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Sequences of siRNAs and primers and probes for qPCR

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Correlation analysis between NCOR1 and NCOR2 expression and AR transcriptional output

Purpose: Castration therapy in advanced prostate cancer eventually fails and leads to the development of castration-resistant prostate cancer (CRPC), which has no cure. Characteristic features of CRPC can be increased androgen receptor (AR) expression and altered transcriptional output. We investigated the expression of nuclear receptor corepressor 1 (NCOR1) in human prostate and prostate cancer and the role of NCOR1 in response to antiandrogens.Experimental Design: NCOR1 protein levels were compared between matched normal prostate and prostate cancer in 409 patient samples. NCOR1 knockdown was used to investigate its effect on bicalutamide response in androgen-dependent prostate cancer cell lines and transcriptional changes associated with the loss of NCOR1. NCOR1 transcriptional signature was also examined in prostate cancer gene expression datasets.Results: NCOR1 protein was detected in cytoplasm and nuclei of secretory epithelial cells in normal prostate. Both cytoplasmic and nuclear NCOR1 protein levels were lower in prostate cancer than in normal prostate. Prostate cancer metastases show significant decrease in NCOR1 transcriptional output. Inhibition of LNCaP cellular proliferation by bicalutamide requires NCOR1. NCOR1-regulated genes suppress cellular proliferation and mediate bicalutamide resistance. In the mouse, NCOR1 is required for bicalutamide-dependent regulation of a subset of the AR target genes.Conclusions: In summary, we demonstrated that NCOR1 function declines with prostate cancer progression. Reduction in NCOR1 levels causes bicalutamide resistance in LNCaP cells and compromises response to bicalutamide in mouse prostate in vivo. Clin Cancer Res; 22(15); 3937–49. ©2016 AACR.

Average mouse body weight for Figure 5; PD analyses of C86 +/- JG98 in vivo; in vitro effects of C86 +/- JG98 on other client proteins

DHT regulated pathways

NCOR1 regulated pathways

AR signature in LNCaP cells

NCOR1 signature

SIAH2 signature in LNCaP cells

Castration-resistant prostate cancer (CRPC) is characterized by reactivation of androgen receptor (AR) signaling, in part by elevated expression of AR splice variants (ARv) including ARv7, a constitutively active, ligand binding domain (LBD)-deficient variant whose expression has been correlated with therapeutic resistance and poor prognosis. In a screen to identify small-molecule dual inhibitors of both androgen-dependent and androgen-independent AR gene signatures, we identified the chalcone C86. Binding studies using purified proteins and CRPC cell lysates revealed C86 to interact with Hsp40. Pull-down studies using biotinylated-C86 found Hsp40 present in a multiprotein complex with full-length (FL-) AR, ARv7, and Hsp70 in CRPC cells. Treatment of CRPC cells with C86 or the allosteric Hsp70 inhibitor JG98 resulted in rapid protein destabilization of both FL-AR and ARv, including ARv7, concomitant with reduced FL-AR- and ARv7-mediated transcriptional activity. The glucocorticoid receptor, whose elevated expression in a subset of CRPC also leads to androgen-independent AR target gene transcription, was also destabilized by inhibition of Hsp40 or Hsp70. In vivo, Hsp40 or Hsp70 inhibition demonstrated single-agent and combinatorial activity in a 22Rv1 CRPC xenograft model. These data reveal that, in addition to recognized roles of Hsp40 and Hsp70 in FL-AR LBD remodeling, ARv lacking the LBD remain dependent on molecular chaperones for stability and function. Our findings highlight the feasibility and potential benefit of targeting the Hsp40/Hsp70 chaperone axis to treat prostate cancer that has become resistant to standard antiandrogen therapy.Significance:These findings highlight the feasibility of targeting the Hsp40/Hsp70 chaperone axis to treat CRPC that has become resistant to standard antiandrogen therapy. Cancer Res; 78(14); 4022–35. ©2018 AACR.

Supplementary Figure 1 Comparison of NCOR1 and NCOR2 knockdown in LAPC4 and LNCaP cells Supplementary Figure 2 NCOR1 regulates motility of LNCaP cells Supplementary Figure 3 AR signature is present among bicalutamide treated genes in both control and NCOR1 depleted cells Supplementary Figure 4 Comparative levels of UGT2B15 and UGT2B17 mRNA expression in LNCaP and C4-2 cells Supplementary Figure 5 Loss of both UGT2B 15 and UGT2B 17 lead to elevated DHT-dependent PSA expression. Supplementary Figure 6 NCOR1 is required for optimal TARP and CHEK1 expression in LAPC4 cells Supplementary Figure 7 AR is recruited to PSA enhancer and INPP4B promoter after NCOR1 depletion Supplementary Figure 8 Examples of variable NCOR1 staining in normal prostate epithelium, PIN, and prostate cancer on tissue microarray.

Supplementary Figure 1 from Androgens Modulate Expression of Transcription Intermediary Factor 2, an Androgen Receptor Coactivator whose Expression Level Correlates with Early Biochemical Recurrence in Prostate Cancer

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

Steroid hormone receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [74, 215, 3]) are nuclear hormone receptors of the NR3 class, with endogenous agonists that may be divided into 3-hydroxysteroids (estrone and 17β-estradiol) and 3-ketosteroids (dihydrotestosterone [DHT], aldosterone, cortisol, corticosterone, progesterone and testosterone). For rodent GR and MR, the physiological ligand is corticosterone rather than cortisol.

Androgen receptor (AR) signaling continues to drive castration-resistant prostate cancer (CRPC) in spite of androgen deprivation therapy (ADT). Constitutively active shorter variants of AR, lacking the ligand binding domain, are frequently expressed in CRPC and have emerged as a potential mechanism for prostate cancer to escape ADT. ARv7 and ARv567es are 2 of the most commonly detected variants of AR in clinical samples of advanced, metastatic prostate cancer. It is not clear if variants of AR merely act as weaker substitutes for AR or can mediate unique isoform-specific activities different from AR. In this study, we employed LNCaP prostate cancer cell lines with inducible expression of ARv7 or ARv567es to delineate similarities and differences in transcriptomics, metabolomics, and lipidomics resulting from the activation of AR, ARv7, or ARv567es. While the majority of target genes were similarly regulated by the action of all 3 isoforms, we found a clear difference in transcriptomic activities of AR versus the variants, and a few differences between ARv7 and ARv567es. Some of the target gene regulation by AR isoforms was similar in the VCaP background as well. Differences in downstream activities of AR isoforms were also evident from comparison of the metabolome and lipidome in an LNCaP model. Overall our study implies that shorter variants of AR are capable of mediating unique downstream activities different from AR and some of these are isoform specific.

Prostate Cancer (PCa) is an androgen dependent disease and patients with metastatic PCa are treated with androgen deprivation therapy (ADT). Although most tumors respond initially, tumors become resistant and are termed castration resistant prostate cancer (CRPC). There is compelling evidence that most of these tumors retain androgen receptor (AR) dependence and some data to suggest that, in some cases, the glucocorticoid receptor (GR) substitutes for AR. AR, itself, is re-activated through a variety of mechanisms including the expression of constitutively active AR splice variants that lack the ligand binding domain (LBD) of AR. Expression of one variant, AR-V7, which contains the amino-terminal domain and DNA binding domain of AR and 16 unique amino-acids, has been correlated with resistance to second line ADT. Although there has been some debate regarding the role of AR-V7, whether it is only a partial substitute for AR or has unique activities, our studies of engineered cell lines treated to express levels of AR-V7 equivalent to AR, clearly show that while the AR isoforms have common targets, they each also have unique targets. Consistent with this, the cistromes of the two show many unique sites as well as common sites. AR-V7 binding is enriched near the transcription start site (TSS) and we have identified a novel de novo binding motif. These findings suggest the possibility of developing a gene signature unique to AR-V7.Because GR activity in PCa has also been suggested as an escape mechanism in response to ADT, and GR binds to the same consensus response elements, we sought to identify a GR signature in PCa, to compare it with the AR and AR-V7 signatures, and to ask whether the AR-V7 and/or GR signatures are enriched in CRPC. Because much of the gene signatures are cell line dependent, we sought to compare GR and AR-V7 action in cells that express both. LN-95 cells express both AR-V7 and GR. MDA-PCa-2b cells, a cell line derived from an African American patient, expresses GR, but not AR-V7. The parental line was infected with a lentivirus that expresses AR-V7 in response to doxycycline. Transcriptomes were determined for AR, GR, and AR-V7 in these lines using RNA-Seq. Overall the magnitude of regulation of gene expression was generally lower than in the LNCaP AR-V7 and VCaP-AR-V7 lines, but there was good overlap of the MDA-PCa-2b AR-V7 regulated with the LNCaP AR-V7 regulated genes. Genes induced by GR overlapped with AR and isoform common genes, but did not overlap with AR-V7 specific genes. A comparison of AR-V7 specific genes common to the LNCaP and VCaP models as well as to publicly available data sets for LN-95 and 22RV1 AR-V7 signatures, show a strong correlation with CRPC compared to primary tumors when analyzed in the Grasso data set.

Activation and transcriptional reprogramming of AR in advanced prostate cancer frequently coincides with the loss of two tumor suppressors, INPP4B and PTEN, which are highly expressed in human and mouse prostate epithelium. While regulation of AR signaling by PTEN has been described by multiple groups, it is not known whether the loss of INPP4B affects AR activity. Using prostate cancer cell lines, we showed that INPP4B regulates AR transcriptional activity and the oncogenic signaling pathways Akt and PKC. Analysis of gene expression in prostate cancer patient cohorts showed a positive correlation between INPP4B expression and both AR mRNA levels and AR transcriptional output. Using an Inpp4b−/− mouse model, we demonstrated that INPP4B suppresses Akt and PKC signaling pathways and modulates AR transcriptional activity in normal mouse prostate. Remarkably, PTEN protein levels and phosphorylation of S380 were the same in Inpp4b−/− and WT males, suggesting that the observed changes were due exclusively to the loss of INPP4B. Our data show that INPP4B modulates AR activity in normal prostate and its loss contributes to the AR-dependent transcriptional profile in prostate cancer.

Background: Recurrences are delayed for years with endocrine therapy (ET), but resistance ultimately evolves with the development of ER+ metastatic breast cancer (MBC). ESR1 mutations are acquired in MBC undergoing ET in about 20-40% of patients. We have previously demonstrated that AR overexpression confers resistance to both tamoxifen and aromatase inhibitor treatments. We discovered that AR protein is up-regulated in tumors expressing ESR1 mutations, and herein explore the functional consequences of AR-ESR1 mutant co-expression on metastatic progression of breast cancer. Objective: We hypothesize that ET resistance mechanisms driven by AR overexpression and acquisition of ESR1 mutations co-evolve under the selective pressure of therapy to drive distant metastasis. We investigated whether AR engages in novel genomic binding interactions with mutant ER to promote tumor progression. Methods: Expression profiling of CRISPR-Cas9 engineered cell lines was integrated with chromatin immunoprecipitation-sequencing (ChIP-Seq) to define the genomic distribution of ER and AR binding sites. Genomic distribution of AR-ER co-occupied binding sites from MBC patient tumors, and a gene expression signature associated with patient outcomes was generated using using Cox Regression analysis to calculate Hazard Ratios for recurrence free survival from gene expression in the KMPlotter Breast Cancer Dataset and signature validation by Kaplan Meier analysis in the ER+ METABRIC patient cohort. Results: AR protein was post-transcriptionally elevated in ESR1 mutant cells, including distant metastatic tumors from ESR1 mutant xengraft models. Transcriptome data showed significant elevation of Cancer Hallmark pathways, including epithelial-mesenchymal transition (EMT) and estrogen response, but down-regulation of androgen response genes. However, both ER and AR functions were constitutively activated, independent of their respective ligands in ESR1 mutant-expressing models. AR was disproportionally re-distributed to selected enhancer genomic regions, but also heterochromatin in ESR1 mutant compared to cells with WT ER in the presence of estrogen. In addition, AR-ER co-occupied sites were increased in enhancer regions in ESR1 mutant cells, and these sites best discriminated distal ER enhancer regions in non-responder patients and MBC. . The expression of genes co-bound by AR and ER were significantly associated with shorter recurrence-free and overall survival in ER+ breast cancer. Treatment with AR agonists blocked distant metastasis of ESR1 mutant PDX models in vivo, in part due to reduction in ER protein and ER binding to select genomic sites. Conclusions: Our results demonstrate that unliganded AR collaborates with ESR1 mutations to regulate genes associated with the metastasis of ER+ breast cancer. We hypothesize that in an ESR1 mutant genomic background, AR acquires ER co-activator functions that are blocked with AR agonists, suggesting a unique hormonal therapeutic vulnerability in tumors with ESR1 mutations. Citation Format: Suzanne Fuqua, Thomas L Gonzalez, Guowei Gu, Sarah Herzog, Yassine Rechoum, Amanda Beyer, David Edwards, Hangqing Lin, BAsil Paul, Ralf Kittler, Nancy Weigel, Sandra Grimm, Cristian Coarfa, Tasneem Bawa-Khalfe. Androgen and ESR1 mutant receptors mechanistically collaborate for overexpression of genes associated with poor outcomes in ER-positive metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-09.

The androgen receptor (AR) is a hormone activated transcription factor which is important for the growth and progression of prostate cancer (PCa). Metastatic PCa is treated with androgen deprivation therapy (ADT) based regimen but eventually, these tumours develop resistance through multiple mechanisms that reactivate AR. These include expression of constitutively active AR splice variants that lack a ligand binding domain such as AR-V7. To characterize the common and unique activities of AR and AR-V7, we engineered LNCaP and VCaP cell lines that express AR-V7 in response to doxycycline and performed RNA-Seq. Many genes were found to be regulated by both AR and AR-V7, but there were also genes regulated by only one isoform. Moreover, both isoforms altered mRNA splicing of numerous target genes, in some cases without altering overall levels of mRNA. In many cases, the alterations in splicing are AR isoform specific. To understand the differential splicing by AR isoforms, CD44 mini-gene splicing assay and studies of endogenous target genes are being done. The CD44 mini-gene contains an MMTV promoter and is a multi-functional molecule containing two variable exons, v4 and v5 that can be either included or excluded during the splicing process. Both AR and AR-V7 induce transcription from an MMTV promoter. Co-transfection of AR or AR-V7 with the CD44 mini-gene in Hela cells demonstrated that CD44 splicing was altered by AR and its variant AR-V7. R1881 treated samples showed a shift to RNA products resulting from the skipping of both variable exons. Treatment with R1881 showed a decrease in the PSI (Percent Spliced Inclusion) as compared to obtained in the absence of R1881 or AR-V7. In contrast, AR-V7 minimally induced single exon skipping as compared to vehicle. These observations suggest that AR and its variants can have marked effects on RNA splicing to produce radically different proteins with different splicing/inclusion ratio. Analysis of the RNA-Seq data revealed that multiple types of splicing events are affected by the AR isoforms. For instance, whether an isoform favours exclusion or inclusion of a particular exon is event specific. A comparison of splicing in VCaP and LNCaP models showed that only a subset of events were common to the two models. One of the most differentially regulated events in both lines is an exon inclusion/exclusion event in PGAP2. Both isoforms increase the inclusion/exclusion ratio. However, further analysis showed that AR-V7 preferentially increased the levels of inclusion, but did not affect the basal levels of skipping. Also, AR increased the inclusion/skipping ratio through repression of expression of the skipped product. Similar analyses for other splicing events are ongoing. Our studies will provide a better understanding for the molecular basis for isoform specific actions and the role of AR/AR-V7 in the regulation of mRNA transcription and splicing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The precise molecular alterations driving castration-resistant prostate cancer (CRPC) are not clearly understood. Using a novel network-based integrative approach, here, we show distinct alterations in the hexosamine biosynthetic pathway (HBP) to be critical for CRPC. Expression of HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be significantly decreased in CRPC compared with localized prostate cancer (PCa). Genetic loss-of-function of GNPNAT1 in CRPC-like cells increases proliferation and aggressiveness, in vitro and in vivo. This is mediated by either activation of the PI3K-AKT pathway in cells expressing full-length androgen receptor (AR) or by specific protein 1 (SP1)-regulated expression of carbohydrate response element-binding protein (ChREBP) in cells containing AR-V7 variant. Strikingly, addition of the HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to CRPC-like cells significantly decreases cell proliferation, both in-vitro and in animal studies, while also demonstrates additive efficacy when combined with enzalutamide in-vitro. These observations demonstrate the therapeutic value of targeting HBP in CRPC., The molecular alterations driving anti-androgen resistance in prostate cancer are unclear. Here, the authors show, using a network-based approach, that inhibition of the hexosamine biosynthetic pathway is necessary to develop resistance and that increasing the activity of the pathway enhances the anti-androgen response.

Castration-resistant prostate cancer (CRPC) is characterized by reactivation of androgen receptor (AR) signaling, in part by elevated expression of AR splice variants (ARv) including ARv7, a constitutively active, ligand binding domain (LBD)-deficient variant whose expression has been correlated with therapeutic resistance and poor prognosis. In a screen to identify small-molecule dual inhibitors of both androgen-dependent and androgen-independent AR gene signatures, we identified the chalcone C86. Binding studies using purified proteins and CRPC cell lysates revealed C86 to interact with Hsp40. Pull-down studies using biotinylated-C86 found Hsp40 present in a multiprotein complex with full-length (FL-) AR, ARv7, and Hsp70 in CRPC cells. Treatment of CRPC cells with C86 or the allosteric Hsp70 inhibitor JG98 resulted in rapid protein destabilization of both FL-AR and ARv, including ARv7, concomitant with reduced FL-AR- and ARv7-mediated transcriptional activity. The glucocorticoid receptor, whose elevated expression in a subset of CRPC also leads to androgen-independent AR target gene transcription, was also destabilized by inhibition of Hsp40 or Hsp70. In vivo, Hsp40 or Hsp70 inhibition demonstrated single-agent and combinatorial activity in a 22Rv1 CRPC xenograft model. These data reveal that, in addition to recognized roles of Hsp40 and Hsp70 in FL-AR LBD remodeling, ARv lacking the LBD remain dependent on molecular chaperones for stability and function. Our findings highlight the feasibility and potential benefit of targeting the Hsp40/Hsp70 chaperone axis to treat prostate cancer that has become resistant to standard antiandrogen therapy. Significance:These findings highlight the feasibility of targeting the Hsp40/Hsp70 chaperone axis to treat CRPC that has become resistant to standard antiandrogen therapy. Cancer Res; 78(14); 4022–35. ©2018 AACR.

Progesterone receptors (PRs) are phosphorylated on multiple sites, and a variety of roles for phosphorylation have been suggested by cell-based studies. Previous studies using PR-null mice have shown that PR plays an important role in female fertility, regulation of uterine growth, the uterine decidualization response, and proliferation as well as ductal side-branching and alveologenesis in the mammary gland. To study the role of PR phosphorylation in vivo, a mouse was engineered with homozygous replacement of PR with a PR serine-to-alanine mutation at amino acid 191. No overt phenotypes were observed in the mammary glands or uteri of PR S191A treated with progesterone (P4). In contrast, although PR S191A mice were fertile, litters were 19% smaller than wild type and the estrous cycle was lengthened slightly. Moreover, P4-dependent gene regulation in primary mammary epithelial cells (MECs) was altered in a gene-selective manner. MECs derived from wild type and PR S191A mice were grown in a three-dimensional culture. Both formed acinar structures that were morphologically similar, and proliferation was stimulated equally by P4. However, P4 induction of receptor activator of nuclear factor-κB ligand and calcitonin was selectively reduced in S191A cultures. These differences were confirmed in freshly isolated MECs. Chromatin immunoprecipitation analysis showed that the binding of S191A PR to some of the receptor activator of nuclear factor-κB ligand enhancers and a calcitonin enhancer was substantially reduced. Thus, the elimination of a single phosphorylation site is sufficient to modulate PR activity in vivo. PR contains many phosphorylation sites, and the coordinate regulation of multiple sites is a potential mechanism for selective modulation of PR function.

Context Although a number of studies have demonstrated the importance of constitutively active androgen receptor variants (AR-Vs) in prostate cancer, questions still remain about the precise role of AR-Vs in the progression of castration-resistant prostate cancer (CRPC). Objective Key stakeholders and opinion leaders in prostate cancer convened on May 11, 2017 in Boston to establish the current state of the field of AR-Vs. Evidence acquisition The meeting "Mission Androgen Receptor Variants" was the second of its kind sponsored by the Prostate Cancer Foundation (PCF). This invitation-only event was attended by international leaders in the field and representatives from sponsoring organizations (PCF and industry sponsors). Eighteen faculty members gave short presentations, which were followed by in-depth discussions. Discussions focused on three thematic topics: (1) potential of AR-Vs as biomarkers of therapeutic resistance; (2) role of AR-Vs as functionally active CRPC progression drivers; and (3) utility of AR-Vs as therapeutic targets in CRPC. Evidence synthesis The three meeting organizers synthesized this meeting report, which is intended to summarize major data discussed at the meeting and identify key questions as well as strategies for addressing these questions. There was a critical consensus that further study of the AR-Vs is an important research focus in CRPC. Contrasting views and emphasis, each supported by data, were presented at the meeting, discussed among the participants, and synthesized in this report. Conclusions This article highlights the state of knowledge and outlines the most pressing questions that need to be addressed to advance the AR-V field. Patient summary Although further investigation is needed to delineate the role of androgen receptor (AR) variants in metastatic castration-resistant prostate cancer, advances in measurement science have enabled development of blood-based tests for treatment selection. Detection of AR variants (eg, AR-V7) identified a patient population with poor outcomes to existing AR-targeting therapies, highlighting the need for novel therapeutic agents currently under development.

Steroid hormone receptors are hormone-activated transcription factors. Their activities are regulated by site specific phosphorylation. Conversely, steroids and their receptors act at many levels to regulate cell signaling and their target transcription factors in many tissues, including brain. Thus, the actions of steroids and other cellular regulators are intricately intertwined.

Despite recent developments in treatment strategies, castration-resistant prostate cancer (CRPC) is still the second leading cause of cancer-associated mortality among American men, the biological underpinnings of which are not well understood. To this end, we measured levels of 150 metabolites and examined the rate of utilization of 184 metabolites in metastatic androgen-dependent prostate cancer (AD) and CRPC cell lines using a combination of targeted mass spectrometry and metabolic phenotyping. Metabolic data were used to derive biochemical pathways that were enriched in CRPC, using Oncomine concept maps (OCM). The enriched pathways were then examined in-silico for their association with treatment failure (i.e., prostate specific antigen (PSA) recurrence or biochemical recurrence) using published clinically annotated gene expression data sets. Our results indicate that a total of 19 metabolites were altered in CRPC compared to AD cell lines. These altered metabolites mapped to a highly interconnected network of biochemical pathways that describe UDP glucuronosyltransferase (UGT) activity. We observed an association with time to treatment failure in an analysis employing genes restricted to this pathway in three independent gene expression data sets. In summary, our studies highlight the value of employing metabolomic strategies in cell lines to derive potentially clinically useful predictive tools.

Steroid hormone receptors (SHRs) are hormone-activated transcription factors involved in numerous cellular functions and in health and disease. Their activities depend on the cellular level of the receptor, the presence of coregulator proteins and the cell signaling pathways that are active in the cell. SHRs and their coregulators are phosphorylated on multiple sites by a wide variety of kinases. Each site may contribute to multiple functions and the net effect of an individual phosphorylation depends on the activating kinase. Here we discuss functions of known SHR phosphorylation sites, kinase regulation, evidence of translational relevance, and cross-talk between SHRs and cell signaling pathways. Understanding how cell signaling pathways regulate SHRs might yield novel therapeutic targets for multiple human diseases.

The evidence that androgen blockade-resistant prostate cancer, termed castration resistant, remains androgen receptor (AR) dependent is compelling. AR is re-activated through multiple mechanisms including expression of constitutively active splice variants that lack hormone binding domains (HBDs). This highlights the need to develop therapies that target regions other than the HBD. Because the p160 coactivators interact most strongly with the amino-terminus of AR, we examined the consequences of disrupting this interaction. We identified two overlapping SRC-1 peptides that interact with AR, but not with progesterone receptor. These peptides reduce AR and AR variant AR-V7 dependent induction of an AR responsive reporter. Using mammalian two hybrid assays, we found that the peptides interrupt the AR/SRC-1, AR/SRC-2 and AR N/C interactions, but not SRC-1/CARM-1 interactions. Consistent with the SRC-1 dependence of induced, but not repressed genes, in LNCaP cells, the peptides inhibited hormone dependent induction of endogenous target genes including PSA and TMPRSS2, but did not block AR dependent repression of UGT2B17 or inhibit vitamin D receptor activity. Simultaneous detection of SRC-1 peptides and PSA by double immunofluorescence in transfected LNCaP cells clearly demonstrated a strong reduction in PSA levels in cells expressing the peptides. The peptides also inhibited the AR dependent expression of PSA in castration resistant C4-2 cells. Moreover they inhibited androgen dependent proliferation of LNCaP cells and proliferation of C4-2 cells in androgen depleted medium without affecting AR negative PC-3 cells. Thus, the p160 coactivator binding site is a novel potential therapeutic target to inhibit AR activity.

// Justin M. Roberts 1 , Rebeca San Martin 1 , D. Badrajee Piyarathna 1, 2 , James G. MacKrell 3 , Guilherme V. Rocha 3 , Jeffery A. Dodge 3 , Cristian Coarfa 1, 2 , Venkatesh Krishnan 3 , David R. Rowley 1 and Nancy L. Weigel 1, 4 1 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA 2 Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX, USA 3 Lilly Research Labs, Eli Lilly and Company, Indianapolis, IN, USA 4 Scott Department of Urology, Baylor College of Medicine, Houston, TX, USA Correspondence to: Nancy L. Weigel, email: nweigel@bcm.edu Keywords: vitamin D, prostate cancer, CYP24A1, TMPRSS2:ERG, VCaP Received: November 30, 2016 Accepted: May 04, 2017 Published: May 18, 2017 ABSTRACT Whether vitamin D is chemopreventive and/or has potential therapeutically in prostate cancer is unresolved. One confounding factor is that many prostate cancers express a TMPRSS2:ERG fusion gene whose expression is increased both by androgens and by vitamin D receptor (VDR) activation. Two challenges that limit VDR agonist use clinically are hypercalcemia and the cooperation of VDR with ERG to hyper-induce the 1α,25-dihydroxyvitamin D 3 metabolizing enzyme, CYP24A1, thus reducing VDR activity. Using the VCaP TMPRSS2:ERG positive cell line as a model, we found that a nonsecosteroidal CYP24A1 resistant VDR agonist, VDRM2, substantially reduces growth of xenograft tumors without inducing hypercalcemia. Utilizing next generation RNA sequencing, we found a very high overlap of 1,25D(OH) 2 D 3 and VDRM2 regulated genes and by drawing upon previously published datasets to create an ERG signature, we found activation of VDR does not induce ERG activity above the already high basal levels present in VCaP cells. Moreover, we found VDR activation opposes 8 of the 10 most significant ERG regulated Hallmark gene set collection pathways from Gene Set Enrichment Analysis (GSEA). Thus, a CYP24A1 resistant VDR agonist may be beneficial for treatment of TMPRSS2:ERG positive prostate cancer; one negative consequence of TMPRSS2:ERG expression is inactivation of VDR signaling.

Androgen ablation therapy represents the first line of therapeutic intervention in men with advanced or recurrent prostate tumors. However, the incomplete efficacy and lack of durable response to this clinical strategy highlights an urgent need for alternative treatment options to improve patient outcomes. Targeting the molecular chaperone heat shock protein 90 (Hsp90) represents a potential avenue for therapeutic intervention as its inhibition results in the coordinate blockade of multiple oncogenic signaling pathways in cancer cells. Moreover, Hsp90 is essential for the stability and function of numerous client proteins, a number of which have been causally implicated in the pathogenesis of prostate cancer, including the androgen receptor (AR). Here, we examined the preclinical activity of ganetespib, a small molecule inhibitor of Hsp90, in a panel of prostate cancer cell lines. Ganetespib potently decreased viability in all lines, irrespective of their androgen sensitivity or receptor status, and more effectively than the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Interestingly, while ganetespib exposure decreased AR expression and activation, the constitutively active V7 truncated isoform of the receptor was unaffected by Hsp90 inhibition. Mechanistically, ganetespib exerted concomitant effects on mitogenic and survival pathways, as well as direct modulation of cell cycle regulators, to induce growth arrest and apoptosis. Further, ganetespib displayed robust antitumor efficacy in both AR-negative and positive xenografts, including those derived from the 22Rv1 prostate cancer cell line that co-expresses full-length and variant receptors. Together these data suggest that further investigation of ganetespib as a new therapeutic treatment for prostate cancer patients is warranted.

The authors provide the rationale, design, and description of a unique teaching award that has enhanced Baylor College of Medicine's teaching environment and become highly valued by the promotions and tenure (PT) committee in determining a faculty member's readiness for promotion. This award is self-nominating and standards based. The primary purpose for development of the award was to provide the Baylor community and the PT committee a method to understand and value the scholarship of teaching to the same degree that they understand and value the scholarship of discovery.The authors also present results from an internal evaluation of the program that included a survey and interviews. Between the inception of the award in 2001 and the internal review conducted in 2010, the award could have had an influence on the promotion of 130 of the recipients. Of the 130, 88 (65.6%) received this award before gaining their current rank (χ (1) = 16.3, P.001). Stakeholders, including department chairs and members of the PT committee, agreed that this award is valuable to those seeking promotion. Individual recipients stated that the award is good for the institution by encouraging reflection on teaching; increasing the recognition, importance, and value of teaching; encouraging the improvement of teaching skills; and providing a better understanding to others about what medical teachers really do. Of the 214 open-ended responses to survey questions of award recipients, more than half the comments were about the value of the award and its positive effect on promotion.

Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.

Background: Aromatase inhibitors (AIs) have emerged as the therapy of choice for the treatment of estrogen receptor alpha (ERα)-positive breast cancer. However, many patients develop resistance to AI treatment. Although the involvement of the ERα in AI resistance is well established, the role of the androgen receptor (AR) is not known. It has been estimated that about 60%-70% of ERα-positive breast cancer co-express the AR, and that AR agonists can either inhibit or stimulate breast cancer cell proliferation. Thus it is important to determine if there are biomarkers predicting AR's effects in breast tumors. We have previously shown a role for AR-overexpression in tamoxifen resistance in ERα-positive MCF-7 breast cancer cells; here we hypothesized that AR overexpression might similarly be involved in resistance to the AI anastrazole (Anas). Materials and Methods: Stable transfection of MCF-7 cells was performed to generate cell lines that express the aromatase gene (MCF-7 BK Arom) and then co-transfected with an AR expression vector (MCF-7 AR Arom). Aromatase and AR expression levels were evaluated by western blot analysis, and the enzyme activity was evaluated using aromatase activity assays. Proliferation was tested using anchorage independent soft agar assays and MTT in the presence of the androgen substrate androstenedione (AD), or AD plus Anas. ERα and AR transcriptional activities were tested with ERE-luciferase reporter assays. Localization of ERα and AR within the cells was visualized using immunofluorescence microscopy. Results: ERα-positive MCF-7 cells were stably transfected with either aromatase, or aromatase plus AR. MCF-7 aromatase clones overexpressing AR were resistant to the growth inhibitory effects of Anas when stimulated with the androgen AD. Resistance was not mediated through changes in aromatase expression or activity. The growth of several of the AR Arom-overexpressing cells was stimulated with treatment of Anas alone, suggesting that Anas was acting as an agonist. As expected, AD treatment stimulated ERα transcriptional activity, but Anas was unable to block AD-stimulated activity in AR Arom-overexpressing cells using ERE-Luciferase reporter assay. Anas was able to enhance AR and ERα colocalization in AR-overxpressing cells. Resistance was not associated with activation of known mechanisms of resistance, such as HER2, IGF-1R, or MAPK. However AR-overexpressing cells had higher constitutive phosphorylation of Akt. Accordingly, resistance to Anas was blocked using an Akt1/2 inhibitor. Conclusion: Using a model of ERα-positive breast cancer cells expressing exogenous aromatase and AR, we have demonstrated that AR overexpression confers resistance to the AI Anas. These results suggest that in patients recurring on hormonal therapy whose tumors express elevated levels of AR, targeted therapy to Akt might restore hormone sensitivity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-07.

BACKGROUND The active metabolite of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D) reduces the growth of several prostate cancer cell lines, most commonly by inducing a cell-cycle arrest in G1. This is mediated, in part, through down-regulation of c-Myc, a positive regulator of the transcription factor, E2F. There is evidence that prostate cancer cells lacking functional retinoblastoma protein (Rb), a negative regulator of E2F activity, are poorly responsive to 1,25D treatment. Since up to 60% of prostate cancers demonstrate a loss of heterozygosity for Rb, we sought to determine whether Rb is required for the growth inhibitory effects of 1,25D. METHODS Using siRNA, Rb was reduced in C4-2 prostate cancer cells, and the response of cells to 1,25D treatment or depletion of c-myc measured by [3H]-thymidine incorporation and flow cytometry. The effects of 1,25D treatment on E2F levels and activity, and E2F target gene expression were also measured. RESULTS 1,25D treatment and c-Myc depletion both cause a G1 arrest inhibiting C4-2 cell proliferation independently of Rb. 1,25D reduces c-Myc expression and causes a decrease in E2F and E2F target genes. Bcl-2, an E2F target and positive regulator of C4-2 cell growth, also is down-regulated by 1,25D independently of Rb. CONCLUSIONS Redundant growth inhibitory pathways compensate for the loss of Rb, and tumors lacking functional Rb may be responsive to 1,25D. Prostate 71: 98–110, 2011. © 2010 Wiley-Liss, Inc.

Vitamin D receptor (VDR) agonists have been shown to reduce the growth of several prostate cancer cell lines. However, the effects of VDR activation have not been examined in the presence of the recently identified androgen-regulated TMPRSS2:ERG gene fusions, which occur in a high percentage of prostate cancers and play a role in growth and invasiveness. In a previous microarray study, we found that VDR activation induces TMPRSS2 expression in LNCaP prostate cancer cells. Here we show that the natural VDR agonist 1α,25-dihydroxyvitamin D3 and its synthetic analog EB1089 increase expression of TMPRSS2:ERG mRNA in VCaP prostate cancer cells; this results in increased ETS-related gene (ERG) protein expression and ERG activity as demonstrated by an increase in the ERG target gene CACNA1D. In VCaP cells, we were not able to prevent EB1089-mediated TMPRSS2:ERG induction with an androgen receptor antagonist, Casodex, although in LNCaP cells, as reported for some other common androgen receptor and VDR target genes, Casodex reduces EB1089-mediated induction of TMPRSS2. However, despite inducing the fusion gene, VDR agonists reduce VCaP cell growth and expression of the ERG target gene c-Myc, a critical factor in VDR-mediated growth inhibition. Thus, the beneficial effects of VDR agonist treatment override some of the negative effects of ERG induction, although others remain to be tested.

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.

Steroid receptors (SRs) are hormone-activated transcription factors important for a wide variety of cellular functions. Post-translational modifications of SRs, including phosphorylation, ubiquitination, acetylation, and sumoylation regulate their expression and function. The remarkable number of phosphorylation sites in these receptors and the wide variety of kinases shown to modulate phosphorylation influence the integration between cell-signaling pathways and SR action. These phosphorylation sites have been identified in all of the functional domains with the majority being located within the amino-terminal portions of the receptors. The regulation of function is receptor specific, site specific, and often dependent on the cellular context. Numerous roles for site-specific phosphorylation have been elucidated including sensitivity of hormone response, DNA binding, expression, stability, subcellular localization, dimerization, and protein-protein interactions that can determine the regulation of specific target genes. This review summarizes the current knowledge regarding receptor site-specific phosphorylation and regulation of function. As functional assays become more sophisticated, it is likely that additional roles for phosphorylation in receptor function will be identified.

Evidence that the androgen receptor (AR) is not only important in androgen-dependent prostate cancer, but also continues to play a role in tumors that become resistant to androgen deprivation therapies, highlights the need to find alternate means to block AR activity. AR, a hormone-activated transcription factor, and its coactivators are phosphoproteins. Thus, we sought to determine whether inhibition of specific cell signaling pathways would reduce AR function. We found that short-term inhibition of p42/p44 MAPK activity either by a MAPK kinase inhibitor, U0126, or by depletion of kinase with small interfering RNA caused target gene-specific reductions in AR activity. AR enhances histone H3 acetylation of target genes that are sensitive to U0126 including prostate-specific antigen and TMPRSS2, but does not increase histone H3 acetylation of the U0126-resistant PMEPA1 gene. Thus, although AR induces transcription of many target genes, the molecular changes induced by AR at the chromatin level are target gene specific. Long-term treatment (24–48 h) with U0126 causes a G1 cell cycle arrest and reduces AR expression both through a decrease in AR mRNA and a reduction in AR protein stability. Thus, treatments that reduce p42/p44 MAPK activity in prostate cancer have the potential to reduce AR activity through a reduction in expression levels as well as by target gene-selective inhibition of AR function.

FOXO (Forkhead box O1 transcription factors) factors interact with and modify the activity of other transcription factors, including nuclear hormone receptors. However, not all of the structural domains within the FOXO proteins that mediate these functional interactions have been clearly defined. To address this issue, we used a constitutively active (nuclear) mutant of FOXO1a (designated FOXOA3) and within FOXOA3 made additional mutations to alter the putative nuclear hormone interacting domain (NID), minimal activation domain (MAD), DNA-binding domain (DBD), and the N terminus. We document that FOXOA3 enhanced the hormone-dependent transcriptional activity of liganded progesterone receptors A (PGRA) on a glucocorticoid response element-responsive promoter, PGRA on the insulin-like growth factor-binding protein 1 promoter, and estrogen receptor α on an estrogen response element-responsive promoter. The effects of FOXOA3 on PGRA were dependent, in part, on an intact NID, the MAD, and N-terminal domain. In striking contrast, a FOXOA3 DNA-binding mutant (FOXOA3-mDBD) modulated PGRA, PGRB, and ESR1 activities by distinctly different mechanisms, markedly elevating ligand-independent activity of these nuclear hormone receptors even in the double mutant lacking the MAD. Furthermore, both FOXOA3 and FOXOA3-mDBD enhanced the activity of a transcriptionally defective PGRA lacking its AF1 transactivation domain, indicating that this region of the receptor is not essential in this context. Since FOXOA3, FOXOA3-mDBD, and FOXOA3-mNID all bound PGRA in a GST pull-down assay, it appears that the LXXLL (leucine–X–X–leucine–leucine) motif within the NID is not critical for FOXOA3 interactions with PGRA, but may modify the recruitment of other co-regulatory molecules. Collectively, the results show that FOXOA3 exerts co-regulatory functions independent of DNA binding and that the DNA-binding defective form of FOXO1a is transcriptionally active as a co-regulator of these nuclear hormone receptors.

Steroid receptors (SR), which are ligand activated transcription factors, and their coactivators are phosphoproteins whose activities are regulated by cell signaling pathways. Many of the identified phosphorylation sites in these proteins contain Ser/Thr-Pro motifs suggesting that they are substrates for cyclin dependent kinases and/or for mitogen activated protein kinases. An analysis of the roles of cyclins and their kinases in regulating receptor action has revealed that there are both stimulatory and inhibitory actions of cyclins, that some of the actions are independent of the partner kinases and that these activities are receptor specific. Consistent with this finding, the limited analyses of receptor activity as a function of cell cycle reveal distinct patterns of activation. SR often regulate cell proliferation. Thus, the cross-talk between cyclins and their kinases and the SR provides a means for integrating the actions of the SR with the cell cycle status of cells.