Activation of androgen receptor (AR) is crucial for prostate cancer growth. Remarkably, also castration-resistant prostate cancer (CRPC) is dependent on functional AR, and several mechanisms have been proposed to explain the addiction. Known causes of CRPC include gene amplification and overexpression as well as point mutations of AR. We report here the pharmacological profile of ODM-201, a novel AR inhibitor that showed significant antitumor activity and a favorable safety profile in phase 1/2 studies in men with CRPC. ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a castration-resistant VCaP xenograft model. In contrast to other antiandrogens, ODM-201 shows negligible brain penetrance and does not increase serum testosterone levels in mice. In conclusion, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC.

Using the DNA-binding domain of androgen receptor (AR) as a bait in a yeast two-hybrid screening, we have identified a small nuclear RING finger protein, termed SNURF, that interacts with AR in a hormonedependent fashion in both yeast and mammalian cells. Physical interaction between AR and SNURF was demonstrated by coimmunoprecipitation from cell extracts and by protein-protein affinity chromatography. Rat SNURF is a highly hydrophilic protein consisting of 194 amino acid residues and comprising a consensus C 3 HC 4 zinc finger (RING) structure in the C-terminal region and a bipartite nuclear localization signal near the N terminus. Immunohistochemical experiments indicated that SNURF is a nuclear protein. SNURF mRNA is expressed in a variety of human and rat tissues. Overexpression of SNURF in cultured mammalian cells enhanced not only androgen, glucocorticoid, and progesterone receptor-dependent transactivation but also basal transcription from steroid-regulated promoters. Mutation of two of the potential Zn 2؉ coordinating cysteines to serines in the RING finger completely abolished the ability of SNURF to enhance basal transcription, whereas its ability to activate steroid receptor-dependent transcription was maintained, suggesting that there are separate domains in SNURF that mediate interactions with different regulatory factors. SNURF is capable of interacting in vitro with the TATA-binding protein, and the RING finger domain is needed for this interaction. Collectively, we have identified and characterized a ubiquitously expressed RING finger protein, SNURF, that may function as a bridging factor and regulate steroid receptor-dependent transcription by a mechanism different from those of previously identified coactivator or integrator proteins.Androgen receptor (AR) that mediates the biological actions of physiological androgens is a member of the superfamily of ligand-inducible transcription factors (59). Like other nuclear receptors, AR contains three major structural and interchangeable domains: the N-terminal transactivation domain, the central DNA-binding domain (DBD) that associates with specific androgen response elements (AREs) in target genes, and the C-terminal ligand binding domain (LBD) that binds physiological and synthetic androgens. Upon ligand binding, AR acquires a new conformational state (39), which enables the receptor to interact with AREs and converts the protein to a transcriptional activator. Molecular analyses have shown that the N-terminal half of AR, similar to that of other steroid receptors, contains sequences responsible for the activation function AF-1 (11,34,37,47,54,59,65). In addition to AF-1, another activation function (AF-2) has been identified in the LBDs of various nuclear receptors, including AR (11, 51). Besides encompassing nuclear localization signal (NLS) (38,65,75), the functional role of the hinge region residing between the DBDs and LBDs of steroid receptors has remained elusive.Nuclear receptors have been shown to contact the basal transcription machinery,...

Cross-modulation between androgen receptor (AR) and NF-kappaB/Rel proteins was studied using various androgen- and NF-kappaB-regulated reporter genes under transient transfection conditions. In COS-1 cells, elevated expression of RelA (p65) repressed AR-mediated transactivation in a dose-dependent manner, whereas NFkappaB1 (p50), another major member of the NF-kappaB family, did not influence transactivation. The repression of AR appeared to involve the N-terminal region of the protein between residue 297 and the DNA-binding domain. RelA-mediated transrepression could not be overcome by increasing the amount of AR. Transcriptional interference between RelA and AR was mutual in that cotransfected AR was able to attenuate transactivation by RelA in a dose- and steroid-dependent fashion. An excess of RelA was able to rescue the repression to some extent. Immunological analyses of RelA and AR protein levels indicated that transrepression was not due to reciprocal decrease in their amounts. Neither did AR increase the concentration of IkappaBalpha, which can sequester and inactivate RelA. Electrophoretic mobility shift assays using extracts from cotransfected cells and purified recombinant proteins showed that AR and RelA did not significantly influence each other's DNA binding activity. Nevertheless, protein-protein interaction experiments demonstrated a weak association between AR and RelA. Collectively, these data suggest that the mutual repression in intact cells is due to formation of AR-RelA complexes that are held together by another partner or to competition for a coactivator required for transcription.

We have characterized a novel partner for androgen receptor (AR), termed ARIP3, that interacts with the DNA-binding domain/zinc finger region of AR and is predominantly expressed in the testis. Rat ARIP3 is a nuclear protein comprising 572 amino acids. It modulates AR-dependent but not basal transcription, suggesting that ARIP3 acts as an AR transcriptional coregulator. Except for the C-terminal AR-interacting domain, ARIP3 contains distinct regions that are also present in two recently described proteins, a protein inhibitor of activated Stat3 and an RNA helicase II-interacting protein (Gu/RH-II binding protein). Conserved structural features of these proteins indicate the existence of a gene family involved in the regulation of various transcription factors. Collectively, ARIP3 belongs to a novel nuclear protein family and is perhaps the first tissuespecific coregulator of androgen receptor.

Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination.

Androgen receptor (AR) brings about a ligand-dependent inhibition of low-affinity neurotrophin receptor (p75) promoter constructs in cultured cells, with the greatest inhibition being achieved with a reporter gene containing 1050 nucleotides (nt) of the promoter. The receptor domain critical for trans-repression localizes to the same region (amino acids 147-296) as that mandatory for transactivation. In contrast to trans-activation, AR does not interact directly with specific DNA elements to elicit trans-repression of p75 promoter constructs, although an intact DNA-binding domain of the receptor is required for both actions. In a search for interacting partners, both extensively purified full-length AR and AR-DNA binding domain were found to inhibit c-Jun/AP-1 site interaction without themselves binding to the AP-1 element. Prior binding of c-Jun to the AP-1 element protected the complex from the receptor's interference. Repression was not mutual, as c-Jun did not inhibit AR-androgen response element interaction or trans-activation through an androgen response element-containing promoter. The 1050-nt-long p75 promoter sequence does not contain an AP-1 element; an AP-1-like site in the vector backbone mediates the trans-repression by the AR in recipient cells. Intriguingly, an AR form with a large N-terminal deletion (the delta 46-408 mutant) behaved as a transcriptional activator of the p75 promoter through a mechanism that was also independent of specific DNA binding. Collectively, these data indicate that, in a proper context, AR is able to elicit both transrepression and trans-activation without interacting directly with specific DNA elements. Sequences responsible for the down-regulation of p75 mRNA by androgens in vivo are, however, not located in the proximal 1050 nt of the p75 promoter.

Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids. In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro. The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome. However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins. ANPK is a nuclear protein that is widely expressed in mammalian tissues. Its overexpression enhances AR-dependent transcription in various cell lines. In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK. The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo. In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation. INTRODUCTIONThe androgen receptor (AR) mediates the biological actions of physiological androgens (Quigley et al., 1995). DNA-bound transcription factors, such as AR and other nuclear receptors, have been postulated to stimulate the efficiency of transcription by affecting directly or indirectly the assembly of basal transcription factors into the preinitiation complex, thereby increasing the rate of transcription initiation (Tjian and Maniatis, 1994;Horwitz et al., 1996;Beato and Sanchez-Pacheco, 1996). However, regulation of receptor function also involves cross-talk with other signaling pathways and interactions with other transcription factors and coregulatory proteins (Beato et al., 1995). Although nuclear receptors may contact directly some members of the basal transcription machinery (Ing et al., 1992;Blanco et al., 1995;Hadzig et al., 1995;Schulman et al., 1995;McEwan and Gustafsson, 1997) or TATA-binding protein-associated factors (Jacq et al., 1994;Schwerk et al., 1995; Mengus et al., 1997), they appear to employ preferentially coregulators to interact with the transcription machinery (Beato and Sanchez-Pacheco 1996;Horwitz et al., 1996). Examples of the coregulators include RIP-140 (Cavaillés et al., 1995), TIF1 (Le Douarin et al., 1995), TRIP1/SUG1 (Lee et al., 1995;vom Baur et al., 1996), ARA 70 (Yeh and Chang, 1996), CBP/p300 (Chakravarti et al., 1996;Hanstein et al., 1996;Kamei et al., 1996), and SRC-1 (Oñ ate et al., 1995) and its variants or related proteins, such as GRIP1, TIF2, AIB1, and TRAM-1 (Hong et al., 1996;Voegel et al., 1996;Anzick et al., 1997;Takeshita et al., 1997). Most of the these coregulators have been discovered through protein-protein interaction screening ...

Nuclear receptors, including the androgen receptor (AR), regulate target cell transcription through interaction with auxiliary proteins to modify chromatin structure. We describe herein a novel AR-interacting protein, termed ARIP4, that has structural features typical of the SNF2-like protein family. With regard to the Snf2 domain, the closest homolog of ARIP4 is the ATRX protein.ARIP4 is a nuclear protein and comprises 1466 amino acids. It interacts with AR in vitro and in cultured yeast and mammalian cells. ARIP4 can be labeled with 8-azido- [␥-32 P]ATP and exhibits DNA-dependent ATPase activity. Like several ATP-dependent chromatin remodeling proteins, ARIP4 generates superhelical torsion within linear DNA fragments in an ATP-dependent manner. With a stably integrated target promoter, ARIP4 elicits a modest enhancement of AR-dependent transactivation. In transient cotransfection assays, ARIP4 modulates AR function in a promoterdependent manner; it enhances receptor activity on minimal promoters, but does not activate more complex promoters. ARIP4 mutants devoid of ATPase activity fail to alter DNA topology and behave as trans-dominant negative regulators of AR function in transient assays. INTRODUCTIONThe androgen receptor (AR) belongs to the superfamily of nuclear receptors that are ligand-activated transcription factors capable of regulating transcription of genes containing appropriate response elements, usually within or around the proximal promoter regions (Beato et al., 1995;Quigley et al., 1995;Perlmann and Evans, 1997). After hormone binding, the receptors associate with their cognate DNA motifs and modulate transcription initiation. Nuclear receptors may interact directly with the basal transcription factors associated with RNA polymerase II, such as TFIIB (Ing et al., 1992;Blanco et al., 1995;Hadzic et al., 1995), TFIIF (McEwan and Gustafsson, 1997), and TFIIH (Lee et al., 2000), or elicit their actions indirectly via auxiliary regulatory proteins, called coactivators and corepressors (Torchia et al., 1998;Freedman, 1999;McKenna et al., 1999;Glass and Rosenfeld, 2000).DNA is folded in the nucleus into a tight chromatin structure that often renders important regulatory sequences inaccessible for sequence-specific transcription factors, including steroid receptors (Kingston et al., 1996;Näär et al., 2001). As a consequence, different chromatin-modifying complexes are required to counteract this repressive effect (Bjö rklund et al., 1999;Kingston and Narlikar, 1999;Lemon and Freedman, 1999;Vignali et al., 2000). The protein complexes can be classified into two main categories: 1) ATP-dependent chromatin-remodeling complexes, which use the energy of ATP hydrolysis to alter the association of histones with DNA; and 2) complexes that alter chromatin by covalent modification of its components. These modifications include histone acetylation, methylation, phosphorylation, and ADP-ribosylation. Yeast SWI/SNF was the first ATP-dependent complex shown to facilitate the function of gene regulatory proteins in a...