**1. Introduction**

Tristetraprolin (TTP, ZFP36) is an RNA binding protein that binds to AU-rich elements (AREs) within the 3- -untranslated region (3- -UTR) of mRNAs and promotes the degradation of these mRNAs [1,2]. AREs are found within the 3- -UTR of many short-lived mRNAs, such as cytokines and proto-oncogenes mRNAs [3]. TTP functions as a tumor suppressor by destabilizing the mRNA of critical genes implicated in both tumor onset and progression [4,5]. *TTP* is widely expressed, with particularly high levels in spleen,

**Citation:** Jeon, D.Y.; Jeong, S.Y.; Lee, J.W.; Kim, J.; Kim, J.H.; Chu, H.S.; Jeong, W.J.; Lee, B.J.; Ahn, B.; Kim, J.; et al. FOXO1 Is a Key Mediator of Glucocorticoid-Induced Expression of Tristetraprolin in MDA-MB-231 Breast Cancer Cells. *Int. J. Mol. Sci.* **2022**, *23*, 13673. https://doi.org/ 10.3390/ijms232213673

Academic Editors: Barbara De Filippis, Marialuigia Fantacuzzi and Alessandra Ammazzalorso

Received: 7 October 2022 Accepted: 4 November 2022 Published: 8 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

lymph nodes, and thymus [6]. However, *TTP* expression is significantly decreased in various cancers [7]; its downregulation correlates with increased expression of proto-oncogenes and may contribute to cancer processes. Re-expression of TTP in cancer cells has a growth inhibitory effect [8–10]. The expression of *TTP* in cancer cells is induced by p53 [11] and inhibited by Myc [12]. Notably, nearly all types of cancers have abnormalities in the p53 pathway [13], and c-Myc is often activated in human cancers [14]. Together, these features may lead to a widespread decrease in the expression of *TTP* in human cancers. Thus, enforced expression of *TTP* may represent a new therapeutic avenue for cancer prevention and treatment.

Previously it has been reported that glucocorticoids (GCs) induce the expression of *TTP* in cells [15–18]. However, the mechanisms underlying the GC-mediated TTP induction in cancer cells remain unclear. GCs are steroid hormones synthesized and released by the adrenal glands in response to physiological cues and stress [19]. GCs regulate fundamental body functions in mammals, including control of cell growth, development, metabolic homeostasis, cognition, mental health, immune homeostasis, and apoptosis [20–23]. Various synthetic GCs (e.g., prednisolone, aldosterone, dexamethasone, and betamethasone) have been developed by the pharmaceutical industry and serve as treatments for various diseases. Both natural and synthetic glucocorticoid hormones exert their biological effects predominantly via the glucocorticoid receptor (GR; NR3C1) [24], a ligand-activated transcription factor that is constitutively and ubiquitously expressed throughout the body [25]. GR functions by regulating the expression of GC responsive genes in a positive or negative manner. Upon ligand binding, GR shuttles into the nucleus [22] and binds to DNA sequences called glucocorticoid response elements (GREs) as a homodimeric complex [26]. The binding of GR homodimers to GRE sequences leads to an enhancement of gene expression [27]. GR also interacts with DNA as a monomer by binding to GRE half-sites and positively or negatively influences the transcription of target genes by interacting with promoter-bound STAT5, activator protein 1 (AP-1), or NF-κB transcription factors [28,29]. Independent of binding to GRE, monomeric GR can also regulate gene expression in a mechanism known as tethering, which involves physical interaction of monomeric GR with another transcription factor, such as AP-1 and NF-κB [30]. GCs also exert rapid non-genomic effects that do not require transcription processes or protein synthesis [31]. Interestingly, GCs not only increase the expression of *FOXO* [32–34] but also increase FOXO activity [35,36] in a variety cells.

The FOXO family of transcription factors comprises four closely related members— FOXO1, FOXO3, FOXO4, and FOXO6—that are direct downstream targets of AKT [37,38]. FOXOs play central roles in regulating normal hematopoiesis and are integral mediators of AKT actions in cellular growth and survival [38,39]. In the absence of active AKT, FOXOs localize to the nucleus, where they regulate the transcription of genes involved in cellcycle arrest, apoptosis, and reactive oxygen species (ROS) detoxification. Previous studies have reported that GCs increase *FOXO1* level in a variety of cells, including hepatocytes, cardiomyocytes, and tenocytes [32–34]. A reduction in FOXO1 protein protects against beta cell death induced by the synthetic GC dexamethasone, suggesting that FOXO1 activation mediates the pro-apoptotic effects of GCs [40].

Several compounds and cytokines have been identified to induce *TTP* expression [4,5]. However, until now, there has been no report regarding the identification of compounds from library screening. The purpose of this study was to identify natural compounds that induce the expression of *TTP* in cancer cells by screening a natural compound library using a cell-based reporter assay. Among the 1019 natural compounds in the library, we identified one molecule, of which the enantiomers are betamethasone 21-phosphate (BTM-21-P) and dexamethasone 21-phosphate (DXM-21-P), as the best inducer of *TTP* in the cell-based reporter assay. We show here, for the first time, that GCs such as BTM-21-P, DXM-21-P, and DXM induce the expression of *TTP* in a FOXO1-dependent manner, even in p53 mutant breast cancer cells. The inhibition of GR or FOXO1 by inhibitors abrogated the effects of GCs with respect to *TTP* induction. We also found that GC-induced *TTP* is required for

the growth inhibitory effect of GC on breast cancer cells. Together, these studies identify a novel signaling pathway by which GCs induce *TTP* expression in a FOXO1-dependent manner, representing a possible novel pharmacological approach to treat p53 mutant breast cancer cells.

#### **2. Results**
