Prostate cancer is the second most common type of cancer diagnosed in men worldwide and the second leading cause of male cancer-related deaths in the U.S. (1). The American Cancer Society estimates about 268,490 new cases of prostate cancer and about 34,500 deaths from prostate cancer in the U.S. this year (1). According to the American Cancer Society data (cancer.org), patients with local or regional stage prostate cancer have nearly a 100% 5-year survival rate; however, the survival rate is only 30% for men diagnosed with distal metastasis.

Currently, localized prostate cancer is primarily treated with surgical removal of the gland or radiation therapy if a patient’s condition is not permissive for surgery. Distal metastasis occurs in high-risk patients, including locally advanced (positive surgical margin) or high-grade (Gleason sum score ≥ 8) tumors, which is the sole cause of death from prostate cancer (2). This short review work will discuss the current treatment options and recent development of anti-androgen receptor (AR) therapeutic approaches for metastatic prostate cancer ( Table 1 and Figure 1 ).

Metastatic prostate cancers are initially treated with androgen deprivation therapy (ADT) because prostate tissue (benign or malignant) expresses androgen receptor (AR) protein that is critical for prostate cancer development and progression (3, 4). Castration by surgery or medical treatment reduces androgen hormones, resulting in prostatic atrophy and cancer regression (5). This approach was developed eighty years ago in 1941 (3, 4). Since then, prostate cancer treatment has been mainly focused on reducing androgen levels and blocking androgen-induced AR activation (5). However, prostate cancers often relapse and progress to a stage termed as castration-resistant prostate cancers (CRPC) (67, 68), and the majority of these CRPCs still depend on the AR signaling for growth and progression (the AR addictiveness) (69, 70).

The mechanisms for CRPC progression include AR gene mutation, amplification, transcriptional splicing, and crosstalks with cellular signal pathways, plus de novo androgen synthesis by the malignant prostate cells (5). Therefore, clinical therapies use anti-androgens (Flutamide, Bicalutamide, Enzalutamide, Apalutamide, and Darolutamide) to competitively suppress androgen-induced AR activation or CYP17A1 inhibitor (Abiraterone) to reduce androgen production in prostate cancer tissues (5). So far in the clinic, these therapies provided certain clinical benefits of survival extension in CRPC patients (71). However, with the widespread use of Enzalutamide and Abiraterone, a subset of CRPC patients developed neuroendocrine progression, termed as anti-AR treatment-induced NEPC (t-NEPC) (72, 73), accounting for more than 25-30% mortality of CRPC fatality (74). There were multiple mechanisms involved in NEPC progression, including attenuated control of transcriptional factors, metabolic alterations, aberrant activation of cellular kinases, long noncoding RNAs, transcriptional splicing, and epigenetic modifications (75–87). It is postulated that extensive stress of AR inhibition under the long-term ADT condition forced an epigenetic reprogramming of CRPC cells into neuroendocrinal trans-differentiation (88–93). Treatment option for NEPC patients is limited in the clinic and the salvage platinum-based chemotherapy only provided very little survival benefit (75).

The AR protein is a nuclear receptor expressed in benign and malignant prostate tissues, critical for prostate physiological functionality and prostate cancer progression (94, 95). As a transcriptional factor, the AR protein modulates gene expression after being activated by androgens via binding on its C-terminal ligand-binding domain (95). Given that hormone therapy, including ADT and anti-androgens for the last eighty years, has been failed to be a curable approach for metastatic prostate cancers, eliminating the AR protein in prostate cancer cells recently emerged as a realistic solution for a potentially curable result.

In contrast to the CTD, the AR NTD has very few mutations without truncation (130). For example, the cBioportal database showed only 9 (0.145%) point-mutations identified from the NTD regions in 6334 prostate cancer specimens. There are two transactivation unit (TAU-1, aa100-370) and TAU-5 (aa360-485) motifs within the AR NTD (131). The TAU-1 motif is critical for the full-length AR activation after ligand binding, while the TAU-5 motif functions as a constitutive active motif for truncated AR protein (e.g., AR-V7) (132, 133). Especially, the TAU-1/TAU-5 motifs are rarely mutated or deleted in prostate cancer patients, making them a feasible target for prostate cancer therapy (130).

EPI series compounds are the first class of AR NTD inhibitors. The first compound EPI-001 was identified by screening a library of marine sponge extracts to inhibit AR NTD transactivation activity (134). EPI-001 binds to the TAU-5 motif and inhibits AR NTD activity at a relatively high dose (>25 μM in cell culture models) (135, 136). EPI compounds also suppressed tumor growth in VCaP and LNCaP95 cell-derived xenograft models at 100-200 mg/kg/day doses (135, 137). Although the older EPI compounds did not affect AR protein levels (the full length and AV variants), the new analog EPI-7170 suppressed AR-V7 expression in CRPC cells (138). EPI-002 (commercial name Ralaniten) is one of the four EPI-001 stereoisomers, and its pro-drug EPI-506 (Ralaniten acetate) was failed in a phase-I clinical trial due to excessive pill burden and poor oral bioavailability (139, 140). The newest analog, EPI-7386, showed 20-fold higher anti-androgenic potency than Ralaniten (141), and it is being tested in clinical trials in combination with Enzalutamide (NCT05075577/NCT04421222).

QW07 is a small synthetic molecule identified as an AR NTD-specific inhibitor via an AR-NTD-driven luciferase high-throughput screening (54). QW07 suppressed the activity of AR full-length and splicing variants at 5-8 μM in prostate cancer cells, which is more potent than EPI-001 (54). QW07 binds with the AR NTD directly and suppresses AR recruitment onto the target gene promoter. In animal xenograft experiments, QW07 inhibited tumor growth derived from prostate cancer 22RV1 and VCaP cells at a dose of 40 mg/kg/day, similar to EPI-001. However, QW07 did not affect AR protein expression (the full length or splicing variants).

As a transcription factor, the AR proteins translocate into the nuclear compartment after being activated by the androgens (5). In the nuclear, AR protein interacts with the androgen response elements in the gene promoter region to modulate gene expression. The AR protein has one nuclear localization sequence or signal (NLS) in each domain, the NTD region (aa294-556), the DBD-hinge region (aa617-633), and the LBD region (aa666-919) (142–144). In the absence of androgens, the AR protein is exported from the nuclear compartment via its nuclear export signal (NES, aa743-817) within the LBD region (145). In CRPC tissue or cells that androgen levels are deficient due to androgen deprivation therapy, the NLS in the NTD region is responsible for AR nuclear localization (143). Blocking AR nuclear translocation with a potent NLS inhibitor is feasible to suppress prostate cancer development and progression by shutting down AR-modulated gene expression.

EPPI and CPPI are small molecules identified as inhibitors of AR nuclear translocation in Dr. Z Wang’s lab using a 2GFP-AR fusing protein-based high-throughput screening approach (55). Both EPPI and CPPI at 25 μM inhibited AR nuclear localization in prostate cancer cells, which was reversed when the androgen level (R1881) was over 1.0 nM level, a physiological androgen concentration (56). Also, CPPI at a 50 mg/kg/day dose suppressed tumor growth in LNCaP but not PC-3 cell-derived xenograft models with or without castration, indicating an AR-specific effect (56). Further analysis revealed that CPPI blocked AR nuclear import and promoted AR degradation in the nuclear compartment through MDM2-dependent proteasome mechanism in CRPC cells (C4-2 and LNCaP95) and xenograft tumor models, leading to sharp retardation of tumor growth (57). No effect was observed for CPPI or EPPI on the AR variant proteins (57).

IMPPE (SID3712502) was another small molecule identified from the 2GFP-AR fusing protein screening assay with a robust inhibitory effect at 2.0 μM concentration on AR nuclear translocation and its downstream target PSA gene expression, plus downregulation of AR gene expression at a higher concentration of 10 μM (55). Further study found that IMPPE inhibited both full-length and LBD-lacking AR activity at a relatively high dose (>10 μM) and suppressed 22RV1 but not PC-3 cell-derived xenograft tumor growth at a dose of 25 mg/kg/day in castrated nude mice (58).

JJ-450 is an IMPPE scaffold analog with higher potency and better physicochemical properties (59). JJ-450 at 10 μM concentration inhibited both the transcriptional activities of the full-length and splicing variant AR proteins in CRPC cells by blocking AR binding to its target gene promoter without affecting AR protein levels (59). In CRPC xenograft models derived from 22RV1 and VCaP cells, JJ-450 at 10 mg/kg/day dose suppressed xenograft tumor growth by 60%, slightly better than Enzalutamide (59). Especially, JJ-450 was found to block the nuclear translocation and activity of the AR F876L mutant protein identified from Enzalutamide-resistant CRPC patients and LNCaP cells after long-term exposure to Enzalutamide (60–62).

The AR DBD-Hinge region has P-box and D-box motifs responsible for dimerization and DNA binding after androgen stimulation (146). Using a virtual in-silico drug design approach (63–65), a surface-exposed region (aa579-610) on the AR DBDH domain was discovered as a potential target site by small-molecule compounds, including VPC-14228 and VPC-14449 (66). These two compounds at 10 μM concentration selectively suppressed AR (full-length and splicing variant proteins) but not ER or GR activity by blocking AR interaction with the target gene promoters without affecting AR nuclear translocation and protein stability (66). In LNCaP cell-derived xenograft experiments, VPC-1449 at 100 mg/kg/day dose suppressed tumor growth at a similar extent as Enzalutamide (10 mg/kg/day) (66).

The AR protein is critical for prostate cancer progression by transcriptionally modulating gene expression after activation by androgens via binding on its LBD. Metastatic prostate cancers are initially treated with androgen deprivation or castration therapies (surgical or medical) based on the findings reported about 80-years ago. However, this androgen removal approach is not curative for prostate cancers, and the diseases often relapse and progress to the CRPC stage. Since most of these CRPCs are still AR addictive, current clinical therapies mainly focus on blocking androgen to bind with the AR LBD (AR antagonists) or reducing androgen production (CYP17a1 inhibitors) in non-testis tissues, including prostate cancer tissues. However, treatment resistance eventually develops in part due to AR gene mutation and mRNA splicing events (e.g., AR-V7) in virtually all CRPC patients. Furthermore, after long-term treatment with AR antagonists, up to 20% of CRPC patients will develop an even more aggressive subtype, neuroendocrinal prostate cancer (NEPC). Therefore, the androgen removal and blockage approach are non-curative and leads to a more aggressive disease.

To overcome this obstacle of treatment resistance, research has shifted from androgens to the AR protein in the last 20 years ( Figure 1 ). The initial approach was the antisense oligonucleotides (ASO) targeting the AR mRNA to reduce AR protein production in prostate cancer cells. Due to the inhibitory nature of the ASO approach on protein production, tumor growth was only suppressed but not eradicated in xenograft models. In contrast, our group used the siRNA approach that efficiently eliminated the AR protein from prostate cancer cells. Nanoparticle-loaded AR siRNA resulted in xenograft tumor regression and eradication owing to robust cell death after AR protein removal in prostate cancer cells. Unfortunately, this AR siRNA project was stalled due to a failure in the patent application.

Targeting AR protein stability has emerged in recent years as the hotspot in developing new therapeutics for advanced prostate cancers, and several small molecules were reported to reduce AR protein stability. The curcumin analog ASC-J9, Ailanthone, HG122, and CUDC-101 induced AR protein degradation in prostate cancer cells. However, the AR or prostate cancer tissue specificity is not established with these small molecules. The PROTAC technique for AR-specific degradation showed a promising result. The AR PROTAC ARV-110 is tested as a combinational treatment with Abiraterone in a clinical trial. However, these AR CTD-targeting PROTACs utilized AR LBD ligands, and therefore, they are inactive on AR CTD splicing variants, a critical mechanism for treatment resistance in CRPC patients. Interestingly, some other agents specifically targeted the AR-V7 variant for degradation, including Niclosamide, CUDC-101, Thailanstatins, Rutaecarpine, Indisulam, and Nobiletin. Combining AR antagonists, PROTAC molecules, and AR-V7 inhibitors might provide synergistic effects in the clinic.

Targeting AR NTD is another approach to bypass AR CTD splicing defects. The first generation of AR NTD inhibitor EPI compounds was failed in clinical trials due to excessive bill burden. The second generation of EPI compound with 20-fold higher potency is being tested as a combinational treatment with Enzalutamide in a clinical trial. UT-34 targets the AR NTD and is also waiting for a clinical test.

AR nuclear translocation is an important event for its activity as a transcription factor. Two novel compounds, IMPPE and JJ-450, were recently developed to block AR nuclear translocation. These two compounds showed a very permissive result in animal models. In addition, an AR DBD blocking agent VPC-14449 was reported to suppress AR interaction with its target gene promoter in the nuclear compartment and was found to suppress tumor growth in mice. These compounds are all needed for clinical testing.

AR activity is only temporally suppressed during prostate cancer treatment by androgen deprivation and AR antagonists. Due to these treatment stresses, prostate cancer cells used other cellular signal pathways and/or splicing variants for AR reactivation, resulting in treatment resistance. Therefore, complete removal of the AR protein from prostate cancer cells will eliminate all events of AR reactivation after ADT and anti-AR therapy. Especially in the early phase of treatment, most prostate cancer cells are still AR-dependent. Simultaneously removal of the AR protein and androgens will result in robust cell death, leading to a possible curative result or long-term disease-free survival. In addition, early reduction of the AR protein in the androgen-responsive phase of prostate cancer will reduce the likelihood of transcriptional reprogramming (88, 93, 147). Also, tissue-specific delivery of the AR protein degradation agents will restrict potential side effects.

All authors participated in drafting the manuscript. All authors contributed to the article and approved the submitted version.

This work was partially supported by a grant from KUMC Lied pilot program and DoD PCRP PC190026 to Benyi Li, MD/PhD.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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