Multifactorial ERβ and NOTCH1 control of squamous differentiation and cancer
Yang Sui Brooks, … , Karine Lefort, G. Paolo Dotto
Yang Sui Brooks, … , Karine Lefort, G. Paolo Dotto
Published April 17, 2014
Citation Information: J Clin Invest. 2014;124(5):2260-2276. https://doi.org/10.1172/JCI72718.
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Research Article Oncology

Multifactorial ERβ and NOTCH1 control of squamous differentiation and cancer

Abstract

Downmodulation or loss-of-function mutations of the gene encoding NOTCH1 are associated with dysfunctional squamous cell differentiation and development of squamous cell carcinoma (SCC) in skin and internal organs. While NOTCH1 receptor activation has been well characterized, little is known about how NOTCH1 gene transcription is regulated. Using bioinformatics and functional screening approaches, we identified several regulators of the NOTCH1 gene in keratinocytes, with the transcription factors DLX5 and EGR3 and estrogen receptor β (ERβ) directly controlling its expression in differentiation. DLX5 and ERG3 are required for RNA polymerase II (PolII) recruitment to the NOTCH1 locus, while ERβ controls NOTCH1 transcription through RNA PolII pause release. Expression of several identified NOTCH1 regulators, including ERβ, is frequently compromised in skin, head and neck, and lung SCCs and SCC-derived cell lines. Furthermore, a keratinocyte ERβ–dependent program of gene expression is subverted in SCCs from various body sites, and there are consistent differences in mutation and gene-expression signatures of head and neck and lung SCCs in female versus male patients. Experimentally increased ERβ expression or treatment with ERβ agonists inhibited proliferation of SCC cells and promoted NOTCH1 expression and squamous differentiation both in vitro and in mouse xenotransplants. Our data identify a link between transcriptional control of NOTCH1 expression and the estrogen response in keratinocytes, with implications for differentiation therapy of squamous cancer.

Authors

Yang Sui Brooks, Paola Ostano, Seung-Hee Jo, Jun Dai, Spiro Getsios, Piotr Dziunycz, Günther F.L. Hofbauer, Kara Cerveny, Giovanna Chiorino, Karine Lefort, G. Paolo Dotto

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Figure 8

Elevated ERβ expression suppresses proliferation of skin, lung, and oral SCC cells.

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Elevated ERβ expression suppresses proliferation of skin, lung, and oral...
(A) Alamar blue cell density assays. Cell lines derived from skin (SCC13), H/N (Cal33, FaDu, SCCO11, SCCO13, SCCO28), and lung (H520, H2170, HCC95, SK-MES1, SW900) SCCs were infected with either an ERβ-expressing lentivirus (SCC13, SCCO13, and SCCO28) or retrovirus (all other cell lines) versus corresponding empty vector controls, followed, 48 hours later, by G418 selection. Stably infected cells were plated in 96-well plates (2000 cells/well). Alamar blue fluorescence intensity assays were performed in triplicate every 2 days as indicated. Data are presented as mean fold change of fluorescence intensity ± SD over day 1. *P < 0.05. (B) Clonogenicity assays. Skin (SCC13) and H/N (SCCO13 and SCCO28) SCC cells infected with an ERβ-expressing lentivirus versus empty vector were plated at limited density on triplicate dishes (103 cells/60 mm dish), and colony formation was measured by crystal violet staining 10 days later. *P < 0.05. (C and D) Spheroid assays. Skin and H/N (C) and lung (D) SCC cells were infected with an ERβ-expressing retrovirus or lentivirus versus empty vector as in A. Stably infected cells were plated in duplicate in Matrigel suspension in 8-well chambers (2000 cells/well). Spheroid numbers were quantified 10 days later by digital acquisition of the whole well images and ImageJ software analysis. *P < 0.05. (E) Representative images of spheroids formed by SCCO11 cells infected with control versus ERβ-expressing retroviruses. Original magnification, ×2.5. Photographs of spheroids formed by other SCC cells are shown in Supplemental Figure 7B.