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 9

Elevated ERβ expression induces NOTCH1 expression and differentiation.

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Elevated ERβ expression induces NOTCH1 expression and differentiation. (...
(A) SCC cell lines infected with ERβ-expressing viral vectors versus controls as in Figure 8 were analyzed for expression of the indicated proteins by immunoblotting. (B) SCC13 (skin), SW900 (lung), and SCCO13 (oral) SCC cells infected with ERβ-expressing and control viral vectors were treated 24 hours after infection with DAPT (10 μM) or DMSO control followed, 72 hours later, by RT-qPCR analysis of involucrin and keratin 10 differentiation marker expression. *P < 0.05. (C) H2170 lung SCC cells infected with ERβ-expressing versus control vectors were injected intradermally in parallel in the right and left suprascapular regions of NOD/SCID mice (n = 5; 1 × 106 cells per injection). Animals were sacrificed 1 week later, and tumor samples were processed for H&E and immunofluorescence analysis of NOTCH1 expression. For each tumor pair, images were taken under the same capture conditions and are representative of several independent fields. Black scale bar: 500 μm; white scale bar: 100 μm. (D) SCC13 and SCCO13 cells infected with ERβ-expressing versus control vectors were tested by parallel intradermal injections into mice as in C. Animals were sacrificed 3 weeks later, and tumor samples were processed for H&E and immunofluorescence analysis of NOTCH1 and differentiation marker expression as indicated. Analysis of other tumor pairs is shown in Supplemental Figure 8. Black scale bar: 250 μm; white scale bar: 100 μm.