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 5

EGR3, DLX5, and ERβ as regulators of NOTCH1 expression and function in HKCs.

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EGR3, DLX5, and ERβ as regulators of NOTCH1 expression and function in H...
(A) HKCs under sparse conditions were infected with retroviruses expressing EGR3, DLX5, or ERβ versus empty vector controls as indicated, followed 72 hours later (at approximately 80% confluence) by immunoblot analysis for levels of these proteins as well as the full-length NOTCH1 protein (NOTCH1 FL) and the intracellular activated form (NOTCH1 ICD). HKCs under differentiating conditions (100% confluence for 3 days: 100% cf+3D) were analyzed in parallel as point of reference. Similar results were observed at the RNA level and in other experiments with HKCs of independent origin (Supplemental Figure 6, A and B). (B) HKCs with or without infection with EGR3-, DLX5-, and ERβ-expressing retroviruses as in the previous panel were treated with DAPT (10 μM) or DMSO control at 24 hours after infection followed, 48 hours later, by RT-qPCR analysis of involucrin gene expression, with 36β4 for normalization. As an alternative, HKCs stably infected with a shRNA lentivirus against NOTCH1 or empty vector control were superinfected with the EGR3, DLX5, and ERβ expressing retroviruses, followed 72 hours later by RT-qPCR analysis of the same genes. (C) HKCs with or without infection with EGR3-, DLX5-, and ERβ-expressing retroviruses as in A were analyzed, 72 hours after infection, by RT-qPCR of the indicated genes. *P < 0.001.