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 3

FOXA2 knockdown promotes EC cell migration and invasion.

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FOXA2 knockdown promotes EC cell migration and invasion. (A) Western blo...
(A) Western blot shows effective lentiviral shRNA-mediated knockdown of FOXA2 in HEC-1-B cells (one of the EC lines with high endogenous FOXA2 expression, Figure 1C). (B) Immunostaining of FOXA2 (red) with actin control (green) showing expected reduction of nuclear FOXA2 signal in FOXA2KD HEC-1-B cells. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (C) RNA expression analysis, volcano plot showing differentially expressed significant genes (P <0.05) in FOXA2KD HEC-1-B cells compared with empty vector control (1713 upregulated genes [red, fold change ≥ 2] and 2439 downregulated genes [green, fold change ≤ –2]). Dotted vertical lines represent log2 (fold change) threshold of ±1 and dotted horizontal line represents P value threshold of 0.05. Selected genes are shown. (D) Protein expression by Western blot in control and FOXA2KD HEC-1-B cells. (E) Indirect immunofluorescence in control and FOXA2KD HEC-1-B cells; representative images. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (F) Cell migration and invasion assays for control and FOXA2KD HEC-1-B cells. Cells that migrated/invaded from the upper chamber of a Transwell to its lower chamber without (migration) or with a growth factor gradient (invasion) are visualized by crystal violet staining; representative images. Scale bars: 200 μm. (G) Quantitative analysis of migrated or invaded control and FOXA2KD HEC-1-B cells (n = 3). Data shown as mean ± SEM; *P < 0.05; ***P < 0.001, 2-tailed t test. (H) Western blot documenting lentivirus-mediated enforced CDH1 (E-cadherin) expression in FOXA2KD HEC-1-B cells. (I) E-cadherin expression (red) by immunofluorescence in FOXA2KD HEC-1-B cells with CDH1 enforced expression. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (J) Cell invasion and migration assays of FOXA2KD HEC-1-B cells with or without CDH1 reconstitution. Scale bars: 200 μm. (K) Quantitative analysis of migrated and invaded FOXA2KD HEC-1-B cells with or without CDH1 reconstitution (n = 3). Data shown as mean ± SEM; ***P < 0.001, 2-tailed t test.