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Novel assays monitoring direct GR protein activity exhibit high predictive power for ligand activity on endogenous GR gene targets

ABSTRACT: Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and auto-immune diseases. Unfortunately, their use is hampered by many side effects and therapy resistance. Efforts to find more selective glucocorticoid receptor (GR) agonists and modulators (called SEGRAMs), able to separate anti-inflammatory effects via gene suppression from metabolic effects via gene activation, have been unsuccessful so far. In this study, we characterized a set of functionally diverse GR ligands in A549 cells, first using a panel of luciferase-based reporter gene assays evaluating GR-driven gene activation and gene suppression. We expanded this minimal assay set with novel luciferase-based read-outs monitoring GR protein levels, GR dimerization and GR Serine 211 (Ser211) phosphorylation status and compared their outcomes with compound effects on the mRNA levels of known GR target genes in A549 cells and primary hepatocytes. We found that luciferase reporters evaluating GR-driven gene activation and gene repression were not always reliable predictors for effects on endogenous target genes. Remarkably, our novel assay monitoring GR Ser211 phosphorylation levels proved to be the most reliable predictor for compound effects on almost all tested endogenous GR targets, both driven by gene activation and repression. The integration of this novel assay in existing screening platforms may therefore increase chances to find novel GR ligands with an improved therapeutic benefit.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Delphi Van Haver

LAB HEAD: Kris Gevaert

PROVIDER: PXD031319 | Pride | 2022-04-10

REPOSITORIES: Pride

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			Action	DRS
	F004687_2p_50uPAC2G2_trap12_CMB-882_KRGEV_shotgun-1.raw	Raw
	F004689_2p_50uPAC2G2_trap12_CMB-882_KRGEV_shotgun-2.raw	Raw
	F004691_2p_50uPAC2G2_trap12_CMB-882_KRGEV_shotgun-3.raw	Raw
	F004693_2p_50uPAC2G2_trap12_CMB-882_KRGEV_shotgun-4_20210803124018.raw	Raw
	F004695_2p_50uPAC2G2_trap12_CMB-882_KRGEV_shotgun-5.raw	Raw

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Project description:The glucocorticoid receptor (GR) regulates gene expression throughout the human genome in a cell-type-specific manner, governing various aspects of homeostasis. The influence of this transcriptional regulator is seen in multiple pathologies, including cancer. Pharmacological activation of the GR is frequently used to alleviate side-effects caused by therapy for patients with various non-lymphoid solid (i.e. non-hematologic) cancers; however, prior studies have shown that this treatment might also have anti-proliferative action on cancer cells. Nonetheless, the molecular underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the molecular mechanisms of glucocorticoid response in non-lymphoid solid cancers models, focusing on lung cancer. Activation of the GR induces reversible cancer cell dormancy in which cells are tolerant to large array of anti-cancer drugs. This state transition is accompanied by induction of growth factor survival signalling (IGF-1R) and acquired vulnerability to inhibitors of this pathway, both in vitro and in vivo. The observed phenotype is dependent on a single GR target gene - CDKN1C, which encodes for a cell cycle inhibitor protein p57. We have discovered an upstream distal enhancer of CDKN1C, which through GR-induced chromatin looping regulates the expression of this key gene, as confirmed in human tumour specimens. Furthermore, we demonstrate that the SWI/SNF complex composition fine-tunes the GR transcriptional activity at the CDKN1C locus, allowing for precise regulation of cell dormancy induction. Collectively, we show that SWI/SNF-facilitated regulation of CDKN1C underlines GR-induced reversible drug-tolerant state in cancer cells. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anti-cancer therapy related side-effects.

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Novel assays monitoring direct GR protein activity exhibit high predictive power for ligand activity on endogenous GR gene targets

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