In a little more than 10 years, nuclear receptor (NR) coregulators (coactivators and corepressors) have contributed to our present realization that a great level of sophistication exists in transcriptional regulation. Here, we discuss the implications of coregulators as versatile regulatory agents, influencing not only transcriptional initiation but also elongation, splicing, and translation. In addition to this, there is an increasing recognition that they also regulate a variety of biological processes outside of the nucleus. An important concept that we wish to emphasize is that coregulators are both targets and propagators of posttranslational modification (PTM) codes. This underlies a sophisticated epigenetic regulatory scheme from which a complex and dynamic mammalian phenotype emanates.

Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter in vitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain "poised" for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription.

Steroid receptor coactivator 3 (SRC-3/NCoA3/AIB1), is a key regulator of gene transcription and it plays a central role in breast cancer (BC) tumorigenesis, making it a potential therapeutic target. Beyond its function as an important regulator of estrogen receptor transcriptional activity, SRC-3 also functions as a coactivator for a wide range of other transcription factors, suggesting SRC-3 inhibition can be beneficial in hormone-independent cancers as well. The recent discovery of a potent SRC-3 small molecule inhibitor, SI-2, enabled the further development of additional related compounds. SI-12 is an improved version of SI-2 that like SI-2 has anti-proliferative activity in various cancer types, including BC. Here, we sought to identify gene targets, that when inhibited in the presence of SI-12, would lead to enhanced BC cell cytotoxicity. We performed a genome-scale CRISPR-Cas9 screen in MCF-7 BC cells under conditions of pharmacological pressure with SI-12. A parallel screen was performed with an ER inhibitor, fulvestrant, to shed light on both common and distinct activities between SRC-3 and ERα inhibition. Bearing in mind the key role of SRC-3 in tumorigenesis of other types of cancer, we extended our study by validating potential hits identified from the MCF-7 screen in other cancer cell lines.

We report the quaternary structure of core transcriptional complex for the full-length human progesterone receptor-B (PR-B) homodimer with primary coactivator steroid receptor coactivator-2 (SRC-2) and the secondary coactivator p300/CREB-binding protein (CBP). The PR-B homodimer engages one SRC-2 mainly through its activation function 1 (AF1) in N-terminus. SRC-2 is positioned between PR-B and p300 leaving space for direct interaction between PR-B and p300 through PR-B's C-terminal AF2 and its unique AF3. Direct AF3/p300 interaction provides long-desired structural insights into the known functional differences between PR-B and the PR-A isoform lacking AF3. We reveal the contributions of each AF and demonstrate their structural basis in forming the PR-B dimer interface and PR-B/coactivator complex. Comparison of the PR-B/coactivator complex with other steroid receptor (estrogen receptor and androgen receptor) complexes also shows that each receptor has its unique mechanism for recruiting coactivators due to the highly variable N-termini among receptors.

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were 'permanently eradicated' in a genetically engineered tamoxifen-inducible Treg-cell specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the Chemokine (C-C motif) ligand (Ccl) 19/Ccl21/ Chemokine (C-C motif) Receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C Motif Chemokine Ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and Natural Killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish pre-established breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3 deleted Tregs represents a novel approach to completely block tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators.

Introduction: Pathologic remodeling of the brain following ischemic stroke results in neuronal loss, increased inflammation, oxidative stress, astrogliosis, and a progressive decrease in brain function. We recently demonstrated that stimulation of steroid receptor coactivator 3 with the small-molecule stimulator MCB-613 improves cardiac function in a mouse model of myocardial ischemia. Since steroid receptor coactivators are ubiquitously expressed in the brain, we reasoned that an MCB-613 derivative (MCB-10-1), could protect the brain following ischemic injury. To test this, we administered MCB-10-1 to rats following middle cerebral artery occlusion and reperfusion. Methods: Neurologic impairment and tissue damage responses were evaluated on day 1 and day 4 following injury in rats treated with control or 10-1. Results: We show that 10-1 attenuates injury post-stroke. 10-1 decreases infarct size and mitigates neurologic impairment. When given within 30 min post middle cerebral artery occlusion and reperfusion, 10-1 induces lasting protection from tissue damage in the ischemic penumbra concomitant with: (1) promotion of reparative microglia; (2) an increase in astrocyte NRF2 and GLT-1 expression; (3) early microglia activation; and (4) attenuation of astrogliosis. Discussion: Steroid receptor coactivator stimulation with MCB-10-1 is a potential therapeutic strategy for reducing inflammation and oxidative damage that cause neurologic impairment following an acute ischemic stroke.

Members of the steroid receptor coactivator (SRC) family are overexpressed in numerous types of cancers. In particular, steroid receptor coactivator 3 (SRC-3) has been recognized as a critical coactivator associated with tumor initiation, progression, recurrence, metastasis, and chemoresistance where it interacts with multiple nuclear receptors and other transcription factors to enhance their transcriptional activities and facilitate cross-talk between pathways that stimulate cancer progression. Because of its central role as an integrator of growth signaling pathways, development of small molecule inhibitors (SMIs) against SRCs have the potential to simultaneously disrupt multiple signal transduction networks and transcription factors involved in tumor progression. Here, high-throughput screening was performed to identify compounds able to inhibit the intrinsic transcriptional activities of the three members of the SRC family. Verrucarin A was identified as a SMI that can selectively promote the degradation of the SRC-3 protein, while affecting SRC-1 and SRC-2 to a lesser extent and having no impact on CARM-1 and p300 protein levels. Verrucarin A was cytotoxic toward multiple types of cancer cells at low nanomolar concentrations, but not toward normal liver cells. Moreover, verrucarin A was able to inhibit expression of the SRC-3 target genes MMP2 and MMP13 and attenuated cancer cell migration. We found that verrucarin A effectively sensitized cancer cells to treatment with other anti-cancer drugs. Binding studies revealed that verrucarin A does not bind directly to SRC-3, suggesting that it inhibits SRC-3 through its interaction with an upstream effector. In conclusion, unlike other SRC SMIs characterized by our laboratory that directly bind to SRCs, verrucarin A is a potent and selective SMI that blocks SRC-3 function through an indirect mechanism.

Approximately 75% of breast cancers are estrogen receptor alpha (ERα)-positive and are treatable with endocrine therapies, but often patients develop lethal resistant disease. Frequent mutations (10-40%) in the ligand-binding domain (LBD) codons in the gene encoding ERα (ESR1) have been identified, resulting in ligand-independent, constitutively active receptors. In addition, ESR1 chromosomal translocations can occur, resulting in fusion proteins that lack the LBD and are entirely unresponsive to all endocrine treatments. Thus, identifying coactivators that bind to these mutant ERα proteins may offer new therapeutic targets for endocrine-resistant cancer. To define coactivator candidate targets, a proteomics approach was performed profiling proteins recruited to the two most common ERα LBD mutants, Y537S and D538G, and an ESR1-YAP1 fusion protein. These mutants displayed enhanced coactivator interactions as compared to unliganded wild-type ERα. Inhibition of these coactivators decreased the ability of ESR1 mutants to activate transcription and promote breast cancer growth in vitro and in vivo. Thus, we have identified specific coactivators that may be useful as targets for endocrine-resistant breast cancers.

About 200 coactivators play a central role in promoting gene expression mediated by nuclear receptors. This diverse group of proteins are key integrators of signals from steroid hormones and have been implicated in cancer and other diseases.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR -mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR -mutant NSCLC cells.

Enhancers not only activate target promoters to stimulate messenger RNA (mRNA) synthesis, but they themselves also undergo transcription to produce enhancer RNAs (eRNAs), the significance of which is not well understood. Transcription at the participating enhancer-promoter pair appears coordinated, but it is unclear why and how. Here, we employ cell-free transcription assays using constructs derived from the human GREB1 locus to demonstrate that transcription at an enhancer and its target promoter is interdependent. This interdependence is observable under conditions where direct enhancer-promoter contact (EPC) takes place. We demonstrate that transcription activation at a participating enhancer-promoter pair is dependent on i) the mutual availability of the enhancer and promoter, ii) the state of transcription at both the enhancer and promoter, iii) local abundance of both eRNA and mRNA, and iv) direct EPC. Our results suggest transcriptional interdependence between the enhancer and the promoter as the basis of their transcriptional concurrence and coordination throughout the genome. We propose a model where transcriptional concurrence, coordination and interdependence are possible if the participating enhancer and promoter are entangled in the form of EPC, reside in a proteinaceous bubble, and utilize shared transcriptional resources and regulatory inputs.

Triple negative breast cancer (TNBC) has the poorest prognosis of all types of breast cancer and currently lacks efficient targeted therapy. Chemotherapy is the traditional standard-of-care for TNBC, but is frequently accompanied by severe side effects. Despite the fact that high expression of steroid receptor coactivator 3 (SRC-3) is correlated with poor survival in estrogen receptor positive breast cancer patients, its role in TNBC has not been extensively investigated. Here, we show that high expression of SRC-3 correlates with both poor overall survival and post progression survival in TNBC patients, suggesting that SRC-3 can serve as a prognostic marker for TNBC. Furthermore, we demonstrated that bufalin, a SRC-3 small molecule inhibitor, when introduced even at nM concentrations, can significantly reduce TNBC cell viability and motility. However, because bufalin has minimal water solubility, its in vivo application is limited. Therefore, we developed a water soluble prodrug, 3-phospho-bufalin, to facilitate its in vivo administration. In addition, we demonstrated that 3-phospho-bufalin can effectively inhibit tumor growth in an orthotopic TNBC mouse model, suggesting its potential application as a targeted therapy for TNBC treatment.

In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

The impact of chemotherapy on tumor-immune system interaction can be either beneficial or harmful, which is represented by the immunogenic cell death (ICD) paradigm or overexpression of the immunosuppressive protein - programmed death ligand 1 (PD-L1). In this study we explore the impact of steroid receptor coactivator inhibitor, other targeted anti-cancer compounds and traditional chemotherapeutic agents on the expression of PD-L1 in four breast cancer (BC) cell lines. Our results show that these agents induce PD-L1 expression, yet the magnitude of this induction varies substantially across the different compounds. In addition, we utilized the E0771 ER + BC cells as a model to examine in greater detail the relationship between pharmacological pressure, cell stress and the induction of PD-L1. Our results imply that drug induced PD-L1 expression occurs in the broader context of cell-stress, without conferring acquired drug-resistance. Furthermore, a balance between BC cytotoxicity, induction of cell-stress and the overexpression of PD-L1 can be achieved through the selection of appropriate combinations of anti-cancer compounds. Therefore, we propose that drug combination can be employed not only for increasing the direct kill of cancer cells, but also as a strategy to minimize the activation of immunosuppressive and cancer cell pro-survival program responses during drug treatment.

Development of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid-liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

A distinct 12-hour clock exists in addition to the 24-hour circadian clock to coordinate metabolic and stress rhythms. Here, we show that liver-specific ablation of X-box binding protein 1 (XBP1) disrupts the hepatic 12-hour clock and promotes spontaneous non-alcoholic fatty liver disease (NAFLD). We show that hepatic XBP1 predominantly regulates the 12-hour rhythmicity of gene transcription in the mouse liver and demonstrate that perturbation of the 12-hour clock, but not the core circadian clock, is associated with the onset and progression of this NAFLD phenotype. Mechanistically, we provide evidence that the spliced form of XBP1 (XBP1s) binds to the hepatic 12-hour cistrome to directly regulate the 12-hour clock, with a periodicity paralleling the harmonic activation of the 12-hour oscillatory transcription of many rate-limiting metabolic genes known to have perturbations in human metabolic disease. Functionally, we show that Xbp1 ablation significantly reduces cellular membrane fluidity and impairs lipid homeostasis via rate-limiting metabolic processes in fatty acid monounsaturated and phospholipid remodeling pathways. These findings reveal that genetic disruption of the hepatic 12-hour clock links to the onset and progression of NAFLD development via transcriptional regulator XBP1, and demonstrate a role for XBP1 and the 12-hour clock in the modulation of phospholipid composition and the maintenance of lipid homeostasis.

Speckle-type poz protein (SPOP) is an E3-ubiquitin ligase adaptor for turnover of a diverse number of proteins involved in key cellular processes such as chromatin remodeling, transcriptional regulation, and cell signaling. Genomic analysis revealed that SPOP somatic mutations are found in a subset of endometrial cancers, suggesting that these mutations act as oncogenic drivers of this gynecologic malignancy. These studies also raise the question as to the role of wild-type SPOP in normal uterine function. To address this question, we generated a mouse model (Spopd/d) in which SPOP is ablated in uterine cells that express the PGR. Fertility studies demonstrated that SPOP is required for embryo implantation and for endometrial decidualization. Molecular analysis revealed that expression levels of the PGR at the protein and transcript level are significantly reduced in the Spopd/d uterus. While this result was unexpected, this finding explains in part the dysfunctional phenotype of the Spopd/d uterus. Moderate increased levels of the ESR1, GATA2, and SRC2 were detected in the Spopd/d uterus, suggesting that SPOP is required to maintain the proteome for normal uterine function. With age, the Spopd/d endometrium exhibits large glandular cysts with foci of epithelial proliferation, further supporting a role for SPOP in maintaining a healthy uterus. Collectively, studies on the Spopd/d mouse support an important role for SPOP in normal uterine function and suggest that this mouse model may prove useful to study the role of SPOP-loss-of-function mutations in the etiopathogenesis of endometrial cancer.

A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were "permanently eradicated" in a genetically engineered tamoxifen-inducible Treg-cell-specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the chemokine (C-C motif) ligand (Ccl) 19/Ccl21/chemokine (C-C motif) receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C motif chemokine ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and natural killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish preestablished breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3-deleted Tregs represents an approach to completely block tumor growth and recurrence without the autoimmune side effects that typically accompany immune checkpoint modulators.

A subset of CD4 + lymphocytes, regulatory T cells (Tregs), are necessary for central tolerance and function as suppressors of autoimmunity against self-antigens. The SRC-3 coactivator is an oncogene in multiple cancers and is capable of potentiating numerous transcription factors in a wide variety of cell types. Src-3 knockout mice display broad lymphoproliferation and hypersensitivity to systemic inflammation. Using publicly available bioinformatics data and directed cellular approaches, we show that SRC-3 also is highly enriched in Tregs in mice and humans. Human Tregs lose phenotypic characteristics when SRC-3 is depleted or pharmacologically inhibited, including failure of induction from resting T cells and loss of the ability to suppress proliferation of stimulated T cells. These data support a model for SRC-3 as a coactivator that actively participates in protection from autoimmunity and may support immune evasion of cancers by contributing to the biology of Tregs.