Abstract
BackgroundAltered 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.MethodsWe 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.ResultsIn 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.ConclusionsPhospho-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.
Highlights
Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors
Our studies define a ligand-independent role for glucocorticoid receptor (GR) as a homeostatic “sensor” of intrinsic stimuli as well as extrinsic factors rich within the tumor microenvironment (TME) (TGFβ1) that enable potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive triple-negative breast cancer (TNBC)
We showed previously that phosphorylation of GR on Serine 134 is elevated in TNBC relative to other breast cancer subtypes [16]; this p38 MAPK-dependent event was insensitive to GR ligands, but induced in response to cellular stress, including ROS/H2O2, hypoxia, and nutrient starvation [17], as well as loss of attachment and stress-inducing chemotherapies [16] such as paclitaxel (Taxol), a taxane microtubule stabilizing drug routinely used as adjuvant or neoadjuvant chemotherapy in TNBC patients [16, 18]
Summary
Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors. The primary method of classifying breast cancer relies on the presence or absence of estrogen receptor (ER), the ER target gene progesterone receptor (PR), and amplification of the human epidermal growth factor receptor 2 (HER2) [2, 3]. These molecular markers in part dictate the treatment regimen that BC patients receive [4]. When these receptors are present, targeted therapies are used to decrease recurrence rate and improve survival (e.g., tamoxifen inhibits ER and trastuzumab/pertuzumab targets HER2 [5]). Appropriate biomarkers of driver pathways and new therapeutic targets are urgently needed
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