Using biochemistry and biophysics to extinguish androgen receptor signaling in prostate cancer

Abstract

Castration resistant prostate cancer (CRPC) continues to be androgen receptor (AR) driven. Inhibition of AR signaling in CRPC could be advanced using state-of-the-art biophysical and biochemical techniques. Structural characterization of AR and its complexes by cryo-electron microscopy would advance the development of N-terminal domain (NTD) and ligand-binding domain (LBD) antagonists. The structural basis of AR function is unlikely to be determined by any single structure due to the intrinsic disorder of its NTD, which not only interacts with coregulators but likely accounts for the constitutive activity of AR-splice variants (SV), which lack the LBD and emerge in CRPC. Using different AR constructs lacking the LBD, their effects on protein folding, DNA binding, and transcriptional activity could reveal how interdomain coupling explains the activity of AR-SVs. The AR also interacts with coregulators that promote chromatin looping. Elucidating the mechanisms involved can identify vulnerabilities to treat CRPC, which do not involve targeting the AR. Phosphorylation of the AR coactivator MED-1 by CDK7 is one mechanism that can be blocked by the use of CDK7 inhibitors. CRPC gains resistance to AR signaling inhibitors (ARSI). Drug resistance may involve AR-SVs, but their role requires their reliable quantification by SILAC-mass spectrometry during disease progression. ARSI drug resistance also occurs by intratumoral androgen biosynthesis catalyzed by AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase), which is unique in that its acts as a coactivator of AR. Novel bifunctional inhibitors that competitively inhibit AKR1C3 and block its coactivator function could be developed using reverse-micelle NMR and fragment-based drug discovery.

Keywords: aldo-keto reductase; allostery; androgen receptor; cryo-electron microscopy; mass spectrometry; nuclear magnetic resonance; proteomics; splice variants.

Figure 1

Central role of AR signaling in prostate cancer and targets in CRPC. Activation of the AR signaling pathway begins in CRPC with intratumoral testosterone (T) and dihydrotestosterone (DHT) synthesis catalyzed by AKR1C3. DHT then binds to AR sequestered by Hsp90 in the cytosol leading to translocation of the dimerized AR to the nucleus and its binding to androgen response elements (AREs) in the promoters of responsive genes. The sites of action of two ARSI therapies, abiraterone and enzalutamide, are shown. Alternative forms of the AR that are transcriptionally active in the absence of ligand are shown (AR-SV) and phosphorylated forms of AR. The recruitment of coregulators to the transcriptional complex is shown. Proteins in red boxes identify targets for the eradication of AR signaling. 4-AD, 4-androstene-3,17-dione; 5α-AD, 5α-androstane-3,17-dione; DHEA, dehydroepiandrosterone; L, ligand; P, phosphate; SRD5A, steroid 5α-reductase.

Figure 2

AR Structural information.A, domain structure that is common to steroid hormone receptors: hAR, human androgen receptor; hPR, human progesterone receptor; hERα, human estrogen receptor alpha; hG, human glucocorticoid receptor. Numbers refer to the percent sequence identity in the domains where AR is 100%. B, inter- and intradomain interaction in hAR; the intrinsically disordered (ID) NTD contains the FQNLF motif and the AF1 region, which contains Tau-1 and Tau-5; DBD, DNA-binding domain and the LBD, ligand-binding domain also contains the AF2 region. The interaction with a p160 steroid receptor coactivator is shown; C, low-resolution cryo-EM of AR₂•ARE•SRC-3•p300 containing R1881 obtained at 20 Å [from ref (37)].

Figure 3

Domain structure of androgen receptor (AR) and glucocorticoid receptor (GR) showing similar frustration in the NTD of each receptor. For GR, the AF1-containing C-terminal portion of the NTD (yellow) is repressed by the N-terminal 98 a.a. (red), which serves as a regulatory element (R) (40). In AR, the NTD has two functional elements, TAU 1 (red) and TAU 5 (yellow). Like GR, the N-terminal portion of the NTD (red) represses the activity of the functional region (yellow). Unlike GR, where the regulatory region has no activity of its own, acting simply as a repressor, TAU 1 is both a repressor of TAU 5 and is responsible for ligand-dependent activation in AR by interacting with the LBD, (Top). Interdomain coupling where the DBD can activate transcription and the R domain leading to repression in GR similarly interdomain coupling where the DBD can activate transcription and the Tau-1 domain can lead to repression in AR (bottom).

Figure 4

CDK7 phosphorylation of MED1 at T1457 is essential for AR-MED1 interactions at the chromatin to active AR signaling. A, CAK module of TFIIH basal transcription factor containing CDK7 phosphorylates MED1 at T1457, which is required for interaction with AR. CDK7 inhibition by small-molecule THZ1 disrupts MED1-AR interaction through the loss of pT1457. However, the phosphomimetic T1457D MED1 is constitutively bound to AR and is resistant to THZ1 treatment. B, potential mechanism of action of CDK7 inhibition in AR-addicted CRPC. AR binding to distal enhancer or superenhancer results in the recruitment of MED1 and other Mediator subunits, which then facilitates chromatin looping to the target promoter containing basal transcription factor TFIIH containing CDK7 and RNA Pol II. CDK7 then phosphorylates RNA Pol II at S5 and S7 of CTD heptapeptide and MED1 at T1457, which interacts with AR to form a stable AR-MED1 complex at the chromatin. CDK7 may also directly phosphorylate AR at S515 and S81, which may help stabilize the chromatin binding of AR. Treatment with CDK7 inhibitors leads to the collapse of the transcriptional complex at the AR target sites leading to loss of AR target gene expression

Figure 5

A, a work flow for SID-LC-IP-HRMS quantitation of AR variants. The biospecimen is spiked with a SILAC-internal standard for an AR variant, the mixture is subjected to denaturation and immunoaffinity purified using Ab-magnetic beads. Eluents are subjected to SDS-PAGE and in-gel digestion with Glu-C. The unlabeled (biospecimen) peptides and labeled (internal standard peptides) are subjected to 2D-nano-LC-parallel reaction monitoring/MS where they behave identically except for differences in mass, top. B, amino acid sequences of AR-FL and AR-V1-AR-V9 in the region 620 to 682. C, amino acid sequences of AR-FL and AR-V12 in the region 700 to 734.

Figure 6

AKR1C3 acts as a 17β-hydroxysteroid dehydrogenase and as a coactivator of the androgen receptor (AR). AKR1C3 converts weak androgens (4-androstene-3,17-dione and 5α-androstane-3,17-dione) to potent androgens (T and DHT = L) and translocates to the nucleus with AR where it acts as a coactivator. Both the enzymatic function of AKR1C3 and its coactivator function are blocked by GTx-560.