Prostate cancer is the most commonly diagnosed cancer in men all over the world. Localized cancers in the early stages can be well managed by surgical or radiation therapy. Metastatic prostate cancer is treated with androgen deprivation therapy because androgen signaling is essential to the prostate tumor growth and anti-apoptotic ability. However, resistance develops quickly in the clinical course and leads to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) functions as a nuclear receptor to facilitate ligand-dependent transcriptional activation in the nucleus. AR interacts with several tissue-specific transcription factors such as forkhead box protein A1 (FOXA1) and regulates epigenetic status by recruiting epigenetic factors. In addition, AR transcriptional activity is modulated by interacting directly or indirectly with non-coding RNAs such as long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). Notably, enhanced AR signaling in CRPC has been documented in several studies; however, which of these factors are important for the biological function it remains poorly understood. Here, I review our current knowledge of the mechanistic roles of AR involved in prostate cancer progression and discuss the importance of the prostate cancer-associated signals.
- androgen receptor
- prostate cancer
- non-coding RNA
Released hormone to an entire body is responsible for the development of various human diseases and physiology. Androgens, male sex hormones, mediate their effects predominantly by binding to the androgen receptor (AR), a member of the ligand-dependent nuclear receptor superfamily. Two major androgens, testosterone and dihydrotestosterone (DHT), bind and activate AR to regulate target gene expression . Testosterone produced in the testes is the most abundant androgen. After diffusing into cells, testosterone is converted to dihydrotestosterone (DHT) by the enzyme 5α-reductase . DHT directly binds to and activates AR even more tightly than testosterone . Androgens play a key role in the development of the male genital tract favoring differentiation and external genitalia during fetal life and sexual characteristic during puberty and are required for the establishment of adult sexual function. In addition to the classical activities in the male reproductive system, androgens also have anabolic functions in other tissues such as bone, muscle and central nervous systems . Notably, AR has a central role in prostate cancer progression. In this review, we focus on AR functions through epigenetic factors and non-coding RNAs that have been shown to play a role in prostate cancer progression.
2. De-regulation of AR during the progression of prostate cancer
AR is a member of the nuclear receptor superfamily  and plays a key role in androgen signaling (Figure 1A). In the absence of ligand, AR is expressed mainly in the cytoplasm forming a complex with molecular chaperones and co-chaperones from the heat shock protein (Hsp) family. Upon androgen treatment, a conformational change in the complex leads to nuclear translocation of AR. In the nucleus, AR binds as a dimer to specific DNA sequences called androgen responsive elements (AREs), which are found in the vicinity of AR target genes . AR activates gene expressions by modifying the epigenetic condition of AR binding regions . Generally, nuclear receptors including AR have multiple domains called DNA binding domain, a ligand-binding domain (LBD), and an N-terminal domain (NTD), [2, 8, 9]. In the NTD, the transcriptional activation function 1 (AF1) domain promotes transcriptional activation with or without ligand binding , which is associated with enhanced AR function. AF2 domain in the LBD interacts with co-regulators with LXXLL motif . Point mutations mapped to the LBD have been identified to have relevance with the treatment-resistance to drugs targeting AR in prostate cancer [11, 12].
Prostate cancer is one of the leading causes of cancer morbidity and mortality in developed countries. Androgens induce proliferation of prostate epithelial cells or prostate cancer tumor growth . Early diagnosis of prostate cancer is currently based on the measurement of serum prostate-specific antigen (PSA), a representative AR target gene. Treatment of localized prostate cancer is determined based on clinico-pathological factors such as Gleason score, initial PSA level, patient’s age and clinical tumor stage . Because AR and its downstream signaling are essential for the development and progression of both localized and advanced metastatic prostate cancer, hormone therapy is a first-line and initially successful strategy for treating advanced prostate cancer. Androgen deprivation therapy decreases the circulating testosterone levels to a very low amount, a condition called chemical castration of men .
However, most of these tumors relapse and progress to hormone therapy resistant prostate cancer (HRPC) or castration-resistant prostate cancer (CRPC). To overcome CRPC/HRPC, new AR inhibitors have been developed. Abiraterone acetate, a potent inhibitor of CYP17 reduces testosterone synthesis from cholesterol . Despite suppression of circulating testosterone, castration does not decrease androgens enough from the prostate tumor microenvironment and residual androgen levels are well within the range capable of activating AR. Accordingly, therapeutic strategies effectively targeting production of intratumoral androgens are necessary. Clinical studies showed that abiraterone improved overall survival, progression free survival, delayed initiation of chemotherapy and doubled the time to first skeletal event. Enzalutamide (MDV3100) is another novel endocrine treatment with reported significant anti-tumor activity . It is an AR-receptor-signaling inhibitor, blocking nuclear translocation, DNA binding, and co-activator recruitment. Enzalutamide significantly prolonged the survival of men with metastatic CRPC after chemotherapy . Although these new types of drugs bring impressive results, the duration of response is variable, and a majority eventually progress with a rising PSA. While the mechanisms determining resistance have not been fully elucidated, persistent AR activation provides a compelling rationale for developing more strategies to inhibit AR .
Enhanced AR downstream signals are caused by AR gene amplification, point mutation, AR variants (particularly AR-Vs), hypersensitivity to androgens, or intratumoral steroidogenesis [20, 21, 22, 23]. AR mRNA is alternatively spliced to AR-Vs and results in prematurely termination of the full AR protein. Most AR-Vs are missing LBD, however, retain the NTD to drive transcription androgen-independently. Among these variants, AR-V7 is expressed in HRPC/CRPCs most frequently and could be the therapeutic target of tumors resistant to existing therapies directed to androgen/AR [24, 25]. In addition, elevated AR expression increased the reactivity of prostate cancer cells to castrate levels of androgens and promotes resistance to AR-targeting drugs. Increased AR expression in CRPC is often mediated by AR gene amplification. Thus, it is critical to investigate AR downstream-signaling or regulatory mechanisms by AR to understand how CRPC develop among the patients.
3. Investigation of AR-regulators and target genes
Chromatin immunoprecipitation (ChIP) and sequencing studies have revealed locations of AR bindings have been found in prostate cancer cells [26, 27, 28]. AR target genes such as ADP-ribosylation factor GTPase-activating protein 3 (ARFGAP3) , Amyloid Beta Precursor Protein (APP) , Acyl-CoA Synthetase Long Chain Family Member 3 (ACSL3) , claudin-8  and Transforming acidic coiled-coil-containing protein 2 (TACC2) , which promotes tumor growth by regulating cell cycle, intratumoral steroidogenesis, cell structure, have been identified in my research. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism, contributing to prostate cancer progression . Ubiquitin-conjugating enzyme E2 C (UBE2C) was identified to be a CRPC-specific AR target gene and promoted G2/M mitotic cell division in prostate cancer cells . Interestingly, androgen regulates p53 localization by inducing GTPase-activating protein-binding protein 2 (G3BP2), which is an AR target gene . G3BP2 associates with p53 and SUMO E3 ligase RAN binding protein 2 (RanBP2), promoting p53 nuclear export via increased p53 sumoylation. Elevated G3BP2 expression repressed docetaxel-mediated apoptosis and promoted CRPC tumor growth.
3.1. Epigenetic factors
In prostate cancer, deregulation of AR interaction with its coregulators within nucleus is common and activation of coregulators is frequently observed. AR has a role in the ligand-dependent epigenetic changes by interacting with various coregulators such as histone-modifying enzymes. DNA, histones and other proteins formed chromatin as a highly ordered structure. Chromatin forms a unit called the nucleosome consisting of a histone octamer (H2A, H2B, H3 and H4, two pairs of each) and DNA. Both DNA methylation and histone modification patterns have also been investigated in prostate cancer. Histone modifications affect the interaction of DNA with histones, transcription factors or other proteins binding to DNA, thus playing a role in the epigenetic control of biological events. Lysine, arginine, serine and threonine residues enriched in N-terminal histone tails serve as substrates for post-translational modifications such as acetylation, phosphorylation, methylation, ubiquitination, sumoylation and deamination. Histone H3, one of the major histones, is most representative for epigenetic regulation. The methylation of lysine on position 9 (H3K9) and H3K27 is an epigenetic mark of condensed chromatin and silent loci. The methylation of H3K4 and H3K36 is associated with open chromatin structures. Acetylation of lysine residues of H3 is also correlated with enhanced enhancer/promoter activation. .
In a recent report, the potential for BET bromodomain protein inhibitors as a novel epigenetic approach to treatment of CRPC has been shown . In prostate cancer cell lines, BET bromodomain inhibitor, JQ1, was demonstrated to induce apoptosis and down-regulate AR target gene expression. Bromodomain and the extra-terminal (BET) subfamily of human bromodomain proteins (BRDs), with a focus on BRD4, were found to play a major role in AR signaling and interact with AR via bromodomain 1/2. JQ1 inhibits this BRD4-AR bond, resulting in removal of RNA polymerase II from AR target genes . This study suggests for the first time that modulating epigenetic function of AR could be a useful strategy to overcome clinical problems associated with AR signals.
Histone acetylation loosens the nucleosome packing within chromatin to increase DNA accessibility, resulting in the recruitment of chromatin remodeling factors that lead to enhanced transcriptional activity. The BRD has the ability to recognize acetylated lysine residues . This activity allows BRDs to play a vital role in histone acetylation-mediated gene transcriptional regulation in chromatin. Such BRDs included Tripartite motif containing 24 (TRIM24), which interacts with AR and are highly expressed in CRPC . Other studies revealed that E3 ubiquitin ligase substrate binding adaptor speckle-type POZ protein (SPOP) binds to and induces ubiquitination of BRD4 for degradation. In many prostate cancer tissues, SPOP is the most frequently mutated to enhance the expression level of BRD4 .
AR regulates the histone modifications in AR binding sites (ARBSs) and promotes enhancer activity by directly interacting with many co-regulators including steroid receptor coactivators (SRCs) or other histone-modifying enzymes [6, 39] (Figure 1B). Methylations of H3K4 (mono-, di- or tri-methylation) indicate the active promoter/enhancer regions  and promoted by the SET1/MLL histone methyltransferase (HMTase) complex. Menin protein binding to the N-terminus of MLL is important for MLL target gene expressions. Menin directly binds to AR and recruited MLL complex. Menin is highly expressed in CRPC tissues and associated with castration-resistant tumor growth . Importantly, small molecule inhibitors against menin-MLL interaction could be the new useful drugs for CRPC. MLL complex plays an important role for androgen-mediated gene induction and its activity is regulated finely. After androgen stimulation, protein kinase C-related kinase 1 (PRK1) promotes histone H3 threonine 11 phosphorylation (H3T11P) . WD repeat containing protein 5 (WDR5), a subunit of the SET1/MLL complex, associates with H3T11P and then promotes the recruitment of the MLL complex for H3K4 tri-methylation (H3K4me3) in ARBSs . WDR5 is a critical epigenetic integrator and is overexpressed in prostate cancers. PRK1 kinase activity facilitates demethylation of H3K9 by cooperating with lysine-specific demethylase 1 (LSD1) [42, 44]. In addition, Protein kinase C beta 1 (PKCβ1) phosphorylates histone H3T6 to prevent lysine specific demethylase including LSD1 from histone H3K4 demethylation . Moreover, C-terminal binding protein 2 (CTBP2) is an androgen-responsive cofactor of AR. CTBP2 repressed tumor-suppressor genes and AR corepressors in prostate cancer cells, such as Nuclear receptor co-repressor (NCOR) and receptor-interacting protein 140 (RIP140), by binding with AR to the promoter enhancers of these genes. Moreover, global gene-expression analyses revealed a positive effect on androgen-mediated gene expression, and CTBP2 silencing was found to increase AR interactions with corepressors that limit histone modification . Thus, these findings have a clinical relevance to develop new drugs for treatment by regulating epigenetic status .
Histone modification is also important for increased AR expression in CRPC . Recent studies showed that both transcriptional and epigenetic changes are important for AR upregulation in prostate cancer. To introns of the AR gene, recruitments of AR and its associated cofactors such as LSD1, which represses transcription by inhibiting histone H3K4 methylation are induced by androgen. This feedback loop mechanism regulates AR expression negatively by androgen. Interestingly, after long-term incubation in castration level of androgens, AR expression increases in prostate cancer cells and then low levels of androgens can activate AR regulated genes in CRPC without repressing genes such as the AR itself.
3.2. Collaborative transcription factors
Functional ARBSs were not only determined by sequence motifs but also chromatin accessibility. ChIP-sequence (ChIP-seq) analyses using next-generation sequencers have been developed as a high-throughput strategy to identify transcription factor binding regions . For instance, AR ChIP-seq was performed in two LNCaP-derived prostate cancer cell lines with higher AR expression . Interestingly, more ARBSs were obtained in these cell lines when they were treated with low concentrations of androgen. These data indicate that the higher expression of AR sensitizes the receptor binding to genome, thus illustrating the mechanism that the AR signaling pathway is enhanced in CRPC.
Furthermore, by analyzing enriched motifs around ARBSs, AR-associated transcription partners such as forkhead box protein A1 (FOXA1) , ERG, GATA2 , Oct1 , RUNX1  and NKX3–1  have been mapped to the prostate cancer genome and these studies suggested the global role of these factor to activate AR-driven transcriptional program. Among them, a chromatin-opening transcription factor, FOXA1, is able to directly bind to the chromatin to open up the local nucleosomal domain (Figure 1A). In prostate cells, FOXA1 protein has been shown physically interact with the AR protein and plays critical roles in regulating the transcription of prostate genes . Moreover, ARBSs in CRPC tissues were found by ChIP-seq and most of them could not been identified in cell lines. Many adjacent genes were
4. The roles of non-coding RNAs in AR action and prostate cancer biology
Recent advances in DNA sequence technology have demonstrated that more than 90% of the human genome is actively transcribed. The encyclopedia of DNA elements (ENCODE) project has shown that only 2% of these transcripts are translated into proteins . The non-coding RNAs (ncRNAs), that occupy the majority of transcripts in the nucleus, were initially thought as the “dark matter.” Non-coding RNAs are broadly categorized into short and long transcripts. Short non-coding RNAs with a length within 200 nucleotides include such as transfer RNA, microRNA (miRNA), and snoRNA. miRNAs play important roles in cancer by post-transcriptional modification of target mRNA or protein expression. Long non-coding RNAs (lncRNAs) represent most of the transcribed ncRNA in the human genome longer than 200 bp. GENCODE v27 includes 15,778 human lncRNA-related genes, which produce 27,908 lncRNAs .
LncRNAs exhibit similar structure and biogenesis as mRNAs. They are polyadenylated and may function in either nuclear or cytoplasmic compartments. Growing number of evidences have shown that lncRNAs are involved in numerous human diseases including cancer . Global nuclear run-on sequencing (GRO-seq) was developed as a new technology to detect androgen-induced transcripts including lncRNAs . This study has demonstrated that the production of enhancer-templated non-coding RNAs (eRNAs) is important for nucleosome remodeling to induce enhancer/promoter interaction by looping and gene activation. It was also shown that androgen promotes both transcriptional initiation and elongation. These active enhancers are tuned dynamically to modulate gene expression network in prostate cancer. AR is widely recruited to these eRNA-bound enhancer- promoter regions for activating the genes in the vicinity. Interestingly, knockdown of eRNA represses androgen-dependent enhancer promoter interaction and gene activation . DNA nicking activity of topoisomerase I (TOP1) was found to produce robust eRNA for enhancer activation. Furthermore, DNA damage repair machinery is recruited kinetically to the AR-regulated enhancers .
LncRNAs are able to fold into secondary and tertiary structures by which they perform their function (Figure 2A). Direct regulation of AR epigenetic function by lncRNA is strikingly receiving attention among all.
Global transcriptome analysis showed that most of the genome can be transcribed from both sense and antisense strands. More than 1000 pairs of sense/antisense transcripts were obtained and antisense transcription is involved in such bidirectional gene regulation [70, 71]. Sense-transcript is regulated by antisense through several mechanisms. For example, post-transcriptional degradation is caused by antisense transcript. Another mechanism is recruitments of antisense RNA associated transcription factors for epigenetic regulation. In prostate cancer, an lncRNA,
Genome-wide androgen-regulated transcriptome analysis identified a new androgen-responsive lncRNA,
Interestingly, another report has shown that PSF binds to another CRPC-associated lncRNA,
These analyses of lncRNA functions revealed a novel transcriptional regulatory mechanism. Several lncRNAs such as
MicroRNAs (miRNAs) are evolutionally conserved single-stranded small non-protein Generally miRNAs binds to the 3′ untranslated region (UTR) of mRNAs to inhibit their translation. For examples, dysregulation of miRNA expression profiles during the progression of prostate cancer have been discussed . In these studies, miR-21, miR-29a/b, miR-32, miR-99a, miR-148a, miR-125b and miR-141 were found to be androgen-regulated miRNAs and dysregulated in prostate cancer. Upregulated miR-21 enhanced AR-dependent cell proliferation and associated with development of CRPC [83, 84]. Another androgen regulated miRNA, miR-125b targets apoptosis inducing factors regulated by p53 (PUMA and BAK1) . Thus, by repressing these genes, overexpression of miR-125b in tumors collapses the balance between pro- and anti-apoptotic processes. It was shown that miR-148a is also regulated by androgen and highly induced in AR positive prostate cancer cells. miR-148a targets cullin-associated and neddylation dissociated 1 (CAND1), a cell cycle regulator, to promote cell proliferation . Moreover, miR-32 inhibits apoptosis by targeting BIM, a pro-apoptotic member of the BCL2 family . Both miR-32 and miR-148a were overexpressed in CRPC tissues, indicating that these miRNAs have important roles in the promotion of castration-resistance .
DNA methylation is also the representative epigenetic mark adding a methyl group to the 5′ position of cytosine (5-mC). DNA methylation is added or removed in a spatially and temporally defined context throughout the genome including enhancer/promoter regions. DNA methyltransferases (DNMTs) contributes to the process as enzymes. DNMTs include DNMT3A/DNMT3B for de novo and DNMT1 for maintenance of methylation. The ten-eleven translocation (TET) family proteins catalyzed the production of 5-hydroxymethylcytosine (5-hmC), an oxidation product of 5-mC. Several studies have demonstrated that 5-hmC is not only an intermediate product of a demethylation process, but can also function as a stable epigenetic mark .
Interestingly, recent study has demonstrated that miR-29 family and miR-22 are highly induced by androgen in hormone-therapy resistant prostate cancer . In prostate cancer clinical samples revealed that the expression level of miR-29a/b is negatively associated with that of TET2. Interestingly,
Studies of non-coding RNAs in AR signals are mainly reported in the research field of prostate cancer. However, knockout of the miRNA processing enzyme, Dicer 1, Ribonuclease III (DICER), in mice inhibited AR function tissue specifically in muscle. In addition, castration in rats inhibited the expression of a large set of miRNAs in prostate and muscle, suggesting the importance of miRNAs in the physiological functions of androgens in other tissues .
5. Conclusion and perspective
The majority of prostate cancer and CRPC tumors are driven by AR signaling. AR-mediated resistance to hormone therapy can be acquired by multiple mechanisms. AR mutation, amplification and truncated variants have been identified to explain aberrant AR activation in prostate cancer progression. AR coregulators and collaborating transcription factors are essentials for AR to exert its transcriptional activity. Recently the role of non-coding RNAs such as lncRNAs and miRNAs has been realized to play a critical role in AR activation and prostate cancer progression. Such alterations of AR function lead to positive or negative regulation of the growth and invasion ability of cancer cells. Although AR-targeting drugs have been developed, we could not eliminate CRPC due to the adaptive evolution of the disease during the treatment. Combinational therapies are required to overcome CRPC problems. Therefore, it is urgent to find better predictive biomarkers or therapeutic targets which have an efficacy for diagnosing and treating prostate cancer and CRPC.
This work was supported by grants from the JSPS (number 17H04334), Japan; grants from Takeda Science Foundation, Japan.
Conflict of interest
The author declares no conflict of interest.