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 May 1, 2014; First 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 4

Silencing of EGR3, DLX5, and ERβ leads to attenuation of NOTCH1 expression and differentiation.

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Silencing of EGR3, DLX5, and ERβ leads to attenuation of NOTCH1 expressi...
(A) HKCs infected with shRNAs silencing lentiviruses versus control were analyzed 96 hours later by immunoblotting. Gene KD efficiency was assessed by parallel blots of those for NOTCH1 and Keratin 1 expression. Similar results were observed at the NOTCH1 mRNA level and in another experiment with HKCs of independent origin (Supplemental Figure 5). (B) HKCs infected with lentiviruses as in A were superinfected with retrovirus expressing NOTCH1 intracellular domain fused to the human estrogen receptor (rNert), or vector control (Neo). 24 hours later, cultures were treated with OH-tamoxifen (OH-TAM) for 48 hours for nuclear NOTCH1 intracellular domain translocation. Expression of Keratin genes was determined by RT-qPCR (*P < 0.02). Results similar to those were obtained with a second HKC (Supplemental Figure 5C). (C–E) HKCs infected with lentiviruses as in A were grown in duplicate dermal equivalent gels at air-liquid interface for 12 days. The experiment was performed twice. (C) Immunoblot analysis of full-length NOTCH1 expression in reconstituted epidermis with signal quantification (numbers) by densitometric scanning and γ-tubulin normalization. (D) H&E analysis showing defective stratification and cornified layer formation in organotypic cultures with ERβ and DLX5 KD HKCs. Scale bar: 100 μm. H&E images of other cultures and immunofluorescence analysis of differentiation marker expression are shown in Supplemental Figure 5D. (E) Immunofluorescence analysis of NOTCH1 expression in the reconstituted epidermis. For each series, image-capture conditions were the same. Scale bars: 50 μm. Images are representative of 3 independent fields.