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 2

FOXA2 suppresses EC cell proliferation and enhances cell adhesion.

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FOXA2 suppresses EC cell proliferation and enhances cell adhesion. (A) F...
(A) FOXA2 reexpression after lentiviral transduction in Ishikawa (ISK) cells (ISK-FOXA2). (B) Immunofluorescence shows expected FOXA2 nuclear localization, DAPI counterstain. Scale bars: 50 μm. (C) Cell growth analysis showing FOXA2-mediated growth suppression; confluency normalized to t0 (n = 3). Data shown as mean ± SEM; 2-tailed t test. (D) Wound-healing assay at t0 and 60 hours. Scale bars: 250 μm. (E) Wound closure per gap distance (n = 3). Data shown as mean ± SEM; 2-tailed t test. (F) Cell cycle analysis (n = 3). Peaks in blue, yellow, and green show percentage cells in G0/G1, S, and G2/M phase. (G) Xenografts after s.c. injection of 1 million cells in left/right flanks of NOD scid gamma females (n = 4). Tumors harvested 35 days after injection. (H) Growth curves of ISK-EV and ISK-FOXA2 xenografts per caliper measurements (n = 4). Data shown as mean ± SEM; 2-tailed t test. (I) Endpoint xenograft weights at day 35 (n = 4, same tumors shown in G). Data shown as mean ± SEM; 2-tailed t test. (J) Gene Ontology (GO) enrichment analysis of 89 significantly upregulated genes by RNA-Seq (≥4-fold and P < 0.0001) in ISK-FOXA2 cells. GO pathways plotted by gene ratio. Dots sized in proportion to gene numbers in the GO term colored by FDR value per inset. (K) Relative change in mRNA expression of AGR2, CNTN1, and PCDHA1 genes (n = 3). Data shown as mean ± SEM; multiple 2-tailed t tests. (L) Cell adhesion assays, representative phase contrast images of ISK-EV and ISK-FOXA2 cells adhering to extracellular matrix protein panel after 16 hours. Scale bars: 50 μm. (M) Quantitative analysis, cell adhesion assays (n = 9). Data shown as mean ± SEM; multiple 2-tailed t tests. For all panels, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.