Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer

Clinical resistance to the second-generation antiandrogen enzalutamide in castration resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights the unmet medical need for next generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide), and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR:CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm bound to heat shock proteins. Thus, we have identified third generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC

Partial Agonist Activity of the Progesterone Receptor Antagonist RU486 Mediated by an Amino-Terminal Domain Coactivator and Phosphorylation of Serine400

Jun dimerization protein-2 (JDP-2) is a progesterone receptor (PR) coregulatory protein that acts by inducing structure and transcriptional activity in the disordered amino-terminal domain (NTD) of PR. JDP-2 can also potentiate the partial agonist activity of the PR antagonist RU486 by mechanisms that have not been defined. Functional mutagenesis experiments revealed that a subregion of the NTD (amino acids 323-427) was required for the partial agonist activity of RU486 induced by PR interaction with JDP-2. However, this subregion was not required for JDP-2 enhancement of the activity of progestin agonists. Mutation of phosphorylation sites within this region of the NTD showed that phosphorylation of serine 400 was required for the partial agonist activity of RU486 stimulated by JDP-2, but was not required for activity of hormone agonist, either in the presence or absence of JDP-2. Cyclin-dependent kinase 2 (Cdk2)/cyclin A is a novel PR coregulator that binds the NTD and acts by phosphorylating steroid receptor coactivator-1 and modulating steroid receptor coactivator-1 interaction with PR. Cdk2/cyclin A also potentiated the partial agonist activity of RU486; however, phosphorylation of serine 400 was not required, indicating that JDP-2 and Cdk2/cyclin A act by distinct mechanisms. We conclude that PR bound to RU486 and associated with JDP-2 adopts an active conformation in a subregion of the NTD requiring phosphorylation of serine 400 that is distinct from that promoted by progestin agonists. These data underscore the structural flexibility of the NTD of PR, and the ability of steroid ligands together with interacting proteins to affect the conformation and activity of the NTD

Jun Dimerization Protein 2 Functions as a Progesterone Receptor N-Terminal Domain Coactivator

The progesterone receptor (PR) contains two transcription activation function (AF) domains, constitutive AF-1 in the N terminus and AF-2 in the C terminus. AF-2 activity is mediated by a hormone-dependent interaction with a family of steroid receptor coactivators (SRCs). SRC-1 can also stimulate AF-1 activity through a secondary domain that interacts simultaneously with the primary AF-2 interaction site. Other protein interactions and mechanisms that mediate AF-1 activity are not well defined. By interaction cloning, we identified an AP-1 family member, Jun dimerization protein 2 (JDP-2), as a novel PR-interacting protein. JDP-2 was first defined as a c-Jun interacting protein that functions as an AP-1 repressor. PR and JDP-2 interact directly in vitro through the DNA binding domain (DBD) of PR and the basic leucine zipper (bZIP) region of JDP-2. The two proteins also physically associate in mammalian cells, as detected by coimmunoprecipitation, and are recruited in vivo to a progesterone-inducible target gene promoter, as detected by a chromatin immunoprecipitation (ChIP) assay. In cell transfection assays, JDP-2 substantially increased hormone-dependent PR-mediated transactivation and worked primarily by stimulating AF-1 activity. JDP-2 is a substantially stronger coactivator of AF-1 than SRC-1 and stimulates AF-1 independent of SRC-1 pathways. The PR DBD is necessary but not sufficient for JDP-2 stimulation of PR activity; the DBD and AF-1 are required together. JDP-2 lacks an intrinsic activation domain and makes direct protein interactions with other coactivators, including CBP and p300 CBP-associated factor (pCAF), but not with SRCs. These results indicate that JDP-2 stimulates AF-1 activity by the novel mechanism of docking to the DBD and recruiting or stabilizing N-terminal PR interactions with other general coactivators. JDP-2 has preferential activity on PR among the nuclear receptors tested and is expressed in progesterone target cells and tissues, suggesting that it has a physiological role in PR function

Regulation of the Amino-Terminal Transcription Activation Domain of Progesterone Receptor by a Cofactor-Induced Protein Folding Mechanism

We previously identified a small basic leucine zipper (bZIP) protein, Jun dimerization protein 2 (JDP-2), that acts as a coregulator of the N-terminal transcriptional activation domain of progesterone receptor (PR). We show here that JDP-2, through interaction with the DNA binding domain (DBD), induces or stabilizes structure in the N-terminal domain in a manner that correlates with JDP-2 stimulation of transcriptional activity. Circular dichroism spectroscopy experiments showed that JDP-2 interaction caused a significant increase in overall helical content of a two-domain PR polypeptide containing the N-terminal domain and DBD and that the change in structure resides primarily in the N-terminal domain. Thermal melt curves showed that the JDP-2/PR complex is significantly more stable than either protein alone, and partial proteolysis confirmed that JDP-2 interaction alters conformation of the N-terminal domain of PR. Functional analysis of N-terminal domain mutants and receptor chimeras provides evidence that the stimulatory effect of JDP-2 on transcriptional activity of PR is mediated through an interdomain communication between the DBD and the N-terminal domain and that transcriptional activity and functional response to JDP-2 are mediated by multiple elements of the N-terminal domain as opposed to a discrete region

Development and Characterization of a Luciferase Labeled, Syngeneic Murine Model of Ovarian Cancer

Despite advances in surgery and targeted therapies, the prognosis for women with high-grade serous ovarian cancer remains poor. Moreover, unlike other cancers, immunotherapy has minimally impacted outcomes in patients with ovarian cancer. Progress in this regard has been hindered by the lack of relevant syngeneic ovarian cancer models to study tumor immunity and evaluate immunotherapies. To address this problem, we developed a luciferase labeled murine model of high-grade serous ovarian cancer, STOSE.M1 luc. We defined its growth characteristics, immune cell repertoire, and response to anti PD-L1 immunotherapy. As with human ovarian cancer, we demonstrated that this model is poorly sensitive to immune checkpoint modulators. By developing the STOSE.M1 luc model, it will be possible to probe the mechanisms underlying resistance to immunotherapies and evaluate new therapeutic approaches to treat ovarian cancer