Non-Canonical Dimerization of the Androgen Receptor and Other Nuclear Receptors: Implications for Human Disease

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Endocrine-Related A Jiménez-Panizo et al. Androgen receptor 26:8 R479–R497 Cancer dimerization in disease REVIEW Non-canonical dimerization of the androgen receptor and other nuclear receptors: implications for human disease

Alba Jiménez-Panizo1, Paloma Pérez2, Ana M Rojas3, Pablo Fuentes-Prior4 and Eva Estébanez-Perpiñá1

1Department of Biochemistry and Molecular Biomedicine, Institute of Biomedicine (IBUB) of the University of Barcelona (UB), Barcelona, Spain 2Instituto de Biomedicina de Valencia (IBV)-CSIC, Valencia, Spain 3Computational Biology and Bioinformatics Group, Andalusian Center for Developmental Biology (CABD-CSIC), Sevilla, Spain 4Molecular Bases of Disease, Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain

Correspondence should be addressed to P Fuentes-Prior or E Estébanez-Perpiñá: [email protected] or [email protected]

Abstract

Nuclear receptors are transcription factors that play critical roles in development, Key Words homeostasis and metabolism in all multicellular organisms. An important family of ff androgen receptor nuclear receptors comprises those members that respond to steroid hormones, and ff glucocorticoid receptor which is subdivided in turn into estrogen receptor (ER) isoforms α and β (NR3A1 and ff protein structure A2, respectively), and a second subfamily of so-called oxosteroid receptors. The latter ff ligand-binding domain includes the androgen receptor (AR/NR3C4), the glucocorticoid receptor (GR/NR3C1), ff multimerization the mineralocorticoid receptor (MR/NR3C2) and the progesterone receptor (PR/NR3C3). ff prostate cancer Here we review recent advances in our understanding of the structure-and-function ff androgen insensitivity relationship of steroid nuclear receptors and discuss their implications for the etiology syndromes (AIS) of human diseases. We focus in particular on the role played by AR dysregulation in ff hormone resistance both prostate cancer (PCa) and androgen insensitivity syndromes (AIS), but also discuss conditions linked to mutations of the GR gene as well as those in a non-steroidal receptor, the thyroid hormone receptor (TR). Finally, we explore how these recent results might be exploited for the development of novel and selective therapeutic strategies. Endocrine-Related Cancer (2019) 26, R479–R497

Introduction

Nuclear receptors (NRs) form a superfamily of related progesterone receptor (PR/NR3C3). Phylogenetic studies transcription factors that play essential roles in show that AR, GR, MR and PR comprise a subfamily of multicellular organisms through coordination of so-called oxosteroid NRs, which markedly differ from both pivotal signaling networks (Evans & Mangelsdorf 2014). ER isoforms (Bledsoe et al. 2002, Evans & Mangelsdorf One important family of NRs groups together those 2014, Gallastegui et al. 2015, Zennaro & Fernandes-Rosa members that respond to steroid hormones, accordingly 2017, Katzenellenbogen et al. 2018). This phylogenetic termed steroid family. In vertebrates, the steroid NRs separation is also reflected at the level of tertiary and include estrogen receptor (ER) isoforms ERα (NR3A1) quaternary structures, as we will discuss below. and ERβ (NR3A2) together with the androgen receptor We note that some members of other subfamilies (AR/NR3C4), the glucocorticoid receptor (GR/NR3C1), of NRs specifically respond to steroid hormones, for the mineralocorticoid receptor (MR/NR3C2) and the instance, the vitamin D3 receptor (VDR/NR1I1), the bile

-19-0132 Endocrine-Related A Jiménez-Panizo et al. Androgen receptor 26:8 R480 Cancer dimerization in disease acid receptor (FXR/NR1H4), oxysterol receptors α and β advanced into late clinical trials (Sadar 2011, Dalal et al. (LXRα/NR1H3 and LXRβ/NR1H2, respectively) and the 2014, Nadal et al. 2017, Veras Ribeiro Filho et al. 2019). retinoid-related orphan receptors α, β and γ (RORα/NR1F1, Over the years, several physiologically relevant protein RORβ/NR1F2 and RORγ/NR1F3, respectively). However, partners of NRs have been reported to selectively fine-tune for space reasons, we will focus in the current review their actions in various tissues. These factors, collectively almost exclusively on the members of the sequence and termed coregulators, can either enhance or inhibit structurally related ‘classical’ steroid family of NRs, most transcription of target genes and are therefore known notably on the AR. as coactivators or corepressors, respectively (Hermanson Similar to other NRs, the members of the steroid et al. 2002, Perissi & Rosenfeld 2005, Dasgupta et al. 2014). family display a marked modular structure: a variable These NR coregulators underlie the tissue-selective actions N-terminal region called the N-terminal domain (NTD) of NR ligands and are also emerging therapeutic targets is followed by a highly conserved DNA-binding domain per se, although they have proved hard to target so far (Yi (DBD) comprising two zinc finger motifs and a C-terminal et al. 2015, Wang et al. 2016, Ruggero et al. 2018). All NR ligand-binding domain (LBD) that contains the internal domains have been involved in coregulator recruitment ligand-binding pocket (LBP) (Jenster et al. 1995, McEwan and macromolecular complex formation, although the 2012a, Gallastegui et al. 2015, Tien & Sadar 2018, Weikum atomic details still remain elusive (Dasgupta et al. 2014, et al. 2018, Fuentes-Prior et al. 2019, Veras Ribeiro Filho Foley & Mitsiades 2016, Giudici et al. 2016, Lempiäinen et al. 2019). DBD and LBD modules are separated by a et al. 2017). poorly conserved interdomain linker called ‘hinge’, which Although the NTDs are variable in length and harbors a nuclear localization signal (NLS) (Fig. 1 for a intrinsically disordered (i.e., isolated NTDs lack stable schematic representation of the domain organization in secondary (2D) and/or tertiary (3D) structures under steroid NRs). All the NR-comprising domains are proposed physiological conditions), the results of different drug targets, but the LBD remains still the main module biochemical and biophysical studies suggest that they for therapeutic intervention and, in spite of intense undergo at least partial folding (‘helicity induction’) efforts, no molecule targeting other receptor domains has to modulate the formation of competent transcription

Figure 1 Schematic representation of the domain organization in steroid receptors. Note the common modular protein structure of four main elements: the N-terminal domain (NTD), which contains the activating function 1 (AF1), the DNA-binding domain (DBD), a hinge region (H) and the actual ligand- binding domain (LBD), which contains the activating function 2 (AF2) assembled by residues from helices H3, H4, H5 and H12. The so-called ‘F domain’, located at the C-terminal end of the receptor, folds back and covers part of the globular LBD in all oxosteroid receptors, but not in ERα/β, whose F domains differ strongly from the one well-conserved in oxosteroid receptors. Other NRs such as the thyroid receptor (TR) lack an F domain altogether. Note also that the NTDs are highly variable in sequence and length. The AR–NTD can be further dissected into two partly overlapping transcription activation units, TAU1 comprising residues 142–485 and TAU5 including residues 360–528 (Jenster et al. 1995, Hilser et al. 2012). Similarly, studies of translational isoforms with variable lengths of the GR-NTD have also found two coupled but thermodynamically distinct regions (Brinkmann et al. 1989, Michigami et al. 1999, Li et al. 2012). At least in the case of these two oxosteroid receptors, the main determinants for transactivation map to their highly similar AF-1 sites (46% sequence identity; Kumar et al. 2004, McEwan et al. 2007, De Mol et al. 2018).

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0132 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/06/2021 07:02:00AM via free access Endocrine-Related A Jiménez-Panizo et al. Androgen receptor 26:8 R482 Cancer dimerization in disease accompanied by significant conformational changes, nuclear Further, it is worth noting that post-translational translocation of the receptor and its dimerization, which modifications (PTMs) profoundly modulate AR actions, is a critical step within this signaling pathway (Centenera both under physiological and pathological conditions et al. 2008, van Royen et al. 2012, Nadal et al. 2017). Pioneer (Faus & Haendler 2006, Gioeli & Paschal 2012, studies suggested that AR oligomerization precedes binding Treviño & Weigel 2013, van der Steen et al. 2013, to chromatin and demonstrated that DNA binding was Koryakina et al. 2014). redox dependent (Kokontis & Liao 1999). More recently, The intrinsically disordered nature of the long NTD AR dimerization has been shown to enhance chromatin and its unique interactions with the LBD (the ‘N/C interaction and remodeling to modulate gene expression (Jin contacts’) raised over the years the issue of whether et al. 2013). However, it must be stressed that the exact cycle FL-AR dimerizes in a parallel, ‘head-to-head’ manner of AR monomer-to-oligomer transition in the cell has not or alternatively in an anti-parallel, ‘head-to-tail’ been precisely elucidated (Schaufele et al. 2005, Centenera configuration Langley( et al. 1995, 1998, Schaufele et al. et al. 2008, van Royen et al. 2012, Nadal et al. 2017). It is 2005, Centenera et al. 2008, Minges et al. 2013). On the usually assumed that this transition follows exactly the other hand, the crystal structure of rat AR–DBD revealed same steps as in related NRs, although data supporting this a head-to-head homodimer bound to its cognate DNA assumption are incomplete or simply lacking. (Shaffer et al. 2004), in an arrangement quite similar to Recent studies of steroid NR trafficking in living cells that adopted by DBD dimers of GR (Luisi et al. 1991) have dramatically advanced our understanding of this and ER (Schwabe et al. 1993). In this conformation, the process, forcing a paradigm shift in the field. In particular, C-terminal residues of both monomers, corresponding careful quantitation of GR, AR and PR dynamics at the to L627 in human AR, are located far away (about 50 Å). single-molecule level have unexpectedly revealed that the However, the length of the G628–E669 linker does not dimeric forms of these receptors are intermediate states allow for an accurate prediction of the likely arrangement toward their biologically active, tetrameric arrangements of LBD monomers in a hypothetical dimer. In fact, (‘dimers of dimers’) (Presman et al. 2016, Paakinaho et al. limited and contradictory information in support of 2017, Presman & Hager 2017, Presman et al. 2017). Further, AR–LBD dimerization had been presented over the years, it has been established that the regulatory complexes with some authors even suggesting that the LBD–LBD assemble around steroid NRs and undergo a rapid exchange interactions may not significantly contribute to receptor in the time scale of seconds (the ‘hit-and-run’ model; Voss oligomerization (Schaufele et al. 2005, Centenera et al. et al. 2011, Sung et al. 2014, Swinstead et al. 2016, Presman 2008, van Royen et al. 2012, Nadal et al. 2017). & Hager 2017). However, detailed information regarding The long-standing questions of whether the the activities of these signaling complexes in normal and AR–LBD domain dimerized and the relative arrangement tumor cells is still missing. The intricate links between NR of monomers have been answered with the resolution of oligomeric state, affinity for ligands (hormones), binding the crystal structure of human AR–LBD bound to DHT cooperativity and recruitment to specific chromatin sites and a peptide derived from the coregulator, ubiquitin- remain to be worked out (Fuentes-Prior et al. 2019). activating enzyme 3 (UBA3) (Nadal et al. 2017). All the AR–LBD structures previously solved by us and others had captured essentially the same conformation of Quaternary structure of the AR–LBD: monomeric AR–LBD (Matias et al. 2000, Sack et al. 2001, functional implications He et al. 2004, Estébanez-Perpiñá et al. 2005, Bohl et al. Several AR features have complicated elucidation of the 2007, Estebanez-Perpina et al. 2007). In striking contrast, physiologically relevant tertiary and quaternary structures our recent crystal structure features four independent, of full-length AR (FL-AR) and other steroid receptors, helically arranged LBD molecules (Nadal et al. 2017) and it is still a matter of debate for example whether (Fig. 2B). Two of these LBD monomers are arranged in a the biological units are dimers or ‘dimers of dimers’ (see symmetrical ‘core dimer’, while the other two peripheral above and Presman et al. 2016, Nadal et al. 2017, Presman partners are associated in a less compact manner to the & Hager 2017, Presman et al. 2017). In addition to the BF-3 sites of each of these monomers (Fig. 2B). The core conformational flexibility that is a prerequisite for the AR–LBD dimer exhibits a head-to-head arrangement and functional versatility of the individual AR modules, they displays the corresponding coactivator-binding sites are known to interact with each other to precisely regulate (AF-2 pockets) facing opposite directions, thus able to the NR functions in a context-dependent manner. independently interact with coregulators (Fig. 2A).

Helices 5 (H5) from both AR–LBD moieties occupy relevance has been questioned upon careful comparison the center of the dimerization interface (Fig. 3A), which is of currently available GR structures, and alternative GR thus topologically distinct from the ‘canonical’, H10–11- dimeric conformations have been suggested (Bianchetti centered arrangements identified, among others, in ER et al. 2018). Clearly, further experimental work is needed to homodimers and in RXR-mediated heterodimers (Fig. 3C) fully clarify the physiologically relevant conformation(s) (Wurtz et al. 1996, Brzozowski et al. 1997, Bourguet of the GR and other members of the oxosteroid subfamily et al. 2000, Chandra et al. 2008, 2017, Khorasanizadeh to integrate structural, biochemical and cellular studies & Rastinejad 2016, Nadal et al. 2017, Fuentes-Prior et al. (Fig. 3) (Bledsoe et al. 2002, Kauppi et al. 2003, Robertson 2019). The H5-centered dimerization mode of human et al. 2013a,b, Presman et al. 2016, 2017, Paakinaho AR–LBD has been confirmed in solution and in cells Nadal( et al. 2017, Presman & Hager 2017, Weikum et al. 2017, et al. 2017). This non-canonical dimeric conformation Wilkinson et al. 2018). might be shared by other NRs, not only of the oxosteroid Although specific functions have been commonly class (Fuentes-Prior et al. 2019). In fact, a topologically ascribed to the different NR domains (e.g., DNA and equivalent dimer of GR–LBD had been previously reported hormone binding), seminal X-ray crystallography studies (Bledsoe et al. 2002) (Fig. 3B), although its biological of non-steroidal NRs indicate that DBD, hinge and LBD

Figure 3 Crystal structures of homodimeric ligand-binding domains from various nuclear receptors. (A) Overall structure of the AR–LBD core dimer (PDB 5JJM). The two monomers are depicted as cartoons showing helices in blue and loops in pink; helices H5, H9, H10-11 and H12 are marked. Note that the AR–LBD mainly dimerizes through helices H5 of both monomers. The bound hormone (dihydrotestosterone, DHT) and the UBA3 peptide are shown as pink spheres and as a magenta cartoon, respectively. (B) Overall structure of the first reported GR-LBD dimer (PDB 1M2Z). The two monomers are depicted as cartoons showing helices in blue and loops in pink; helices H5, H9 and H10-11 are highlighted. The bound hormone (dexamethasone, DEX) and the coactivator peptide are shown as pink spheres and as a pink cartoon, respectively. Note the topological equivalence to the AR–LBD dimer depicted in panel A, in spite of less intimate contacts at the intermonomer interface. (C) Overall structure of the ER–LBD dimer (PDB 1ERE). The two monomers are depicted as cartoons showing helices in pink and loops in red; helices H9 and H10–11 are indicated. Note that the ER–LBD dimerizes according to the ‘canonical’ mechanism, that is through helices H10–11 of each monomer. The hormone (17beta-estradiol, EST) is shown as blue spheres. (D) Overall structure of the TR–LBD dimer (PDB 2PIN). The two monomers are shown as cartoons with helices in green and loops in pink; helices H9, H10–11 and H12 are labeled. The ligand ((4-(4-hydroxy-3-iodo-phenoxy)-3,5-diiodo-phenyl)-acetic acid, 4HY) is shown as pink spheres. Dashed lines indicate parts of the structure that were not visible in the electron density maps.

Allosteric modulation of AR activity

NRs are allosteric proteins par excellence (Kuriyan 2004, del Sol et al. 2006, McEwan 2012b, Mackinnon et al. 2014), and the AR is no exception in this regard (Estebanez- Perpina et al. 2007, Buzón et al. 2012, Grosdidier et al. 2012). Indeed, FL–AR functions as an allosteric switch alternating between inactive, chaperone-bound/ligand- free states and active, hormone- and coactivator-bound conformations. Ligand binding and the exchange of chaperones by coactivators are allosterically coupled, but the sequence of molecular events and detailed conformational changes associated are only partially understood (Hur et al. 2004, Estébanez-Perpiñá et al. 2005). In particular, hormone binding to the LBP has been shown to trigger allosteric remodeling of the AF-2 and BF-3 interaction surfaces. In this manner, occupancy of the LBP by ligands regulates the dynamics and stability of surfaces Figure 4 that recognize coregulators and is thus allosterically Proximity of functionally relevant areas in non-canonical dimers. (A) coupled to the recruitment of these proteins to the AR Close-up of the AR–LBD dimerization interface. Monomers are colored light pink and blue, respectively, and the DHT molecules are shown as (Estébanez-Perpiñá et al. 2005, Estebanez-Perpina et al. color-coded Van der Waals spheres. Residues are shown as color-coded 2007). Albeit a recent proteomics study has identified the sticks (oxygen, red; nitrogen, blue; carbon, yellow or brown) and labeled. essentially overlapping, agonist-specific interactomes of Note that most residues making important contributions to this interface possess hydrophobic/aromatic side chains (e.g., V685, W752, F755, V758, both AR and GR (Lempiäinen et al. 2017), the detailed Y764 and P802). The location of residue R753 is also noteworthy, as its molecular determinants of NR binding to a large number side chain contacts the DHT molecule bound inside the internal of coregulators are far from being well understood. ligand-binding pocket (LBP) from its ‘own’ monomer, while its main chain atoms form part of the dimer interface, and in particular, interact with Noteworthy, in addition to allosteric rearrangements N757 from the second monomer. In this manner, hormone binding and elicited by hormone binding, the target DNA sequences receptor dimerization appear to be intimately coupled in the AR. (B) to which NRs bind also induce important remodeling Close-up of the AR–LBD surface (as seen in PDB 1XOW), highlighting the proximity of the AF-2 pocket (colored blue; the bound NH2-terminal of the receptor structure (Fuentes-Prior et al. 2019). peptide of the receptor is colored cyan) to the LBP (with bound Most impressively, cognate DNA-binding sequences of methyltrienolone/R1881 colored magenta).

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0132 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/06/2021 07:02:00AM via free access Endocrine-Related A Jiménez-Panizo et al. Androgen receptor 26:8 R485 Cancer dimerization in disease the GR have been shown to function as true allosteric metabolism, it is not surprising that dysregulation of modulators, which are capable of affecting conformation their activities is directly responsible for a number of and regulate receptor activity (Meijsing et al. 2009, Love important human conditions (Huang et al. 2010, Evans et al. 2017, Weikum et al. 2017, Frank et al. 2018). Thus, & Mangelsdorf 2014, Luo et al. 2018). For instance, a change in a single base pair in the GR-binding site the ER is a key driver of 70% of breast cancer subtypes resulted in up to 10-fold higher affinity in the activation that require estrogen hormones for progression, and of a glucocorticoid response element (GRE) reporter gene different point mutations in the ER genes are linked in response to ligand. More recently, it has been reported to acquired resistance to commonly used estrogen- that nucleotides directly flanking the core-binding site not blocking drugs such as tamoxifen (Kojetin et al. 2008, only modulate the 3D structure of this site, but also that Katzenellenbogen et al. 2018, Nasrazadani et al. 2018, of the GR–DBD and even the quaternary conformation Reinert et al. 2018). Regarding oxosteroid receptors, of the dimeric receptor (Schöne et al. 2016). This implies single-residue mutations in GR and MR genes have that GR activity can be modulated by both sequence mainly been associated to loss-of-function phenotypes composition and DNA shape to achieve fine-tuning of (Zennaro & Fernandes-Rosa 2017). However, the most the GR structure and activity downstream of binding. conspicuous association between a nuclear receptor These features are likely to be shared by other members and human disease links the AR to several biomedical of the oxosteroid class of NRs, at the light of the almost conditions, as we briefly discuss below. identical sequences of DBDs and relative conservation of interdomain hinges. This flanking effect could explain Genetic bases of drug resistance in prostate cancer how NRs predicted to recognize similar binding motifs show distinct DNA-binding preferences in vivo. It is AR is encoded by a ubiquitously expressed gene located in feasible that while two related NRs can bind to the same the X chromosome at Xq11-12 and is particularly important DNA motif, the competent conformation required for an in prostate development and homeostasis (Lubahn et al. optimal transcriptional response is only achieved through 1988). When dysregulated, however, AR activity is central specific flanking sequences. In particular, we assume to the onset, development and progression to metastasis that AR–DBD binding to its cognate DNA sequences will of prostate cancer (PCa), the most common cancer trigger intra- and interdomain conformational changes diagnosed in males worldwide (Matias et al. 2000, Gottlieb that would affect the overall quaternary structure of the et al. 2004, Knudsen & Penning 2010, Arora & Barbieri receptor, including its hormone-binding domain. 2018, Centenera et al. 2018, Cioni et al. 2018, Li et al. 2018, The precise molecular mechanisms underlying Luo et al. 2018, Nevedomskaya et al. 2018, Paschalis et al. allosteric transitions in FL–AR, including the equilibrium 2018). In addition, AR mutations are linked to disorders between different conformational states and the impact of male sexual differentiation and development termed of ligand binding to allosteric modulatory sites (e.g., androgen insensitivity syndromes (AIS) (Hughes et al. AF-2, BF-3) on receptor oligomerization, remain to be 2012, Mongan et al. 2015, Gibson et al. 2018) and to the worked out (Fig. 2A) (Nadal et al. 2017). In multidomain rare adult-onset hereditary neurodegenerative disorder AR, both intra- and interdomain allostery may occur known as spinal and bulbar muscular atrophy (SBMA or simultaneously (Fernandez et al. 2017, Fernandez 2018). Kennedy’s disease; OMIM #313200) (Spada et al. 1991, The first may take place within the NTD, DBD or LBD Badders et al. 2018, Cortes & La Spada 2018, Lieberman moieties, while interdomain signal transduction appears 2018, Pennuto & Rinaldi 2018). Finally, AR malfunction to be essential in coordinating AR functions (e.g., through is also associated to androgenic alopecia or loss of scalp the well-studied N-C contacts, but also upon as of yet hair and skin malignancies (Ellis et al. 2001, Clocchiatti uncharacterized DBD–LBD and LBD–DNA interactions). et al. 2018). Similar considerations might apply to other steroid NRs. Several altered genetic and epigenetic mechanisms contribute to the development of metastatic and drug- resistant PCa (Knudsen & Penning 2010, Shen & Abate- Shen 2010, Grasso et al. 2012, Robinson et al. 2015, Dysregulation of steroid receptors and Pritchard et al. 2016, Baumgart & Haendler 2017, human disease Armenia et al. 2018, Cotter & Rubin 2018, Li et al. 2018, Given the essential roles of steroid hormones and Quigley et al. 2018, Ruggero et al. 2018, Karthaus & their cognate NRs in development, homeostasis and Sawyers 2019). For example, tandem duplications of an

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0132 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/06/2021 07:02:00AM via free access Endocrine-Related A Jiménez-Panizo et al. Androgen receptor 26:8 R486 Cancer dimerization in disease upstream enhancer of the AR are found in up to 87% encoding truncated AR isoforms. The identification of of metastatic castration-resistant PCa (mCRPC) cases, these constitutively active AR variants (AR-Vs), which lack compared to <2% of primary prostate cancers (Takeda portions or the entire hormone-sensitive LBD, has added et al. 2018, Viswanathan et al. 2018, Wu et al. 2018). In an unanticipated level of complexity to the AR signaling addition, it has been recently reported that deletion of pathways (Dehm & Tindall 2011, Centenera et al. 2013b, the gene encoding the chromatin remodeler, chromatin Ho & Dehm 2017). Many of these truncated AR-Vs seem helicase DNA-binding protein (CHD1), redistributes the to support androgen-independent expression of AR-target AR cistrome in a manner that favors the expression of genes and therefore androgen-independent growth of PCa-specific oncogenic pathways (Augello et al. 2019). PCa cells (Dehm & Tindall 2011). However, the androgen Not surprisingly, CHD1 loss had been previously reported independency of AR-Vs does not imply that expressing as one of the most common and deleterious genetic cells are totally independent of the presence of androgenic alterations in PCa (Grasso et al. 2012, Huang et al. 2012, hormones. The (patho)physiological implications of the Rodrigues et al. 2015, Zhao et al. 2017) and an early event coexistence of full-length AR with one or several AR-Vs in cancer development (Wedge et al. 2018). in the same cell are still a matter of intense debate. The Further, AR gene duplications and AR-mediated likely formation of AR-Vs (hetero)dimers with FL-AR, chromosomal rearrangements are common events. either through DBD-mediated or N–C interactions may Among them, about half of the patients with PCa feature still result in hormone-dependent cell growth leading to fusions of the AR-regulated gene, TMPRSS2, with different PCa progression (Liu et al. 2014, Uo et al. 2017). Further, fragments of the ETS-related gene (ERG), a member of potential differences in the interactomes of the different the ETS family of transcription factors. The presence of AR-Vs in comparison with that of the full-length receptor these TMPRSS2:ERG fusions defines the predominant have not been explored so far. molecular subtype of PCa (Yu et al. 2010, Park et al. 2014, Reig et al. 2016, Stelloo et al. 2018, Berglund et al. The AR–LBD dimerization interface is a hot spot of 2019). The encoded ERG truncated variants are resistant disease-linked mutations to degradation and transform the AR from a factor promoting lineage-specific differentiation of the prostate Inspection of the 3D structure of the AR–LBD homodimer to one that potentiates de-differentiation into a stem cell- immediately revealed that a large number of hitherto like state (Yu et al. 2010). However, TMPRSS2:ERG fusions unexplained mutations of solvent-exposed residues alone do not appear to be transforming (Casey et al. 2012), identified in PCa (Fig. 5A) and AIS patients cluster at and the precise mechanism(s) by which they contribute the dimerization interface (Nadal et al. 2017; see also to PCa initiation and/or progression are still a matter Fig. 5B). We stress that these mutations might have of debate. For instance, it has been reported that these additional consequences for AR protein structure and fusions synergize with deletion of the tumor suppressor, function, such as impaired interaction with chaperones PTEN, to promote prostatic intraepithelial neoplasia (PIN) in the monomeric state (see above). Accordingly, the (Carver et al. 2009, King et al. 2009, Casey et al. 2012). In situation in vivo does not fit a simplistic dichotomy of this regard, and also connecting to the work by Augello gain-of-function mutations causing PCa, while loss-of- and coworkers cited above, it has been proposed that function variants of the AR gene are linked to AIS (Hay & ETS factors alter the AR cistrome to prime the prostate McEwan 2012). epithelium to respond to aberrant signals such as Nevertheless, there are several correlations between PTEN loss, thus ultimately resulting in prostate-specific for example the nature of the mutant residue and disease transformation (Chen et al. 2013). Carver and coworkers severity, which strongly point to impaired homodimer also observed that two genes strongly associated with formation as a more likely cause of the observed cell migration, ADAMTS1 and CXCR4, were upregulated phenotype. This is in particular the case of repeatedly upon ERG overexpression (Carver et al. 2009). Besides, identified mutations that affect residues such as F755, overexpression of MMP9 and plexin B correlates with the N757, V758, N759, R761 and P767 (Figs 4A and 5A, C). presence of the fusion in samples of PCa patients and has Most notably, the mutant Y764C has been found both been linked to migration and invasion of prostate cancer in PCa and AIS. Furthermore, residue F755 has been cells (Liu et al. 2017). found conservatively replaced by either leucine, in some On the other hand, several studies have described patients with partial AIS, or by another aliphatic residue, the synthesis in vivo of alternatively spliced transcripts valine, in cases of complete androgen insensitivity.

Figure 5 Mutations that affect the non-canonical, AR-like dimerization interface are linked to various conditions. The LBD dimers of all four analyzed receptors are represented as gray cartoons. (A and B) AR–LBD (PDB 5JJM) with the side chains of all mutated interface residues shown with all their non-hydrogen atoms as sticks and surface, colored red for mutations reported in PCa patients (A) or blue for those linked to AIS (B). Mutations have been retrieved from the Androgen Receptor Gene Mutations Database (http://androgendb.mcgill.ca/). (C) GR-LBD (PDB 1M2Z) with the side chains of all mutated interface residues shown with all their non-hydrogen atoms as sticks and surface, colored pink to highlight those residues linked to glucocorticoid hormone resistance. Some of these residues have been associated with primary generalized Chrousos syndrome. (D) Cartoon representation of the TR-LBD dimer (gray, PDB 2PIN) with the side chains of all mutated interface residues shown with all their non-hydrogen atoms as sticks and surface, colored green to highlight those residues associated with thyroid hormone resistance.

The side chain of F755 makes important Van der Waals result in impaired tissue-specific sensitivity to GCs, which interactions with P802 from the second monomer may manifest as GC resistance or hypersensitivity, both of (Fig. 4A), and modeling experiments indicate that a which are associated with significant morbidity (Nicolaides leucine residue at position 755 might still interact with & Charmandari 2015, Nicolaides et al. 2015a,b, Wilkinson this proline, supporting homodimer formation. However, et al. 2018). a less bulky valine would not be able to contact P802, with For instance, in primary generalized GC resistance concomitantly impaired dimerization. A second example (PGGR or Chrousos syndrome, MIM #615962), NR3C1 of strong genotype–phenotype correlation is given by heterozygous variants (point mutations, insertions or mutant p.V758I identified in PCa patients. Again, a fully deletions) result in impaired GR function and thus conservative replacement of an aliphatic residue leading decreased sensitivity to GCs in all organs, although to PCa most likely reflects the higher stability of the with a high degree of variability among different tissues. homodimer formed by mutant AR–LBD molecules, as Alterations in the GR function include dysregulation of the bulkier I758 side chain fills better the intermonomer the GC-mediated negative feedback mechanisms, which space. These findings, among others, lend extraordinary results in compensatory activation of the hypothalamic– support to the (patho)physiological relevance of the AR pituitary–adrenal (HPA) axis. In turn, HPA overactivation homodimer interface and suggest that this area might be leads to hypersecretion of ACTH and cortisol (highly targeted for pharmacological intervention (see below). variable, ranging from severe symptoms to subclinical hypercortisolism) and may result in increased production of other adrenal steroids such as androgens and NR3C1/GR mutations linked to either glucocorticoid mineralocorticoids. Patients affected by this syndrome resistance or hypersensitivity syndromes are thus commonly diagnosed by clinical signs of Glucocorticoids (GCs) are major regulators of many mineralocorticoid and/or androgen excess (hypertension physiological functions, and thus contribute substantially due to hyperaldosteronism and/or hyperandrogenic to tissue homeostasis. Dysfunction of GC-mediated signs), rather than those associated with GC deficiency actions underlies two human pathologic conditions, (Kino & Chrousos 2001, Kino et al. 2002, Nicolaides et al. Cushing syndrome and Addison’s disease, which are due 2010, 2015a,b, Charmandari et al. 2013, Nicolaides & to GC excess or deficiency, respectively. On the other Charmandari 2015, Wilkinson et al. 2018). hand, alterations in GR function (either due to mutations So far, 26 mutations have been described in the human or polymorphisms of the encoding gene, NR3C1, or to NR3C1 gene, most of which are missense mutations other molecular changes along the GR signaling pathway) and affect the GR–LBD (Vitellius et al. 2016, 2018,

Nicolaides & Charmandari 2017) (Fig. 5C). Functional LBP (p.S810L) has been associated with a severe form of studies indicate that most NR3C1 mutations impair several inherited hypertension (Kino & Chrousos 2001, Zennaro steps of GC signaling including hormone recognition, GR & Fernandes-Rosa 2017). Finally, PR mutations are less nuclear translocation, DNA binding and interactions with frequent but they have been associated to an increased the coactivator, GRIP1, which ultimately leads to reduced risk of breast, endometrial and colon cancer (Agoulnik or absent GR-dependent transactivation. Structural et al. 2004). studies have contributed to a better understanding of how conformational changes in the receptor cause GC Impact of mutations affecting the non-canonical resistance, although a systematic analysis of all reported dimerization interface in other non-steroidal NRs mutants is lacking. Computer simulations suggest that the primary cause of GC resistance in Chrousos syndrome We finish this section on the linkage between dysregulation is an overall destabilization of the LBD, as the result of of NR activities and pathologic conditions by presenting single amino acid replacements that affect LBP, AF-2 or the case of a non-steroidal NR that has also been shown both (Hurt et al. 2016). The possible role of impaired to dimerize in an AR-like, non-canonical conformation, receptor multimerization on this syndrome, however, has the thyroid receptor (TR; Estébanez-Perpiñá et al. 2007a, not been explored so far. Jouravel et al. 2009 and Fig. 5D). Vertebrates possess actually In addition, it has been recently reported that NR3C1 two TR isoforms, termed TRα (NR1A1) and TRβ (NR1A2), mutations also cause another pathological condition and which are encoded by two independent genes, THRA called incidentaloma (incidentally discovered bilateral and THRB, respectively. In spite of their close structural adrenal hyperplasia), with clinical features similar to similarity (70% sequence identity), these two isoforms differ those of Chrousos syndrome such as asymptomatic strongly both in expression profile. TRα is ubiquitously hypercortisolism and/or hypertension. Five novel expressed, while TRβ is mainly expressed in liver, pituitary, heterozygous NR3C1 mutations that cause impaired GR inner ear and some brain areas (Chatonnet et al. 2013), but signaling were identified, which represents a relatively in particular because they regulate different sets of genes. high prevalence of 5% in the analyzed cohort. These TRs respond to iodinated biomolecules known as thyroid findings suggest that the overall prevalence of NR3C1 hormones (THs), mostly to the active form 3,3′,5-triiodo- mutations may have been previously underestimated l-thyronine (T3), which is generated in turn from the and advise to perform GR mutation screening in patients prohormone, l-thyroxine (T4) (Holzer et al. 2017). with adrenal incidentalomas (Vitellius et al. 2016, 2018). Since THs have fundamental functions in Although most of the identified NR3C1 mutations mapped development, growth and metabolic homeostasis, at the GR–LBD, other (p.R477S, p.R477C, p.R477H and alterations in TR structure affect these functions at many p.Y478C) were located at the C-terminal end of the second different levels. This is in particular the case of generalized zinc finger, implicated in the dimerization of the receptor. thyroid hormone resistance (GTHR), a syndrome By contrast, in the rare syndrome known as primary characterized by elevated serum TH but impaired action generalized glucocorticoid hypersensitivity (PGGH), of these hormones within the hypotalamic–pituitary– patients exhibit clinical signs of metabolic syndrome thyroid axis and variable tissue hyposensitivity to them without hypercortisolism, due to compensatory (Huber et al. 2003a,b, Onigata & Szinnai 2014). GTHR hypoactivation of the HPA axis (Nicolaides & Charmandari is linked to mutations located in the LBD of TRβ (Weiss 2017). This disease mostly correlates with activating NR3C1 et al. 1993, Adams et al. 1994). Similar to the results polymorphisms while to date, a single patient harboring discussed above for GR, most of these mutations result a point mutation in the NTD (p.D401H) exhibited clinical in a lower affinity for T3 or a defective interaction symptoms of GC hypersensitivity including obesity, type with TR coregulators. Whether are least some of these 2 diabetes and hypertension (Charmandari et al. 2008). variants would impair dimer formation has not been Further, loss-of-function mutations in the NR3C2/MR experimentally confirmed. However, inspection of the gene are responsible for renal pseudohypoaldosteronism 3D structure of the TRβ homodimer (Estébanez-Perpiñá type 1 (MIM #177735), a rare disease of mineralocorticoid et al. 2007a, Jouravel et al. 2009) immediately reveals resistance characterized by sodium and potassium that some of the reported missense mutations actually imbalances that manifests at birth with weight loss and map to the homodimer interface and are therefore dehydration despite elevated plasma levels of aldosterone. likely to disrupt receptor homodimerization (Fig. 5D). Conversely, an activating MR mutation that reshapes its Particularly interesting mutations in this regard affect

Table 1 Mutations within the non-canonical dimerization interface are linked to various human conditions.

Androgen receptor Glucocorticoid receptor Estrogen receptor Thyroid receptor Residue Disease Residue Disease Residue Disease Residue Disease Q671R PCa I673T PCa V685I CAIS V543 I326 A234T THR C687R PAIS Y545 Y328 R243Q THR R243W D691E PAIS D549 X K244 D691V CAIS D696H CAIS D554 F337 I250 THR D696N CAIS/PAIS/M I250T D696V CAIS D696Y CAIS A749T PCa A749D PAIS

W752R PCa W610 W393 V319 R753P CAIS R611L GCR R394H Estrogen R320C THR R753Q CAIS insensitivity R320H F755L PCa Y613 M396 D322H THR F755S PAIS/MAIS F755V CAIS T756A PCa R614 E397 L328S THR N757D PCa Q615 X E324 N757S PAIS/MAIS S760F CAIS A618 H398 P323 S760P PCa S760Y CAIS R761? PCa N619 G400 P323/D324 R761S PAIS M762T PAIS L620 K401 E326 L763F CAIS L621 L402 T327P THR T327A Y764C PCa/PAIS C622 L403 T329N THR Y764H CAIS T329I F765L CAIS F623 F404 L330S THR P767A CAIS P625 P406 G332R THR P767S CAIS G332E D768E CAIS N626 N407 E333D THR D768Y CAIS E773 E631 R412 R338W THR R338L THR W797del CAIS H655 R437 S362 Q799E PCa/PAIS/M Q657 N439 N364 Q803R PAIS E661 E443 T368 R847G PCa E705 G494 X N849K CAIS T496 H210 R856C CAIS R714Q Chrousos R503W BCa syndrome R856H CAIS/PAIS/M

Residues from the non-canonical dimerization interface of the AR–LBD were aligned to those of the corresponding domains in GR, ER and TR. Missense mutations that affect these topologically equivalent residues are given, along with the associated disease. Note in particular that several mutations affect topologically equivalent residues in AR–LBD and TR–LBD. Note also that replacement of the highly conserved residue R753 of AR–LBD, which corresponds to R611 of GR–LBD, R394 of ER–LBD and R320 of TR–LBD, causes hormone resistance in all receptors. GCR, glucocorticoid resistance; THR, thyroid hormone resistance. arginine residues at positions 320 (p.R320C, p.R320H) GC resistance (p.R611L in GR) or estrogen insensitivity and 338 (p.R338W, p.R338L) in TRβ, as mutations of (p.R394H in ER) (Table 1) (Weiss et al. 1993). the topologically equivalent residues in several steroid In summary, residues responsible for non-canonical receptors result in either AIS (p.R753P, p.R753Q in AR), homodimer formation in NRs play a critical functional

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0132 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/06/2021 07:02:00AM via free access Endocrine-Related A Jiménez-Panizo et al. Androgen receptor 26:8 R490 Cancer dimerization in disease role, leading to various pathological conditions when (so-called ‘LBP-focused strategies’). However, the recent dimerization is disrupted. A list of mutations that are advances in the structure and function of NRs discussed likely to interfere with non-canonical homodimer above have largely expanded the space for pharmacological formation of some NRs and their associated conditions intervention. First, we mention that AR chaperones had is given in Table 1. been proposed as potential therapeutic targets against PCa and, after initial failures, heat shock proteins, and in particular Hsp90, are currently considered viable targets to treat PCa (Eskew et al. 2011, Centenera et al. 2012, 2013a, Implications for the development of novel He et al. 2013). Indeed, inhibitors of Hsp90 C-terminal therapeutic strategies domain have been recently shown to inhibit growth of All steroid NRs are major therapeutic targets to treat various PCa cell lines without inducing the expression of several endocrine-related diseases (Moore et al. 2006, other chaperones (Armstrong et al. 2016). Evans & Mangelsdorf 2014, Carroll 2016, Nasrazadani Novel strategies that directly target the NR include et al. 2018). Most notably, the dependence of PCa tumors blocking receptor interactions with specific coactivators on the AR protein and its cognate endogenous hormones (Azad et al. 2015, Guy et al. 2015, Foley & Mitsiades 2016), is the basis for its pharmacological exploitation as a drug directly targeting NR binding to cognate DNA sequences target. Indeed, inhibition of AR functions by means or interfering with DBD dimerization (Dalal et al. 2017), of ligand depletion and/or the use of AR antagonists but also modulating ligand-independent functions (antiandrogens) is the first line of therapeutic intervention (reviewed in Caboni & Lloyd 2013). Also along these against PCa (Chen et al. 2008, Mohler et al. 2012, lines, the AR–NTD has been explored as drug target for Lorente et al. 2015, Aggarwal et al. 2017, Ponnusamy AR function control, in particular for management of its et al. 2017, Narayanan et al. 2018, Nevedomskaya et al. ligand-independent truncated variants (De Mol et al. 2016, 2018). However, recurrent resistant and incurable tumors Imamura & Sadar 2016, Kumar et al. 2016, Monaghan & arise as a result of inappropriately restored AR function McEwan 2016). associated with various genetic and epigenetic accidents. It is believed that conformational changes elicited In particular, emergence of point mutations as a response upon ligand binding are ultimately responsible for to antiandrogen therapy is a quite common event, which the different activity profiles exhibited by therapeutic may result in antiandrogens acting as agonists rather than compounds in different cell types, similar to what has been antagonists with severe clinical consequences (Knudsen shown for other NRs (Srinivasan et al. 2013, Nwachukwu & Penning 2010, Balbas et al. 2013, Joseph et al. 2013, et al. 2016). Given that these signals might propagate Schrecengost & Knudsen 2013, Lorente et al. 2015, Watson through the whole multidomain protein, controlling et al. 2015, Jernberg et al. 2017, Giacinti et al. 2018). The NR functions by targeting novel unexplored sites is an identification of constitutively active, truncated splice AR attractive but challenging alternative. Most importantly, variants (AR-Vs) lacking parts or the entire LBD also poses apart from the classical modulators targeting the LBP important pharmacological challenges (Dehm & Tindall site, synthetic allosteric modulators are currently under 2011, Centenera et al. 2013b, Ho & Dehm 2017, Paschalis investigation, thus substantially broadening the chemical et al. 2018). This is in addition to the cross-reactivity of space for novel antiandrogens (Buzón et al. 2012). In this therapeutic steroidal androgens with other, highly related manner, the AR LBP shall no longer be seen as the only steroid NRs (i.e., GR) in PCa patients, which derives in pocket suitable for pharmacological intervention, but all unwanted side effects that pose additional limits to their NR domains and their protein–protein interaction sites are clinical use (Arora et al. 2013, Karamouzis et al. 2016, envisioned as potential drug targets (Estébanez-Perpiñá Narayanan et al. 2016). Altogether, there is an unmet et al. 2007b, Buzón et al. 2012, Nadal et al. 2017, Badders need for the development of novel therapeutic strategies et al. 2018). In this regard, we recall that both AF-2 and including tissue-selective AR modulators (SARMs; BF-3 sites are druggable and are ideal candidates for off- Estébanez-Perpiñá et al. 2007b, Dalton 2017, Narayanan LBP strategies (Estébanez-Perpiñá et al. 2007b, Buzón et al. 2018) that may overcome the problems encountered et al. 2012, Ravindranathan et al. 2013, Badders et al. by currently used drugs. 2018). Most notably, AF2-targeting compounds have been Most therapeutic strategies to date have focused on recently validated as potent androgen-sparing strategies the development of small-molecule compounds that for SBMA therapy (Badders et al. 2018). Last but not least, compete with receptor binding to natural hormones interfering with the non-canonical dimerization interfaces

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0132 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/06/2021 07:02:00AM via free access Endocrine-Related A Jiménez-Panizo et al. Androgen receptor 26:8 R491 Cancer dimerization in disease of the AR and other oxosteroid receptors (Nadal et al. alters AR binding at lineage-specific enhancers and modulates 2017, Fuentes-Prior et al. 2019) offers hitherto unforeseen distinct transcriptional programs to drive prostate tumorigenesis. Cancer Cell 35 603.e8–617.e8. possibilities for the regulation of NR activities, and thus, Azad AA, Zoubeidi A, Gleave ME & Chi KN 2015 Targeting heat shock for the development of novel pharmaceutical drugs. proteins in metastatic castration-resistant prostate cancer. Nature Reviews: Urology 12 26–36. 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Received in final form 17 June 2019 Accepted 26 June 2019 Accepted Preprint published online 26 June 2019