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 10

ERβ agonists induce NOTCH1 and differentiation marker expression in HKCs and SCC cells with concomitantly attenuated proliferation.

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ERβ agonists induce NOTCH1 and differentiation marker expression in HKCs...
(A) Differentiating HKCs (100% confluence) were treated with 10 nM estradiol (E2), 100 nM ERβ-specific agonist (DPN), 100 nM ERα-specific agonist (PPT), 10 nM estrogen receptor panantagonist (fulvestrant) or DMSO control followed, 72 hours later, by RT-qPCR (*P < 0.05) and immunoblot analysis of the indicated genes/proteins. For detailed blot information, see complete unedited blots in supplemental material. (B) Expression of CYP1B1 was determined by RT-qPCR in lung and keratinocyte-derived SCC cell lines in parallel with HBECs and HKCs, respectively. (C) Alamar blue density assays of SCC cells were performed in triplicate wells and treated with DPN or DMSO (refreshed every other day). Data are presented as mean fold change of fluorescence intensity ± SD (*P < 0.05). (D) Indicated SCC cells were plated in duplicate on Matrigel precoated chambers with spheroid number quantification 10 days later by ImageJ analysis of whole-cell images. Data are duplicates ± SD (*P < 0.05). Photographs and spheroid quantification of other SCC cells are shown in Supplemental Figure 9. (E) Indicated SCC cells were treated with the ERβ-specific agonist DPN at the indicated doses for 10 days. For detailed blot information, see complete unedited blots in supplemental material.