S phase. (F) Gross images of xenograft tumors after s.c. injection of 1 million FP-EV and FP-Foxa2 cells in the left and right flanks of NOD scid gamma female mice (n = 4). Tumors were harvested 35 days after cell injection. (G) Growth curves of FP-EV and FP-Foxa2 xenografts per caliper measurements (n = 4). Data represent mean ± SEM; *P < 0.05, 2-tailed t test. (H) Endpoint xenograft tumor weights at day 35 (n = 4, same tumors shown in F). Data represent mean ± SEM; *P < 0.05, 2-tailed t test. (I) Immunostaining of actin filaments (red) in FP-EV and FP-Foxa2 organoids counterstained with DAPI (blue). Representative midsections of Z-stack images are shown. Scale bars: 50 μm. (J) Quantitative analysis of invasive or round-shaped FP-EV and FP-Foxa2 organoids (n = 5). Data shown as mean ± SEM; ****P < 0.0001, 2-tailed t test. (K) Comparison of FP-EV and FP-Foxa2 organoid proliferation (n = 4). Data shown as mean ± SEM; *P < 0.05, 2-tailed t test.