Glucocorticoid receptors are required effectors of TGFβ1-induced p38 MAPK signaling to advanced cancer phenotypes in triple-negative breast cancer

Abstract

Background Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors. We previously reported that phospho-Ser134-GR (pS134-GR) species are elevated in triple-negative breast cancer (TNBC) and cooperate with hypoxia-inducible factors, providing a novel avenue for activation of GR in response to local or cellular stress. Methods We probed GR regulation by factors (cytokines, growth factors) that are rich within the tumor microenvironment (TME). TNBC cells harboring endogenous wild-type (wt) or S134A-GR species were created by CRISPR/Cas knock-in and subjected to transwell migration, invasion, soft-agar colony formation, and tumorsphere assays. RNA-seq was employed to identify pS134-GR target genes that are regulated both basally (intrinsic) or by TGFβ1 in the absence of exogenously added GR ligands. Regulation of selected basal and TGFβ1-induced pS134-GR target genes was validated by qRT-PCR and chromatin immunoprecipitation assays. Bioinformatics tools were used to probe public data sets for expression of pS134-GR 24-gene signatures. Results In the absence of GR ligands, GR is transcriptionally activated via p38-dependent phosphorylation of Ser134 as a mechanism of homeostatic stress-sensing and regulated upon exposure of TNBC cells to TME-derived agents. The ligand-independent pS134-GR transcriptome encompasses TGFβ1 and MAPK signaling gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TNBC cell anchorage-independent growth in soft-agar, migration, invasion, and tumorsphere formation, an in vitro readout of cancer stemness properties. Both pS134-GR and expression of the MAPK-scaffolding molecule 14-3-3ζ were essential for a functionally intact p38 MAPK signaling pathway downstream of MAP3K5/ASK1, indicative of a feedforward signaling loop wherein self-perpetuated GR phosphorylation enables cancer cell autonomy. A 24-gene pS134-GR-dependent signature induced by TGFβ1 predicts shortened overall survival in breast cancer patients. Conclusions Phospho-S134-GR is a critical downstream effector of p38 MAPK signaling and TNBC migration/invasion, survival, and stemness properties. Our studies define a ligand-independent role for GR as a homeostatic “sensor” of intrinsic stimuli as well as extrinsic factors rich within the TME (TGFβ1) that enable potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive TNBC.

Glucocorticoid Receptor: A Multifaceted Actor in Breast Cancer

“…Among them, it was demonstrated that cellular stress, such as oxidative stress or hypoxia, in primary TNBCs or ERα-negative BC cell lines, increases the phosphorylation of GR on S134, thus potentiating stress signaling mediated by GR activation leading to an increase in the expression of breast tumor kinase BRK, known as protein tyrosine kinase 6 (PTK6), essential for aggressive BC phenotypes [ 115 ]. In addition, TNBCs express high levels of functionally active pS134-GR in comparison to luminal BCs, which could explain why GR expression is correlated with a better prognosis in luminal BCs than in TNBCs [ 116 ]. Recently, research on patients and TNBC cell line-derived xenograft models, revealed that GR activation at distant metastatic sites, due to an increase in GC levels, promotes BC colonization and reduces the overall survival by upregulating the expression of ROR-1 kinase, a receptor tyrosine kinase-like orphan receptor-1, previously shown to be implicated in BC [ 13 , 117 , 118 ].…”

“…Moreover, they showed that in the absence of GR ligands, GR is transcriptionally activated in TNBCs through its phosphorylation on S134 by p38, following the homeostatic sensing of intrinsic stress or extrinsic factors (like TGFβ1). Phospho-S134-GR activates the p38 MAPK stress-signaling pathway, leading to TNBC cell anchorage-independent growth and migration [ 116 ].…”

Cancer and Stress: Does It Make a Difference to the Patient When These Two Challenges Collide?

“…These include S45, S113, S141, S203, S211, S226, S236, S267, S404, S134 and T8 and some of them are associated with inhibition of GC signalling [ 112 , 128 ]. A recent study on Triple-Negative Breast Cancer (TNBC) also showed that TGFβ promoted ligand-independent, p38 MAPK—induced S134 GR phosphorylation, which resulted in migration and invasion [ 129 ].…”

90 YEARS OF PROGESTERONE: Steroid receptors as MAPK signaling sensors in breast cancer: let the fates decide

“…(Perez Kerkvliet et al 2020). Thus, targeting phospho-SR species may be possible using existing agents or newer, more selective SR antagonists paired with the appropriate MEK/MAPK or CDK signaling pathway inhibitors (Table 1).…”