Multifactorial ERβ and NOTCH1 control of squamous differentiation and cancer
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
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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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 3

Essential role of EGR3, DLX5, and ERβ in RNA PolII recruitment to the NOTCH1 locus and/or pause release.

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Essential role of EGR3, DLX5, and ERβ in RNA PolII recruitment to the NO...
(A) ChIP analysis of HKCs under growing versus differentiating conditions for levels of PolII occupancy of the NOTCH1 locus and associated levels of active histone marks of active promoter (H3K4me3, H3K27ac) and/or enhancer (H3K4me1, H3K27ac) regions. All ChIP samples were examined in parallel by PCR amplification of a negative control locus (NL) in chromosome 4, devoid of active chromatin marks and utilized for similar normalization purposes in previous studies with a number of cell types. Enrichment folds were calculated and plotted as in Figure 2 (*P < 0.05). Enrichment folds in the immunoprecipitates with nonimmune IgG were in all cases less than 1. Similar patterns of PolII binding were obtained with HKCs of independent origin; see also Supplemental Figure 4B. (B) HKCs transfected with siRNA against EGR3, DLX5, or ERβ versus siRNA controls were processed 96 hours later (at 100% confluency) for ChIP assays of levels of PolII occupancy of the NOTCH1 locus. For ease of representation, levels of PolII binding in cells with KD of the individual genes are separately shown, utilizing PolII levels in control cells as the same point of reference (*P < 0.05). (C) HKC samples utilized in B were analyzed in parallel by ChIP assays for levels of active histone marks at the NOTCH1 promoter and enhancer 1, 2, 4, and 5 regions (*P < 0.05). Results similar to those in A–C were obtained with HKCs of independent origin; see also Supplemental Figure 4D.