Steroid Receptors As MAPK Signaling Sensors in Breast Cancer: Let the Fates Decide

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Journal of Molecular A R Dwyer, T H Truong et al. Steroid receptors as MAPK 65:1 T35–T48 Endocrinology sensors THEMATIC REVIEW 90 YEARS OF PROGESTERONE Steroid receptors as MAPK signaling sensors in breast cancer: let the fates decide

Amy R Dwyer1,*, Thu H Truong1,*, Julie H Ostrander1,2 and Carol A Lange1,2,3

1Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA 2Department of Medicine, Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, Minnesota, USA 3Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, USA

Correspondence should be addressed to C A Lange: [email protected]

*(A R Dwyer and T H Truong contributed equally to this work)

This review forms part of a special section on 90 years of progesterone. The guest editors for this section are Dr Simak Ali, Imperial College London, UK and Dr Bert W O’Malley, Baylor College of Medicine, USA

Abstract

Steroid hormone receptors (SRs) are classically defined as ligand-activated transcription Key Words factors that function as master regulators of gene programs important for a wide range ff glucocorticoid receptors of processes governing adult physiology, development, and cell or tissue homeostasis. ff progesterone receptors A second function of SRs includes the ability to activate cytoplasmic signaling pathways. ff breast cancer Estrogen (ER), androgen (AR), and progesterone (PR) receptors bind directly to ff map kinases membrane-associated signaling molecules including mitogenic protein kinases (i.e. c-SRC ff cancer stem cells and AKT), G-proteins, and ion channels to mediate context-dependent actions via rapid activation of downstream signaling pathways. In addition to making direct contact with diverse signaling molecules, SRs are further fully integrated with signaling pathways by virtue of their N-terminal phosphorylation sites that act as regulatory hot-spots capable of sensing the signaling milieu. In particular, ER, AR, PR, and closely related glucocorticoid receptors (GR) share the property of accepting (i.e. sensing) ligand-independent phosphorylation events by proline-directed kinases in the MAPK and CDK families. These signaling inputs act as a ‘second ligand’ that dramatically impacts cell fate. In the face of drugs that reliably target SR ligand-binding domains to block uncontrolled cancer growth, ligand-independent post-translational modifications guide changes in cell fate that confer increased survival, EMT, migration/invasion, stemness properties, and therapy resistance of non-proliferating SR+ cancer cell subpopulations. The focus of this review is on MAPK pathways in the regulation of SR+ cancer cell fate. MAPK-dependent phosphorylation of PR (Ser294) and GR (Ser134) will primarily be discussed in light of the need to target Journal of Molecular changes in breast cancer cell fate as part of modernized combination therapies. Endocrinology (2020) 65, T35–T48

-19-0274 Journal of Molecular A R Dwyer, T H Truong et al. Steroid receptors as MAPK 65:1 T36 Endocrinology sensors

Introduction Closely related to PR within the SR superfamily, the glucocorticoid receptor (GR) is a ligand-activated Of the 48 ligand-activated and ‘orphan’ (i.e. ligands transcription factor responsible for integrating the remain unidentified) nuclear receptors (NRs), six are physiologic, pathological, and therapeutic functions of related members of the SR superfamily whose steroid hormonal (cortisol, endogenous corticosteroids) and hormone ligands are primarily derived from cholesterol synthetic glucocorticoids like dexamethasone. GR is metabolism: estrogen receptor (ERα, ERβ), progesterone ubiquitously expressed and drives the expression of receptor (PR), glucocorticoid receptor (GR), androgen glucocorticoid-responsive genes in a cell/tissue- and receptor (AR), and mineralocorticoid receptor (MR). PGR is promoter-specific manner after glucocorticoid binding most well-studied as an estrogen-responsive gene, and PR to the GR C-terminal ligand-binding pocket (Miranda expression is used as a clinical biomarker of intact estrogen et al. 2013, Kumar & Thompson 2019). Consequently, GR signaling or functional ER. There are two predominant activation regulates diverse gene programs controlling PR isoforms: full-length PR-B and N-terminally truncated metabolism, immune system, and CNS function to PR-A that is missing the first 164 amino acids found maintain cellular homeostasis. Like other steroid hormone in full-length PR-B, termed the B-upstream segment receptors, GR consists of three functional domains: (BUS). Although PR-A and PR-B share structural and intrinsically disordered N-terminal (NTD), DNA binding sequence identity downstream of the BUS, this segment (DBD), and ligand binding (LBD). NTD and LBD contain contains regulatory components that account for major transcription Activation Function regions, AF1 and AF2, differences in PR-binding partners, promoter selectivity, respectively (Kumar & Thompson 2005). AF2 is strictly and transcriptional activity between these two isoforms. ligand-dependent, while AF1 can act constitutively in A third uterus-specific isoform, termed PR-C, is a further the absence of the LBD (Oakley & Cidlowski 2013), a truncated PR receptor composed of only the hinge region property shared by both PR (Goswami et al. 2014) and AR and hormone binding domain (HBD) that is incapable (Dehm et al. 2008). Like other SRs, functionally distinct of binding DNA but inhibits PR transactivation. PR-C AF domains of GR mediate transcriptional activation signaling occurs specifically in the fundal myometrium by recruiting coregulatory complexes that remodel during the onset of labor and is likely mediated by direct chromatin, target initiation sites, and stabilize RNA competition for C-terminal binding partners (Condon polymerase II for gene transcription (Miranda et al. 2013). et al. 2006). PR-A and PR-B are generally co-expressed Similar to PR isoform structure, a further complexity of in PR+ tissues such as the breast or uterus, but are not GR action is that a large cohort of GR isoforms can be always found together in the same cells (Aupperlee et al. created from a single gene (known as NR3C1) by alternate 2005). Along with ER and Her2, total PR levels, rather splicing and at least eight alternative translational start than individual PR isoforms, are measured clinically sites within the GR N-terminal domain sequence (Turner to classify the luminal A (ER+/PR+) and luminal B & Muller 2005, Duma et al. 2006, Presul et al. 2007). The (ER+/PR-low/null) breast cancer subtypes. However, multiple GR isoforms are expressed in a highly tissue- increasing studies have implicated differential PR isoform specific manner and, when expressed as individual expression in the etiology of breast cancer (Mote et al. 2002, isoforms experimentally, GR isoforms regulate remarkably 2015, Rojas et al. 2017, Lamb et al. 2018). Furthermore, distinct transcriptomes (Lu & Cidlowski 2005). PRs dramatically influence ER transcriptional responses Within the NTD of all SRs, serine, threonine, and lysine (Daniel et al. 2015, Mohammed et al. 2015, Singhal et al. residues can be post-translationally modified (Fig. 1). Our 2018); PR-A can transrepress both ER and PR-B (Abdel- prior studies have primarily defined MAPK-dependent Hafiz et al. 2002), while PR-B is an important ER-binding phospho-PR transcriptomes (Knutson et al. 2012, 2017). partner (Daniel et al. 2015, Mohammed et al. 2015). While the extent to which phosphorylation events modify Measuring PR isoform ratios by immunohistochemistry the GR cistrome is still being studied, such modifications (IHC) has been reported (Sletten et al. 2019) but remains significantly alter GR function Galliher-Beckley( et al. challenging due to the lack of reliable antibodies that are 2011). For example, recent collaborative studies from our capable of accurately distinguishing PR-A vs PR-B since group (Regan Anderson et al. 2016, 2018) have established there are no unique amino acid sequences in PR-A. Thus, GR as an important sensor of both life stress (i.e. a defining the context-dependent actions of PR isoforms collection of classical GR ligands acting via the LBD) as is crucial to understanding how imbalanced PR-A/PR-B well as inducible cellular (hypoxia, reactive oxygen species ratios can be exploited therapeutically in the clinic. (ROS), nutrient starvation), and microenvironmental

to the gradual loss of hormone responsiveness in which SRs shift to hormone-independent or resistant states; this process is well-studied with regard to ER, while a similar paradigm is emerging for both PR and GR (Shen et al. 2001, Irusen et al. 2002, Matthews et al. 2004, Arpino et al. 2008, Giulianelli et al. 2008). One mechanism by which SRs become less dependent upon natural ligands occurs through hormone-induced rapid activation of cytoplasmic signaling pathways that, in turn, phosphorylate and sustain SR activation in diminishing ligand (Fig. 2). For example, constitutive ER/PR complexes exist in breast cancer cell lines and primary breast tumors (Daniel et al. 2015); trace levels of either estrogen (E2) or progestin (P4) induce rapid (3–15 min) activation of p42/p44 MAPKs (ERK1/2) via cytoplasmic or membrane-associated ER/PR/c-SRC complexes that have been shown to regulate Figure 1 breast cancer cell proliferation in vitro (Migliaccio et al. Post-translational modifications to steroid receptors. Multiple posttranslational modification sites are present in the N-terminal domain 1998, Boonyaratanakornkit et al. 2001, Ballare et al. 2003). (NTD) of GR, PR, ER, and AR. PR and AR also have a serine phosphorylation We and others have shown that the SR coactivator, site in the hinge (H) region. The annotated pro-directed sites are known PELP1, functions as a cytoplasmic scaffold for docking to be modified by MAPKs and/or CDKs and alter SR transcriptional activity and co-factor binding. DNA-binding and ligand-binding of multiple SRs including GR (Kayahara et al. 2008, domains are also indicated. Regan Anderson et al. 2016), AR (Yang et al. 2012) and both ER and PR (Vadlamudi et al. 2001, Truong et al. (cytokine) stress, largely acting via p38 MAPK-dependent 2018) as well as the SR coactivator, SRC-3 (Truong et al. phosphorylation of Ser134 (Fig. 2). Herein, we review the 2018). Indeed, a common link between GR signaling in regulation of PR and GR by members of the MAPK family TNBC (discussed subsequently) and PR signaling in ER+ and highlight the functional redundancy of these closely luminal breast cancer is the functional role of PELP1 related SRs as key mediators of advanced luminal (PR) and as a scaffold to bring cytoplasmic kinases and SRs into triple negative (GR) breast cancer phenotypes and therapy- close proximity. In TNBC models, PELP1 expression is resistant tumor progression. A major consequence of the upregulated in response to chemotherapy and cellular integration of SR actions with MAPK signaling pathways stress including hypoxia and ROS production. PELP1 and is the establishment of potent feed-forward signaling GR interactions increase in response to the glucocorticoid loops capable of driving persistent, ligand-independent dexamethasone and creation of ROS by hydrogen SR activation needed for sustained biological responses peroxide (Regan Anderson et al. 2016). Furthermore, relevant to breast cancer cell autonomy and metastatic a transcriptional complex containing PELP1, GR, and dissemination. hypoxia inducible factors (HIFs) is recruited to the breast tumor kinase (Brk/PTK6) promoter; stress-induced Studies of signal transduction Brk/PTK6 signaling promotes breast cancer cell survival, migration, and metastasis (Castro & Lange 2010, Lofgren MAPK signaling pathways promote et al. 2011, Regan Anderson et al. 2013). Similarly, in ligand-independent actions of phospho-SRs ER+ breast cancer, PELP1 scaffolds SRC-3, ER, and PR to Reversible post-translational modifications (PTMs) of SRs promote ER phosphorylation (i.e. at Ser118 and Ser167) include phosphorylation, SUMOylation, acetylation, and and expression of E2-induced genes involved in cell ubiquitination. These PTMs may account for the functional survival and stem/progenitor cell formation (Truong et al. diversity of receptor isoforms. This section will primarily 2018). Notably, expression of PR-B (but not progesterone) focus on ligand-independent actions of PR and GR; the was required for E2-induced expression of a distinct hormonal regulation of these SRs in breast cancer models ER/PR target gene signature that predicts greatly shortened have been reviewed (Diep et al. 2015, Scheschowitsch survival in luminal breast cancer patients (Daniel et al. et al. 2017, Truong & Lange 2018). Ligand-independent 2015). Treatment of breast cancer cells with SI-2, an SRC-3 SR activation has been proposed as a contributing factor inhibitor, disrupts ER/PR/PELP1 complexes and blocks

Figure 2 Phosphorylation of PR and GR establishes a feed-forward signaling paradigm. Phosphorylation of PR Ser294 by p42/p44 MAPKs (A) and GR Ser134 by stress-activated p38 MAPKs (B) modulates SR target gene selection to drive expression of genes that are able to further promote SR phosphorylation. This establishes a feed-forward signaling loop in the absence of steroid ligand resulting in cell survival, migration, EMT, and non-proliferative/stem cell states.

PELP1-induced tumorsphere formation. Importantly, (MAPKs and CDKs) pathways. As discussed subsequently, the PELP1/GR interaction is enhanced by GR Ser134 these signaling inputs to SR N-termini phospho-sites can phosphorylation, while PR Ser294 phosphorylation is dramatically impact cell fate decisions that are highly required for the PELP1/PR/ER interaction. Thus, SRs relevant to breast cancer progression. participate in cytoplasmic signaling complexes that enable them to rapidly sense changes in the signaling Mitogenic signaling inputs to PR phosphorylation microenvironment through direct PTMs. All SRs can be and transcriptional activity phosphorylated at multiple proline-directed sites within the NTD, and PR and AR also contain these sites in the PR transcriptional activity and promoter selection are hinge region (Fig. 1). Specifically, upregulation of MAPK- dramatically influenced by activation of mitogenic and associated signaling pathways (e.g. Akt, ERK1/2, and p38 cell cycle-dependent protein kinases (i.e. MAPK, Akt, MAPK) in cancer states can lead to ligand-independent CK2, and cylin-dependent kinases (CDK)) that alter PR activation of SRs via phosphorylation at their N-terminal phosphorylation (Zhang et al. 1997, Knotts et al. 2001, phosphorylation sites (Fig. 1). Ligand-independent Pierson-Mullany & Lange 2004, Daniel et al. 2007, phosphorylation at Ser118 (via MAPK) or Ser167 (via Akt) Faivre et al. 2008, Hagan et al. 2011, Dressing et al. are two of the most well-studied phosphorylation sites 2014), SUMOylation (Daniel et al. 2007, Daniel & Lange in ER (Stellato et al. 2016). Additionally, PR (Ser294, Ser 2009), or acetylation (Daniel et al. 2010). Modification 345, and Ser400) and GR (Ser134) also undergo ligand- of SRs may not appreciably alter transcriptional activity, independent phosphorylation in response to growth as measured using reporter gene assays in vitro, but is a factor or cytokine induced activation of cytoplasmic kinase major mechanism for regulating endogenous target gene

https://jme.bioscientifica.com © 2020 Society for Endocrinology https://doi.org/10.1530/JME-19-0274 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 03:21:57AM via free access Journal of Molecular A R Dwyer, T H Truong et al. Steroid receptors as MAPK 65:1 T39 Endocrinology sensors selection. PTM sites identified on PR that are known to alter Recently, PRs have been implicated in mechanisms of its transcriptional activity at selected endogenous target early luminal breast cancer dissemination; progesterone genes include phosphorylation (S81 unique to PR-B, S294, induced migration of mammary cells from early lesion, S345, and S400), SUMOylation (K388), and acetylation but not primary tumor samples from BALB-NeuT mice (K183, K638, K640, and K641). These PTMs enable PR to (Hosseini et al. 2016). Breast tumor cells co-associate with induce diverse biological outcomes such as anchorage- local and circulating carcinoma-associated fibroblasts independent growth/survival, tumorsphere formation as (c-CAFs) that can be detected as cancer stem cell/c-CAF a readout of stemness properties, and cell cycle-dependent cell co-clusters in both mouse models of breast cancer gene regulation. These biologies are context-dependent as and the blood of patients with metastatic disease (Ao determined by the presence/absence of activated signaling et al. 2015). Related to these studies, fibroblast growth pathways and the availability of coregulators. factor 2 (FGF-2), produced by CAFs, has been shown to For example, PR Ser294 phosphorylation is ligand- activate PR in breast cancer models (Giulianelli et al. 2008). induced (progestins), but also occurs through ligand- This ultimately leads to transcription of known PR target independent pathways in response to tyrosine kinase genes MYC and CCND1, resulting in increased hormone- growth factor receptor-induced MAPK and/or CDK independent cell proliferation in mouse mammary tumor activation. Phospho-Ser294 PR-B species are deSUMOylated models. Follow-up studies further defined that FGF-2 at Lys388 and are subject to more rapid ligand-dependent promotes interaction between ER and PR at the MYC receptor downregulation relative to dephosphorylated enhancer and proximal promoter regions (Giulianelli et al. and heavily SUMOylated PR species (Daniel et al. 2007). 2019). Notably, over 700 canonical proteins were recruited Lys388 SUMOylation is a transcriptionally repressive to MYC regulatory sequences following FGF-2 induction, modification at PR target genes associated with cancer which included both PR isoforms (PR-A and PR-B) as well as biology that also stabilizes PRs in the presence of progestin the expression of a novel PR splice variant protein product by slowing their 26S proteasome/ubiquitinoylation- PR-BΔ4. These studies suggest that the PR-BΔ4 variant, dependent degradation. However, a number of genes which has an impaired ligand-binding domain and lacks (i.e. including numerous tumor suppressor genes) are the nuclear localization signal, may play a significant role induced by SUMOylated PRs. Co-mutation of PR-B Lys388 in ligand-independent PR actions, given that it is induced (K388R) rescues transcriptional deficiencies of phospho- and activated by FGF-2 but not progestins. The inclusion mutant PR-B S294A receptors in luciferase assays (Daniel of non-canonical PR isoforms such as PR-BΔ4 in studies of et al. 2007). Interestingly, studies with SUMO-deficient ER+ breast cancer may reveal alternate avenues to prevent mutant PR-B (K388R; a biochemical phospho-mimic) or treat endocrine resistance and effectively block luminal demonstrated that SUMO-deficient PR-B exhibited breast cancer progression and metastasis. Namely, CAF- transcriptional hyperactivity at a unique subset of PR target derived FGF-2 represents a potent second ligand that is genes crucial for cell cycle regulation, proliferation, and sensed by PRs in order to sustain ligand-independent PR survival. This occurred independently of progestins and activation in disseminated cancer cell/CAF clusters and was increased by activated CDK2 (Daniel & Lange 2009). reveals a novel strategy to target phospho-PR actions in Additionally, breast cancer cell models expressing K388R otherwise hormone-ablated breast cancer patients. PR exhibited an alternative gene program significantly Stress signaling inputs to GR phosphorylation and associated with Her2 signaling in luminal B (HER2+) transcriptional activity breast cancers and the emergence of endocrine resistance (Knutson et al. 2012). These studies suggest that, in breast Glucocorticoids (i.e. corticosteroid GR agonists) are one cancer cells with increased MAPK or CDK2 signaling, of the most commonly prescribed and effective treatment phospho-Ser294 PR-B (i.e. SUMO-deficient) species regimens for acute inflammatory conditions as well as promote the expression of transcriptional programs that many chronic inflammatory diseases. However, some can contribute to tumor heterogeneity (e.g. emergence of patients become unresponsive to these treatments due to HER2+ cells) during luminal tumor progression (Knutson the development of glucocorticoid resistance. One of the et al. 2012). Crosstalk between Ser294 and Lys388 in mechanisms that accounts for this resistance is decreased PR-A was less apparent relative to that in PR-B (Daniel glucocorticoid signaling, which is correlated with changes et al. 2007). Additional studies are ongoing to define in GR phosphorylation (Bloom 2004, Tsitoura & Rothman the isoform-specific contributions of SUMO-deficient 2004). GR phosphorylation by ubiquitous protein kinases (i.e. K388R mutant) and Ser294-phosphorylated PR-A. has been shown to lead to altered GR transcriptional

https://jme.bioscientifica.com © 2020 Society for Endocrinology https://doi.org/10.1530/JME-19-0274 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 03:21:57AM via free access Journal of Molecular A R Dwyer, T H Truong et al. Steroid receptors as MAPK 65:1 T40 Endocrinology sensors activity and interaction with other coregulatory proteins and aromatase inhibitors (AIs)) inhibit proliferation (Krstic et al. 1997, Galliher-Beckley et al. 2011). GR is and are very effective treatments for ER-positive phosphorylated on serine residues Ser113, Ser134, Ser141, breast cancer. Yet, the benefits of endocrine therapy Ser203, Ser211, and Ser226 by cyclin-dependent kinases, are not sustained long-term in 10% to 41% of patients MAPK, and casein kinase II (Ismaili & Garabedian 2004) who suffer from late recurrence (Pan et al. 2017). It is (Fig. 1). Three of these GR residues (Ser204, Ser211, and hypothesized that non-proliferative cell states, such as Ser226) are located within the AF1 domain, while the quiescence/tumor cell dormancy, cell survival, plasticity, remainder are found in the NTD (Mason & Housley 1993, and epithelial to mesenchymal transition (EMT) as Ismaili & Garabedian 2004). well as cancer stem cells (CSCs), are not well-targeted Phosphorylation of Ser134 by p38 MAPK is the by standard cancer therapies. Indeed, a growing body most well-studied GR PTM event. Ligand-independent of literature suggests that breast CSCs are resistant to phosphorylation of Ser134 in response to stress-activating current therapeutic approaches (Easwaran et al. 2014, stimuli requires p38 MAPK in U2OS osteosarcoma cells Piva et al. 2014). In fact, these non-proliferative cell (Galliher-Beckley et al. 2011) and TNBC models (Regan states are induced or maintained in response to cellular Anderson et al. 2016). In TNBC cells, p38 MAPK inhibition stress signaling and the selective pressure created by blocked Ser134 GR phosphorylation and abrogated the radiation, chemo-, and endocrine therapy that is largely ability of GR to associate with a GRE-containing region cytostatic rather than cytotoxic, ultimately contributing of the BRK (breast tumor kinase) promoter (Regan to therapy-resistant cancer recurrence and metastasis. Anderson et al. 2016). Ligand-independent regulation For example, chemotherapy has been shown to increase of GR transcriptional activity is further supported by CD44+/CD24- CSC populations and tumorsphere studies showing p38 MAPK-induced phosphorylation formation in breast cancer patients (Li et al. 2008). In of Ser134 increased the association of GR with the 14-3- particular, CSCs tend to be non-proliferative, allowing 3zeta signaling proteins on gene regulatory regions in them to readily evade standard therapies and expand chromatin, which simultaneously resulted in reduced over time (Fillmore & Kuperwasser 2008). Recent studies hormone-dependent transcriptional responses (Galliher- utilizing mouse models of prolactin-induced ER+ breast Beckley et al. 2011). Interestingly, p38 MAPK has been cancer showed that treatment with antiestrogens such as shown to be active in alveolar macrophages of asthmatic fulvestrant or tamoxifen reproducibly stimulated breast patients who exhibited poor response to glucocorticoids CSC self-renewal (Shea et al. 2018). These studies suggest when compared to patients with a normal response that antiestrogen therapies may initially slow tumor (Bhavsar et al. 2008). In contrast, p38 MAPK has also been growth, but also concurrently promote unwanted side shown to phosphorylate Ser211 in human leukemic cells, effects by evoking plasticity and regenerative CSC activity leading to increased dexamethasone-driven apoptosis in largely non-proliferative cancer cell compartments. (Khan et al. 2017). This suggests that p38 MAPK kinase The proliferative effects of progestins in normal phosphorylation of GR impacts ligand-independent breast development are well-established. Progestins GR signaling in a cell-specific manner. In breast cancer are proliferative in the breast (McGowan et al. 2007), models, p38 MAPK signaling is emerging as an important and PR-B is the predominant isoform involved in mediator of stem cell biology (Lu et al. 2018, Xu et al. 2018, normal mammary gland tissue expansion during breast Grun et al. 2019) and other non-proliferative states that development after puberty (Mulac-Jericevic et al. 2003). are relevant to cancer biology (discussed subsequently). Mouse models lacking PR-B, but not PR-A, exhibited defects in mammary gland branching and alveologenesis Studies of cancer cell biology (Mulac-Jericevic et al. 2003). Conversely, using knockout mouse models, PR-A has been shown to be required for PR regulation of breast cancer cell proliferation vs normal uterine development and fertility (Conneely et al. stem cell biology 2001). More recently, PRs have been found to promote Most cancer therapies were historically designed to non-proliferative phenotypes that include cell survival target rapidly dividing tumor cells. In the context of and regulation of cancer stem/stem-like cell self-renewal. ER-positive breast cancer, tumor cells are dependent on For example, progestin stimulation of PR+ mammary ER transcriptional activity to promote proliferation. Thus, epithelial cells induced transcription and secretion of endocrine therapies that target ER function (i.e. selective mitogenic factors such as Wnts, Areg, HB-EGF, and ER modulators (SERMs), selective ER degraders (SERDs), RANKL leading to proliferation of neighboring PR- cells

(Fata et al. 2001, Tanos et al. 2013). Separate studies driven secondary tumorsphere formation, an in vitro implicated these same PR signaling outputs (i.e. RANKL and readout of CSC self-renewal (Truong et al. 2019); mutation Wnt4) in the maintenance and expansion of the normal of PR Ser294 to Ala blocked these effects. These findings mammary gland stem compartment (Asselin-Labat et al. indicate that PR Ser294 phosphorylation is a crucial 2010, Joshi et al. 2010). These studies have been extended event that defines both PR-A and PR-B actions in the to show that PR regulates the breast CSC compartment regulation of CSC populations; the increased potency of (Axlund et al. 2013, Cittelly et al. 2013, Hilton et al. 2014, PR-A over PR-B as a driver of breast cancer stemness may Finlay-Schultz & Sartorius 2015, Goodman et al. 2016), be explained by its greater stability (Faivre et al. 2008) and recent work has further shown that PR isoforms have and resistance to deSUMOylation relative to PR-B (Daniel divergent roles in supporting breast CSC properties using et al. 2007). Understanding how SRs contribute to cancer ER+ breast cancer cell line models that either lack both cell fate transitions, plasticity, heterogeneity, and other PRs or express only PR-A, only PR-B, or both isoforms non-proliferative cancer cell biologies is important for (Truong et al. 2019). Measurement of breast CSC markers preventing and targeting therapy resistance. in these models indicated that although PR-A+ breast cancer cells were only weakly proliferative in soft-agar, PR signaling impacts the cell cycle they formed abundant tumorspheres that were enriched for both ALDH+ and CD44+/CD24- cell populations One functional characteristic of CSCs is the ability to relative to PR-B+ cells (which formed fewer but larger maintain a cellular quiescence, reversibly existing in spheres), suggesting a reversible phenotypic flip from the G0 phase of the cell cycle (Bighetti-Trevisan et al. pro-proliferative (PR-B-driven) to non-proliferative CSC 2019). This quiescent state allows for long-term survival (PR-A-driven) programs. Accordingly, S294A PR-A mutant in response to cellular or microenvironmental stress (i.e. models were impaired with regard to ALDH+ tumorsphere chemotherapy, hypoxia, etc.). Signaling molecules that formation, yet exhibited increased proliferation in soft regulate quiescence include cyclin-dependent kinase agar (Truong et al. 2019). These studies suggest tight (CDK) inhibitors (i.e. p21, p27, and p57), as well as the linkage between highly plastic (i.e. proliferative vs CSC) members of the FOXO family of transcription factors. Our regulated cell fates, and implicates PR isoform-specific prior work showed that PRs regulate the cyclin dependent modulation. kinase inhibitor p21 and FOXO1 to promote cellular Divergent roles of PR isoforms may be explained by senescence, the irreversible exit from the cell cycle (i.e. isoform-specific transcriptional control of overlapping permanent accumulation in G0), in ovarian cancer models but distinct gene programs which, in part, is directed by (Diep et al. 2013, 2016). Senescence, FOXO1 and p21 gene PTMs. Notably, phospho-Ser294 species are required for expression, and PR Ser294 phosphorylation were induced both PR-A- and PR-B-induced tumorsphere formation by progestin (ligand-dependent). Chemical inhibition (Knutson et al. 2017, Truong et al. 2019). However, of FOXO1 inhibited PR Ser294 phosphorylation and the phospho-PR-A is preferentially recruited to promoters of PR-dependent senescence phenotype. In these studies, CSC-associated genes (e.g. Wnt4, KLF4, and NOTCH2) PR-B was the dominant isoform required for induction relative to phospho-PR-B; PR isoform recruitment of both FOXO1 and p21 expression as well as progestin- was significantly reduced in phospho-mutant models induced cellular senescence, while PR-A repressed (i.e. S294A). Of note, the effect of PR isoforms on CSC these genes and failed to induce a senescent state upon markers and tumorsphere formation was independent treatment of ovarian cancer cells with progestins. of exogenously added progestin (i.e. ligand-independent Many of the same signaling molecules and transcription in defined media), and the addition of progestin did factors regulate both senescence and quiescence. While a not have a significant effect on tumorsphere number or direct link between PR and CSC quiescence has not yet size. In the context of endocrine therapy resistance, SRs been established in ER+/PR+ breast cancer models, it is may promote tumor progression via ligand-independent possible that context-dependent signals, such as breast vs mechanisms, resulting from the upregulation of growth ovarian cells of origin, cancer type-specific or MAPK/CDK factor and/or stress signaling pathways that occurs signaling pathway activation, and/or ligand-independent following hormone withdrawal (Nicholson et al. 2004, PR phosphorylation, will promote a PR-dependent Need et al. 2015). Relevant to this concept, EGF induces cellular quiescence that contributes to CSC survival. In MAPK-dependent and ligand-independent PR Ser294 support of this, FOXO1 expression increased PR Ser294 phosphorylation, which greatly enhanced PR-A and PR-B phosphorylation and promoted ALDH+ tumorsphere

https://jme.bioscientifica.com © 2020 Society for Endocrinology https://doi.org/10.1530/JME-19-0274 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 03:21:57AM via free access Journal of Molecular A R Dwyer, T H Truong et al. Steroid receptors as MAPK 65:1 T42 Endocrinology sensors formation in both PR-A and PR-B expressing breast cancer proteins (Wu et al. 2004, Regan Anderson et al. 2018). models (Truong et al. 2019). Accordingly, inhibition For example, GR promotes breast tumor kinase (Brk/PTK6) of PR and FOXO1 with onapristone and AS1842856, expression in response to dexamethasone (ligand- respectively, had a synergistic inhibitory effect on CSC dependent), and also in response to hypoxia, cell self-renewal in multiple breast cancer models. Future stress induced by chemotherapy, and anoikis (ligand- studies will examine the cooperative effect of PR isoforms, independent). In TNBC models, hypoxia, paclitaxel, FOXO1, and p21 on CSC quiescence/tumor cell dormancy 5-FU, and non-adherent culture all led to p38-MAPK- in ER+ breast cancer models. dependent GR Ser134 phosphorylation and ligand- independent activation of gene programs implicated in cellular stress-response (Regan Anderson et al. 2016, GR promotes triple negative breast cancer cell 2018). GR-dependent Brk/PTK6 induction protected survival, migration, and invasion cells from chemotherapy-induced cell death and As with PR isoforms, the effects of GR on breast cancer promoted cell migration (Regan Anderson et al. 2018). biology are also highly context dependent and most These studies further our mechanistic understanding relevant to non-proliferative cancer cell fates. Like PR of how GR and glucocorticoids promote plasticity expression, GR expression in ER+ luminal breast cancer in order to protect TNBC cells from cell stress and is associated with a better prognosis. In sharp contrast, chemotherapy-induced cell death and may have clinical however, high GR expression is associated with more implications for the routine use of high-dose steroids aggressive triple negative breast cancer (TNBC) biology prior to chemotherapy. and poor prognosis when compared to patients with Cells sense both local external (TME) and internal low GR expression (Pan et al. 2011). In TNBC models, stress (hypoxia) via activation of JNKs and p38 MAPKs, expression of GR and/or GR-associated target genes also historically known as stress-activated protein kinases (i.e. Brk/PTK6) have been associated with pro-survival or SAPKs (Laderoute et al. 1999, Sumbayev & Yasinska signaling, EMT, and cellular migration/invasion in vitro 2005). Notably, upregulation of the p38 MAPK signaling as well as metastasis in vivo (Lofgren et al. 2011, Regan pathway has emerged as a mechanism by which tumor Anderson et al. 2013, 2016, 2018). Recently, activation of cells increase their metastatic capacity. Mechanistic GR with glucocorticoids was shown to drive breast cancer studies have shown that p38 MAPK signaling promotes metastasis in vivo (Obradovic et al. 2019). the invasive and metastatic capacities of breast tumor Cancer cell survival or resisting cell death is an cells (Limoge et al. 2017). As discussed previously, original hallmark of cancer (Hanahan & Weinberg phosphorylation of Ser134 GR is p38 MAPK-dependent 2000). Cellular mechanisms associated with cell survival in osteosarcoma (Galliher-Beckley et al. 2011) and breast include the regulation of pro- and anti-apoptotic BH3 cancer models (Regan Anderson et al. 2016). The role of family proteins, the extrinsic death receptor signaling hypoxia/HIFs in promoting EMT and increased migration molecules, and the intrinsic caspase and apoptosome of TNBC cell models is well-established (Brooks et al. 2016, proteins that ultimately lead to cell death. Intrinsic Tirpe et al. 2019); our most recent studies indicate that activation of cell death pathways is often initiated by GR is a principle driver of TME- (i.e. cytokine) and stress- cell stress caused by DNA damage, ROS production, induced TNBC migration, specifically via p38 MAPK- or metabolic/nutrient stress. GR is a long-established dependent GR Ser134 phosphorylation (C Perez Kerkvliet sensor of physiologic (i.e. hormonal) stress, and more and CA Lange, unpublished results). While these effects recent data indicate that GR is also an exquisite sensor in TNBC cell models were ligand-independent, others of cellular stress and stress signaling within the TME. have reported that glucocorticoid treatment promoted In response to systemic glucocorticoid exposure, GR metastasis in vivo using patient-derived xenograft (PDX) is known to promote cell death in lymphocytes and models (Obradovic et al. 2019). It is important to note monocytes in order to reduce inflammation Strehl( et al. that GR activation by glucocorticoids has been shown to 2019). However, activation of GR also promotes cell suppress the migration of immune cells, highlighting the survival in a variety of epithelial cell types, including tissue- and cell-specific roles of GR/phospho-GR (Stahn in breast cancer models. In studies of TNBC, ligand- & Buttgereit 2008) and underscoring the need to select dependent and ligand-independent GR activation have patients using appropriate biomarkers of phospho-GR been shown to promote expression of pro-survival signaling in GR+ TNBC patients.

Ligand-independent SRs are important (Perez Kerkvliet et al. 2020). Thus, targeting phospho-SR therapeutic targets species may be possible using existing agents or newer, more selective SR antagonists paired with the appropriate Phospho-SR species are predicted to be valuable MEK/MAPK or CDK signaling pathway inhibitors (Table 1). biomarkers of aggressive breast cancer behavior. Detection Progestins (megestrol acetate or Megase) as well as of phosphorylated receptors or their respective target gene antiprogestins (RU486, onapristone) were effective in signatures may provide a useful means by which to further early clinical trials, particularly when paired with Tam classify more aggressive breast tumors and thus estimate or ICI 162384 (Robertson et al. 1989, 1999, Nishino et al. risk of therapy-resistant recurrence. An important caveat 1994, Klijn et al. 2000), but had significant liver toxicity, to targeting phospho-SR species is that these species likely due, in part, to cross-reactivity with ubiquitously may be insensitive to existing receptor antagonists; expressed GRs. This problem may be overcome by use of phosphorylation events confer partial agonist activity lower dose or timed-release antiprogestin preparations, to SR antagonists (Wardell et al. 2010) and phospho-ER as in the case of Apristor (Context Therapeutics, Inc), a species are strongly implicated in the development of clinical formulation of onapristone. Historical clinical tamoxifen resistance (Likhite et al. 2006, Kastrati et al. studies that clearly implicated progestins (namely MPA, 2019). Notably, RU486 (an antagonist for both PR and a PR agonist) in elevated breast cancer risk (Rossouw GR) does not significantly reduce ligand-independent et al. 2002, MWS 2003, Soini et al. 2016) have renewed phosphorylation of PR or GR. However, at least in vitro, interest in targeting PRs as a part of combination ligand-independent actions of phospho-SRs that are endocrine therapies. As a result, selective antiprogestins not further stimulated by agonists are often sensitive to (onapristone and telapristone acetate) have re-entered selected antagonists. For example, ER-scaffolding actions Phase I-II trials with encouraging results (Cottu et al. 2018, of PR-B appear to be insensitive to type I antagonists (i.e. Lee et al. 2020). Similarly, GR antagonists (i.e. RU486) are RU486) that induce PR Ser294 phosphorylation levels being tested in TNBC clinical trials (Nanda et al. 2016). similar to that of PR agonists (Daniel et al. 2015), but are Of concern is that the science of PR and GR (i.e. in breast blocked by type II antagonists (i.e. onapristone) that also cancer models), while growing, lags far behind that of ER block ligand-induced PR Ser294 phosphorylation (Beck or even AR and fewer ligands are available for pre-clinical et al. 1996). Similarly, high basal TNBC cell migration is modeling of PR or GR-driven actions relative to a wealth only weakly stimulated by the GR agonist dexamethasone, of selective receptor modulators for ER or AR. Before but completely blocked by the GR antagonist, RU486 selective SR modulators can be successfully combined

Table 1 PR and GR post-translational modifications and associated ligand-dependent (LD) and ligand-independent (LI) biological outcomes.

Steroid Kinase receptor PTM pathway Biological outcome PR S81 CK2 Proliferation, survival, migration, and transcriptional regulation (Hagan et al. 2011) PR S294 (Phosphorylation) MAPK, CDK Nuclear localization (LI; Qui & Lange 2003), turnover, transcriptional regulation (LD; Daniel & Lange 2009, Shen et al. 2001), proliferation (Daniel et al. 2007), stemness (Truong et al. 2019), senescence (Diep et al. 2013), and cell cycle progression (Moore et al. 2001) PR S345 (Phosphorylation) MAPK, CDK2 Proliferation, migration (Faivre et al. 2008, Dressing et al. 2014) PR K388 (SUMOylation) – Transcriptional regulation and slows PR turnover (Abdel-Hafizet al. 2002, Daniel et al. 2007) PR S400 (Phosphorylation) CDK2 Cell cycle progression and LI transcription, (Zhang et al. 1997, Pierson- Mullany & Lange 2004, Wardell et al. 2010) PR S676 (Phosphorylation) CDK2 Transcriptional activity (Knotts et al. 2001) PR K638-K641 (KxKK, Acetylation – Disrupts nuclear translocation and delays MAPK-induced Ser345 and consensus sequence) Ser294 phosphorylation (Daniel et al. 2010) GR S134 (Phosphorylation) p38 Response to stress-activating stimuli/p38 MAPK, hypoxia (Regan Anderson et al. 2016), stress-induced survival, migration, and stemness (Perez Kerkvliet et al. 2020) GR S211 (Phosphorylation) p38 Apoptosis (LD; Khan et al. 2017) GR S404 (Phosphorylation) GSK-3 Turnover and resistance to LD apoptosis (Galliher-Beckley et al. 2008)

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Received in final form 16 March 2020 Accepted 25 March 2020 Accepted Manuscript published online 25 March 2020