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Abstract

Androgen receptor plays a pivotal role in prostate cancer progression, and androgen deprivation therapy to intercept androgen receptor signal pathway is an indispensable treatment for most advanced prostate cancer patients to delay cancer progression. However, the emerging of castration-resistant prostate cancer reminds us the alteration of androgen receptor, which includes androgen receptor mutation, the formation of androgen receptor variants, and androgen receptor distribution in cancer cells. In this review, we introduce the process of androgen receptor and also its variants’ formation, translocation, and function alteration by protein modification or interaction with other pathways. We dissect the roles of androgen receptor in prostate cancer from molecular perspective to provide clues for battling prostate cancer, especially castration-resistant prostate cancer.

Keywords

Androgen receptor variants translocation dimerization cell signals

Introduction

Prostate cancer (PCa) has been the most prevalent disease in man for a long time, and it has been estimated that 180,890 new PCa cases account for 21% of all cancer diagnosis in 2016.¹ In the course of battling this kind of disease, androgen deprivation therapy (ADT) is an indispensable treatment for most advanced PCa patients to delay cancer progression since 1940s as established by Huggins and Hodges.² Nowadays, surgical castration quitted the historical stage, and various types of hormone therapy drugs are applied in clinical stage, such as MDV3100³ and abiraterone acetate.⁴ Although patients developed castration-resistant prostate cancer (CRPC) despite castrated testosterone levels after 24–36 months, ADT induced biochemical and clinical responses in more than 90% of patients, and more therapeutic effect can be achieved by combining hormone therapy and chemotherapy in CRPC.⁵ Increasing evidence indicates that even if the disease progresses into castration-resistant stage, maintained androgen receptor (AR) activity is observed and continued androgen suppression is recommended.^6,7 Several resistance mechanisms are found to be related with AR or AR mutation, amplification, splice variants, and AR alterations.⁸ Furthermore, in addition to genomic action of AR, AR exhibits anti-apoptotic effect on bone and other cell types via a non-genomic action.^9,10 In this review, we introduce the process of AR formation, the action it performs, and the modulator affect to bring back the biological network of AR in view.

Genetic alterations of AR: mutations and variants

AR also known as dihydrotestosterone receptor (DHTR) or nuclear receptor subfamily 3, group C, member 4 (NR3C4) is located on Xq12. AR spans ~180 kb of DNA containing 8 exons (recently, a novel AR exon, termed exon 9, was discovered,¹¹ which is mentioned below); normally, exon 1 encodes for N-terminal domain (NTD) which is also named activation function 1 (AF1), which is responsible for regulating AR transcription. Exons 2 and 3 encode for DNA-binding domain (DBD), which determines the binding to specific DNA region (termed androgen response element (ARE)) and regulates gene expression. Exons 4–8 encode for hinge region and ligand-binding domain (LBD); the hinge region is responsible for AR dimerization, and LBD can bind with ligand leading to AR conformation changes subsequently active in its downstream.¹² The four regions constitute the full-length AR, which is a 110-kDa protein composed of 920 amino acids (Figure 1). As a ligand-activated transcription factor, full-length AR can be activated by binding its ligand such as testosterone or dihydrotestosterone.¹³

Figure 1.

The structure of androgen receptor. AR is a 110-kDa protein composed of 920 amino acids and divided into four regions: N-terminal domain (NTD), DNA-binding domain (DBD), hinge region, and ligand-binding domain (LBD). Several amino-acid mutations lead to AR function alteration, such as L701H and T878A. Nuclear localization signal (NLS) and nuclear export signal (NES) are necessary for AR nuclear import and export. P-box and D-box are responsible for the specific recognition of the DNA response element and gene expression regulation.

Genetic aberrations of the AR caused by mutations, rearrangements, and polymorphisms result in variations in mutant receptor (Figure 2(1)), and these alterations may have distinct reactivity in response to androgen or diverse functions compared to wild-type AR.¹³ Mutations in the AR can result in loss-of-function or gain-of-function AR alterations. Loss-of-function alterations can cause androgen insensitivity syndrome (AIS), and gain-of-function alterations were largely found in PCa, breast cancer, and larynx cancer. More than 159 AR mutations have been found in PCa tissue,¹⁴ and AR mutations with a frequency of 8%–25% were found in CRPC. Some AR mutations like L701H permit the AR activated by glucocorticoids or progesterone and therefore confer to ADT resistance.¹⁵ To know AR mutation in more detail, readers can refer to the study by Eisermann et al.¹³

Figure 2.

AR formation, translocation, and dimerization. (1) Genomic-level variable factors affect AR production. ① Rearrangements, ② mutations, and ③ polymorphisms result in variation in mutant androgen receptor. (2) Spliceosome excision of the intron sequences and re-ligation of the adjacent exons lead to AR-variant formation. Androgen induces AR nuclear translocation, and some other factors can modulate nuclear trafficking of AR in distinct mechanisms. (I) SNURF facilitates AR import to nuclei by interacting with AR through a region overlapping with the NLS. (II) AR-V7 induces AR nuclear translocation in heterodimer manner. (III) Tip60 acetylates AR and promotes AR localization in the nucleus. Three forms of protein dimerization have been described for the AR: (a) androgen-induced N-terminal/C-terminal (N/C) interaction ((a1) intramolecular N/C interaction; (a2) intermolecular N/C interaction), (b) LBD-LBD dimerization, and (c) DNA-dependent dimerization mediated through the DBD.

AR amplification/overexpression accounts for 80% high levels of AR expression in CRPC,¹⁶ and increased expression of AR and multiple downstream genes suggest partial reactivation of AR transcriptional activity.¹⁷ Compared with AR amplification, AR variants were relatively comprehensively studied. Some AR variants have truncated C-terminal, lacking the LBD, constitutively inducing the expression of AR-regulated gene, and promoting tumor progression, even under castrate conditions.¹⁸ Two mechanisms such as AR gene rearrangement and increased precursor messenger RNA (pre-mRNA) splicing are proposed for contributing to at least 20 variants¹⁹ (Table 1). It was demonstrated that AR variant resulted from a 35-kb intragenic duplication of AR exon 3 and flanking sequences in 22Rv1 cells was related to its resistance.²⁰ Structural alterations in the AR gene are also linked to stable ARv567es expression as gain-of-function splicing alterations in PCa cell lines, xenografts, and clinical specimens.²¹ The AR pre-mRNA transcribes from DNA sequence by the nuclear RNA polymerase II (RNAPII) and then undergoes a number of processing steps to yield messenger RNA (mRNA).²² Spliceosome (which is composed of a core of five small nuclear ribonucleoproteins, termed U1, U2, U4, U5, and U6, and an additional ~200 protein factors) assemble to the pre-mRNA splicing regions,²³ excision the intron or specific region and re-ligation of the adjacent exons or remaining sequence (Figure 2(2)). Other mechanisms, such as proteolytic degradation of full-length AR by calpain-2, can also lead to AR-variant production.²⁴ In PCa, two well-characterized AR variants, AR3 and ARv567es, were found to promote PCa progression.^25,26 But they could be detected in normal prostate tissues, suggesting that the mere presence of them was not likely to be pathogenic.²⁷ AR variants played a complicated role in PCa as overexpression of AR45 reduced the proliferation of LNCaP cells.²⁸ Other variants were less studied, and their expression and function in PCa demand further research.

Table 1.

The characteristic of AR variants.

AR variants	Alternative names	Transcript structure	Mainly variant region^a	Predicted molecular weight (kDa)	Reference
AR-FL	N/A	1/2/3/4/5/6/7/8/9	Full-length AR	110	Androgen receptor (AR)²⁹ and Sun et al.³⁰
AR-V1	AR4	1/2/3/CE1	630-648: AVVVSERILRVFGVSEWLP649-920: Missing	71	Hu et al.³¹
AR-V2	N/A	1/2/3/3/CE1	629-687: GKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGAAVVVSERILRVFGVSEWLP689-920: Missing	76	Hu et al.³¹
AR-V3	AR1/2/2b, AR6	1/2/CE4	590-642: GFFRMNKLKESSDTNPKPYCMAAPMGLTENNRNRKKSYRETNLKAVSWPLNHT644-920: Missing	71	Hu et al.¹¹, Hu et al.,³¹ and Guo et al.³²
AR-V4	AR1/2/3/2b, AR5	1/2/3/CE4	629-681: GFFRMNKLKESSDTNPKPYCMAAPMGLTENNRNRKKSYRETNLKAVSWPLNHT683-920: Missing	75	Hu et al.³¹
AR-V5	N/A	1/2/3/CE2	629: D630-920: Missing	69	Hu et al.³¹
AR-V6	N/A	1/2/3/CE2	630-634: GSRVS635-920: Missing	70	Hu et al.³¹
AR-V7	AR3	1/2/3/CE3	629-644: EKFRVGNCKHLKMTRP645-920: Missing	71	Hu et al.³¹
AR-V8	N/A	1/2/3/intron 3	629-638: GFDNLCELSS638-920: Missing	70	Watson et al.³³
AR-V9	N/A	1/2/3/CE5	629-651: DNLPEQAAFWRHLHIFWDHVVKK651-920: Missing	72	Watson et al.³³
AR-V10	N/A	1/2/3/intron 3	627-665: PSSGTNSVFLPHRDVVRTGCRSNSGYHSCSCEYHDYCFL665-920: Missing	73	Watson et al.³³
AR-V11	N/A	1/2/3/intron 3	629-648: GKILFFLFLLLPLSPFSLIF648-920: Missing	71	Watson et al.³³
AR-V12	ARv567es	1/2/3/4/8/9	725-734: DCERAASVHF735-920: Missing	81	Sun et al.³⁰
AR-V13	N/A	1/2/3/4/5/6/9	817-819: NHT820-920: Missing	90	Hu et al.¹¹
AR-V14	N/A	1/2/3/4/5/6/7/9	870-877: ITPDAMYL878-920: Missing	96	Hu et al.¹¹
AR45	N/A	2/3/4/5/6/7/8	1-532: Missing533-539: MILWLHS	43	Androgen receptor (AR)²⁹

CE: cryptic exons; N/A: not available.

Mainly descripts the different regions in C-terminal sequence, and protein may differ from AR-FL in several amino acids in other regions.

Nuclear translocation of AR

Detecting the expression of AR in various cell lines revealed a cell-line-specific subcellular distribution. AR was predominantly located in nucleus when transfection occurs in HeLa cells, whereas mainly located in cytoplasm when transfection occurs in COS-1 cells.³⁴ It is unknown whether AR location would be in cell-specific manner in PCa cell line. As a large protein whose molecular weight is more than 40 kDa (threshold for passive diffusion across the nuclear pore complex), AR must be actively transported through the nuclear pore complex to enter or leave the nucleus.³⁵ In the presence of hormone, AR was located in the nucleus. The signal responsible for nuclear import is the presence of amino-acid residues 608–625 (or in the DBD and hinge region, 617–633³⁶), which is functionally called as the nuclear localization signal (NLS).³⁴ Ligand-binding AR leads to conformation alteration, and then, nuclear import factor importin-alpha functions as a receptor that recognizes and binds to specific NLS motifs on cargo proteins.³⁷ Nuclear AR can be exported to the cytoplasm, and upon ligand withdrawal, it is indicated that AR contains both nuclear localization signal (NLS) and nuclear export signal (NES). NES (a region in LBD) is necessary for AR nuclear export and is dominant over the NLS in the absence of hormone.^35,38 Recently, the region of 50–250 of the NTD of AR was found to promote cytoplasmic localization.³⁹

Besides androgen induces AR nuclear translocation, some other factors can modulate nuclear trafficking of AR (Figure 2(I)–(III)). The small nuclear Really Interesting New Gene (RING) finger protein SNURF can interact with AR through a region in the NLS and facilitates AR nuclear import and retards its cytoplasm translocation on hormone withdrawal.⁴⁰ Another protein Tip60 can increase the acetylated AR and AR translocation into the nucleus even without androgen; in contrast, Tip60 knockdown induced AR export to the cytoplasm, accompanied with suppression of the growth of AR-positive PCa cells by inducing cell-cycle arrest at the G1 phase.⁴¹ Later, two major AR-Vs, AR-V7 and ARv567es, were found to induce AR nuclear translocation to subsequently activate AR target genes and confer castration-resistant cell growth.²⁵ Different with AR-FL which requires Hsp90 and importin-beta for nuclear translocation, the AR splice variant is independent of these classical signals. Variant consisting of AR NTD and DBD is sufficient for nuclear localization and activating AR target genes in the absence of androgen.⁴² The NES region of AR was found to interact with splicing factor Prp8, and Prp8 knockdown induced AR accumulation in nucleus; human PCa specimens with high Gleason scores were detected to have relatively low Prp8 expression, and it is difficult to determine whether Prp8 knockdown also increases AR ubiquitination.⁴³

Modifications of the AR action

Activated AR binds to the ARE or negative ARE (nARE) via DBD in nucleus to regulate gene expression.^44,45 The binding depends on AR conformation, and modification of AR implies that AR elicits or promotes/inhibits gene expression. AR conformational alteration or post-translational modification by phosphatase or cofactors can lead to cancer progression or regression.

AR dimerization

Three kinds of AR dimerization have been proposed, including dimerization through DBD depending on DNA binding, the N-terminal/C-terminal (N/C) interaction in the NTD and LBD, and LBD-LBD dimerization (Figure 2(a1), (a2), (b), and (c)).⁴⁶ Similar with other steroid receptors, AR functions as a dimer binding to DNA response elements consisting of two hexamers (5′-AGAACA-3′) separated by a 3-base spacer.³⁸ The first zinc finger residing in the proximal box (amino acids 577–581; GSCKV) is responsible for the recognition of DNA response element, and the second zinc finger containing a region called the distal box (amino acids 596–600; ASRND) is involved in DNA-dependent dimerization between receptor monomers,^38,46 targeting the second zinc finger module represses AR transactivation.⁴⁷ Thus, AR dimerization through the DBD is essential for DNA binding and gene regulation. The D-box also regulates gene expression through androgen-induced N/C interaction manner.⁴⁸ Studies suggest that N/C interaction can occur intramolecularly and intermolecularly; androgen can induce intramolecular N/C interaction immediately, and once AR was translocated to the nucleus, the intermolecular N/C interaction can occur. N/C interaction stabilizes the AR by interrupting ligand dissociation and preventing its degradation and meanwhile enhances AR activity by maintaining it in active state.⁴⁸ It was reported that another nuclear receptor glucocorticoid receptor (GR) upregulated AR but also inhibited AR transcriptional activity by inhibiting DHT-induced N/C-terminal domain interactions.⁴⁹ For AR, the evidence for LBD-LBD interactions is limited. This article reveals that AR-LBD shares a number of conservation amino acids with the GR-LBD, and interaction between GR monomers and equivalent residues in AR-LBD may occur;⁴⁶ heterodimer may format between AR and GR and subsequently alter AR functions. Previous study suggested that C-terminally truncated AR can form homodimers and induce the expression of certain AR target genes, and heterodimers with full-length AR were able to activate the androgen-dependent genes under androgen-deprived conditions.⁵⁰ The study by Chen et al.²⁵ proved that AR-Vs can form heterodimers/homodimers with each other via DBD/DBD interactions and that they can also form heterodimers with AR-FL via DBD/DBD and N/C interactions. Several molecules had been found to be associated with AR dimerization, such as peroxiredoxin-1 enhances AR N/C interaction and promotes its function,⁵¹ whereas Chinese traditional medicine cryptotanshinone blocks AR dimerization formation and consequently inhibits AR activation.⁵²

AR phosphorylation

In addition to regulation by ligand-induced AR dimerization, AR activity is also regulated by serine (S), threonine (T), and tyrosine (Y) residue phosphorylation. More than 30 phosphosites were found: S16, S81, S83, S94, S96, S213, S256, Y223, Y225, S256, S258, Y267, Y269, T282, S293, S308, Y309, Y348, Y359, Y363, Y364, Y365, Y395, S405, S424, S515, Y534, Y535, Y551/552, Y534, S578, S651, S791, T850, and Y916.^53–57 AR activity can be promoted or inhibited by phosphorylation (Table 2). Phosphorylation can affect AR activity from several mechanisms. First, it can influence AR translocation and plexin-B1 can phosphorylate AR at Serine 81, leading to translocation of AR into nucleus, increase in the expression of androgen-dependent genes, and activation of the transcriptional activity of AR;⁵⁸ as S650 was proximal to the DNA-binding domain that contains a nuclear export signal, c-Jun N-terminal kinase (JNK) and p38 phosphorylated S650 inhibiting AR being transported to nucleus. Second, AR phosphorylation can influence AR-binding DNA; Ack1-induced phosphorylation of Y267 and Y363 resulted in AR reporter activation; and recruiting AR to specific gene enhancer,⁵⁹ some phosphosite may involve with multiple mechanisms; the study by Karaca et al.⁶⁰ indicated that Y267 was responsible for nuclear translocation and DNA binding. Third, phosphorylation of AR influences its stability, S308 phosphorylated by cyclin-dependent kinase 5 (CDK5) resulted in AR stabilization,⁶¹ whereas S213 phosphorylated by Mdm2 led to AR and AR-V7 ubiquitination and protein degradation,⁶² and p-Akt was reported to inactivate AR by phosphorylating AR at Ser213 and Ser791, which can lead to ligand dissociation and AR degradation.⁶³ Thus, phosphorylated AR may be a marker for prediction and target for therapy.^64,65

Table 2.

The character of AR phosphosites.

Phosphosite	AR activity	Kinases	Reference
S81	+	CDK9, CDK1, p52, Plexin-B1	Williamson et al.,⁵⁸ Gordon et al.,⁶⁶ Amin et al.,⁶⁷ and Mehraein-Ghomi et al.⁶⁸
S213	−	Akt, PIM1S	Koryakina et al.⁵⁴ and McClurg et al.⁶³
Y223	+	Fer, Mdm2	Li et al.⁶² and Rocha et al.⁶⁹
Y267	+	Ack1	Mahajan et al.⁵⁹ and Karaca et al.⁶⁰
T282	+	Aurora-A	Shu et al.⁷⁰
S293	+	Aurora-A	Shu et al.⁷⁰
S308	+	CDK1, CDK5	Lindqvist et al.⁶¹ and Koryakina et al.⁷¹
Y363	+	Ack1	Mahajan et al.⁵⁹
S405^a	−		Lagarde et al.⁷²
S515	+	CDK1	Willder et al.⁷³
Y534	+	SFK, PTHrP	Gelman⁷⁴ and DaSilva et al.⁷⁵
S578	−	PAK6	Liu et al.⁷⁶
S650	−	JNK, p38	Gioeli et al.⁷⁷
S791	−	Akt	McClurg et al.⁶³
T850	+	PIM1L	Linn et al.⁷⁸

CDK: cyclin-dependent kinase; PAK6: p21-activated kinase 6; JNK: c-Jun N-terminal kinase; SFK: Src family kinase; PTHrP: parathyroid hormone-related protein.

The S405 phosphosite was created by a missense mutation in exon 1 of the AR, which resulted in the substitution of arginine for serine at amino-acid position 405.

AR coregulators

AR interacts with a large, disparate class of molecules known as coregulators, which either potentiate (coactivators) or suppress (corepressors) expression of AR target genes⁷⁹ (Figure 3). Until now, 274 AR-associated coregulator genes were found.⁸⁰ Of these, 50 coregulators were deregulated in PCa. Such deregulation often correlates with aggressive disease and is one of the mechanisms by which AR bypasses ADT.⁸¹ We would discuss the coregulators according to their functions. Corepressors bind with AR leading to AR function inhibition, and corepressors can interact with AR NTD, DBD, or LBD. We consider that corepressors exerted inhibition through at least three patterns. First, corepressors competed with androgen to bind with AR. For instance, flightless I (FLII) was a member of the gelsolin superfamily of actin-remodeling proteins that formed a complex with AR through the LBD of AR, and FLII suppressed AR transactivation activity in a competitive binding manner with ligand.⁸² Other corepressor ARA70alpha interacted with AR-DBD and LBD and may compete with androgen binding with AR.⁸³ Also, corepressors competed with coactivators to inhibit AR activity, and two of the well-characterized AR corepressors, the nuclear receptor corepressor (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptor (SMRT/NCoR2), competed for the same AR-binding surfaces with coactivators including the p160 family;⁸¹ other coregulator small heterodimer partner-interacting leucine zipper protein (SMILE) was found to compete with coactivator SRC-1 (belongs to p160 family) to inhibit AR transactivation.⁸⁴ Third, the corepressor Daxx inhibited the DNA-binding activity of AR through interacting with the AR-DBD.⁸⁵ Remarkably, corepressor may have contrary function in androgen-dependent prostate cancer (ADPC) and CRPC and beta-Arrestin2 has been identified to act as a corepressor by binding AR and affecting the activity and expression of AR in ADPC; however, data also showed that beta-arrestin2 contributed to cancer cell proliferation and viability in CRPC via the downregulation of forkhead box protein O1 (FOXO1) activity and expression.⁸⁶

Figure 3.

Coregulators modulate AR activity through various mechanisms. (A) Corepressors binding with AR lead to AR function inhibition mainly through ① competing with androgen to bind with AR (FLII formed a complex with AR through AR-LBD); ② NCoR1 competes for the same AR-binding surfaces with key coactivators including the p160 family; ③ Daxx inhibits the DNA-binding activity of AR through interaction with the AR-DBD; ④ beta-Arrestin2 acts as a corepressor of AR signaling by binding to AR and affects the activity and expression of AR in ADPC and meanwhile contribute to the cell viability and proliferation of CRPC via the downregulation of FOXO1 activity and expression. (B) Coactivator interacts with AR and enhances its function. (a) SRC-1 represents p160 family that interacts with AR both at the NTD and the LBD. (b) MDC1 facilitates the association between AR and histone acetyltransferase GCN5. (c) HnRNPH1 consolidates AR-ARE binding. (d) TBLR1 selectively activates AR target genes that lead to growth inhibition. (e) Non-coding RNAs such as PCAT8 and PCGEM1 bind to the AR and strongly enhance its activity.

Coactivator interacts with one or more regions of the AR, thus enhancing its function, and steroid receptor coactivators (SRCs) such as SRC-1 and SRC-3 were extensively investigated and implicated in PCa progression.⁸⁷ AR bounds with ARE and recruited different complexes, and SRC interacted with AR at both the NTD and the LBD and catalyzed the addition of an acetyl group to specific lysine residues of histone, subsequently loosening the nucleosomal structure and altering the chromatin structure to enhance AR activity.⁸⁸ In contrast to p160 family, glutathione S-transferase (GST) pull-down demonstrated that 254–532 amino-acid residues of AR were responsible for interaction between mediator of DNA damage checkpoint protein 1 (MDC1) and AR, and MDC1 facilitated the association between AR and histone acetyltransferase GCN5, thus increasing histone H3 acetylation level on cis-regulatory elements of AR target genes, including cell-cycle negative regulator p21 and PCa metastasis inhibitor Vinculin, resulting in suppression of PCa.⁸⁹ In addition to MDC1, transducin β-like-related protein 1 (TBLR1) was a coactivator of AR and selectively activated AR target genes to inhibit PCa cell growth.⁹⁰ Recently, research attested the possibility of hnRNPH1 binding to AR/ARE complex in a hormone-dependent and independent manner, and siRNA silencing of hnRNPH1 reduced AR-ARE binding in the presence or absence of DHT; moreover, siRNA silencing of hnRNPH1 sensitized PCa cells to bicalutamide and inhibited prostate tumorigenesis.⁹¹ Other coactivators as well as lysine deacetylases (KDACs) and lysine acetyltransferases (KATs) played important role in PCa progression, thus targeting AR coregulators may be a promising strategy to treat PCa.⁹²

Alternative coregulators that modified AR activity were the non-coding RNAs which include miRNA and long non-coding RNA. Yang et al.⁹³ report that two lncRNAs were highly overexpressed in aggressive PCa, PRNCR1 (also known as PCAT8) and PCGEM1, bound to the AR and strongly enhanced both ligand-dependent and ligand-independent AR-mediated gene activation and proliferation in PCa cells. Shih et al.⁹⁴ reviewed many other lncRNAs associated with PCa that played oncogenic roles by modulating AR activity through various mechanisms.

Regulation of AR expression

More than 90% of patients expressed AR; AR expression especially the nuclear AR expression affected cancer cell physiology and was able to predict disease outcome.^95,96 AR expression imbalance was involved in PCa progression. We reviewed the factors which may cause AR production and degradation imbalance.

AR expression was affected by DNA methylation, and H3K27me3 enrichment at the AR promoter region is associated with AR silencing, which appears to be reversible when enhancer of zeste homolog 2 (EZH2) is inhibited;⁹⁷ however, co-treatment with DNA methyltransferase and histone deacetylase inhibitors 5-aza-2′-deoxycytidine (Aza-dC) and sodium butyrate (NaB) was most effective in demethylation and re-expression of the AR gene.⁹⁸ The methylation pattern of AR promoter influenced cancer cell renewal, proliferation, and differentiation.⁹⁹ AR gene was upregulated by paired box 2 (PAX2), which decreased the DNA methylation of AR gene and promoted androgen-independent prostate cancer (AIPC) cell growth.¹⁰⁰ In contrast, AR gene expression in PCa was directly suppressed by itself through recruitment of lysine-specific demethylase 1 to the enhancer in the second intron of AR and demethylation of H3K4me1 and H3K4me12.¹⁰¹ Apart from methylation, DNA (cytosine-5)-methyltransferase 1 (DNMT1) was shown to be associated with the AR promoter located 22 bps from the transcription start site and cooperated with E2F1 inhibiting AR gene expression in a methylation-independent manner.¹⁰² Shi et al.¹⁰³ recently identified a miR-124 binding site in the first 436 bases of the AR 3′-UTR, proving that miR-124 negatively regulated AR, and other miRNAs such as miR-34a, miR-34c, miR-135b, miR-185, miR-421, miR-449, miR-634, miR-9, miR-297, and miR-299-3p were able to suppress AR expression by binding to AR 3′-UTR,⁹⁴ highlighting the role of non-coding RNA in AR expression regulation.

After translating into protein, AR protein was affected by the balance of degradation and stabilization; AR protein was often degraded by ubiquitination, and ubiquitination is the enzymatic process in which ubiquitin proteins attach to another protein via the C-terminal glycine residue.¹⁰⁴ The ubiquitination of AR can be influenced by various proteins, such as DNA damage binding protein 2 (DDB2), parathyroid hormone-related protein (PTHrP), ubiquitin-specific protease 26 (USP26), and p300.^75,105–107 The proteins are mainly divided into three types. The ring finger protein 6 (RNF6) was an ubiquitin E3 ligase which induces AR ubiquitination;¹⁰⁸ also, prostate transmembrane protein androgen induced 1 (PMEPA1) controlled AR ubiquitination by the reciprocal negative feedback through NEDD4-E3 ligase,¹⁰⁹ and we classify the proteins interacted with ubiquitin to regulate AR ubiquitination as the first type. The second type was presented as proteins that bind with AR to influence AR ubiquitination, and the phosphatase 1 catalytic subunit (PP1alpha) was the second type of protein that directly binds with AR-LBD leading to inhibition of AR ubiquitination;¹¹⁰ the inhibition recalls deubiquitination enzyme USP26 assembled with AR and other cofactors leading to counteract AR ubiquitination.¹⁰⁶ The cofactor p300 was also found binding with AR and leads to acetylation of AR, thereby precluding its polyubiquitination and degradation.¹¹¹ The third type of protein is DBB2, PTHrP, or Lyn; these proteins interacted with DDB1, epidermal growth factor receptor (EGFR), or Hsp90 to positively or negatively regulate AR ubiquitination.^75,105,112

Target AR expression inhibits cancer progression, and drugs to promote AR ubiquitination have been explored. Galeterone and VNPT55 induced proteasomal degradation of AR and AR-V7, led to significant apoptosis of PCa cells, and inhibited tumor growth.¹¹³ Other chemicals 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and C-4 heteroaryl 13-cis-retinamide were detected by its reaction on AR expression;^107,114 however, these chemicals may take a long time to be applied as drugs in clinical stage.

AR and cell signals

It was known that AR exerted effects on cells through genomic action that regulate the expression of genes such as prostate-specific antigen (PSA), FK506-binding immunophilin 51 (FKBP51), and NK homeobox family member 3 (NKX3.1). In this article, we would dissect the role of AR mainly from the relationship between AR and cell signal transduction such as phosphatidylinositol 3-kinase (PI3K)/Akt, nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and transforming growth factor β (TGFβ).

PI3K/Akt

PI3K/Akt plays a critical role in PCa cell growth and survival. Studies have shown that the effect of PI3K/Akt in prostate cells is mediated through androgen signaling. Sharma et al.¹¹⁵ found that PI3K inhibitors, LY294002 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN), negatively regulated the PI3K/Akt pathway and repressed AR activity, and further experiments showed that PTEN negatively regulated AR activity by controlling β-catenin degradation mediated by glycogen synthase kinase 3 beta (GSK3β). However, a direct interaction between Akt and AR was confirmed by co-immunoprecipitation, and increased phosphorylation of Akt (Ser-473 and Thr-308) was linked with phosphorylation at Ser213 and Ser791 and AR degradation.^63,116 Paliouras and Diamandis¹¹⁷ used a constructed cell line, a myriad of stimulations, and inhibitor treatments to reveal mechanisms that AKT both positively and negatively regulate PSA expression, and they proposed a distinct Akt-AR interaction in ADPC and AIPC. More recently, studies demonstrated that inhibited PI3K-AKT-mammalian target of rapamycin (mTOR) or AR signaling axes led to the activation of another pathway. In mouse PCa induced by PTEN loss, knocking out AR or treating with enzalutamide enhances AKT signaling via downregulating FKBP5 and subsequently leads to the reduction in AKT negative regulator pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP)¹¹⁸ (Figure 4(a)). However, antiandrogen-induced phosphorylation of AKT in LNCaP (PTEN loss and androgen-dependent cell line), but not in C4–2 cells (PTEN loss and androgen-independent cell line).¹¹⁹ Liu prompted that PI3K/AKT inhibitors presented various effects on AR gene and protein expression; off-target effects to AR gene expression could lead to the various results in distinct PCa cell lines, one reason maybe that inhibitors affected other signaling pathway in PCa cells dependent on their genetic backgrounds.¹²⁰ Contrary results also indicated a positive correlation between Akt and AR.^121,122 Nevertheless, preclinical study illustrated the potent anticancer activity of novel PTEN-targeted drugs in combination with hormonal therapy.¹²³

Figure 4.

AR and cell signals. The relationship between AR and (a) PI3K/Akt, (b) NF-κB, (c) MAPK, and (d) TGFβ/Smad pathway.

NF-κB

A functional reporter assay using NF-κB-luciferase and an electromobility gel shift assay have showed that DHT suppressed NF-κB activity,¹²⁴ and study based on constructed cell line PC3-AR (PC3 transfected with AR) showed that AR activation caused a decrease in RelA, a subunit of NF-κB, reduced AR nuclear localization and transcriptional activity, and subsequently suppressed tumor growth.¹²⁵ Opposite results indicated that NF-κB p65-RelA activity was increased in PC3-AR cells and inhibited upon AR knockdown in C4-2, VCaP, or PC3-AR cells.¹²⁶ These data suggest context-depending variations in the role of AR for NF-κB expression. The requirement of NF-κB in interleukin (IL)-4-induced AR activation indicated NF-κB as an upstream of AR.¹²⁷ Overexpression of NF-κB activated the AR promoter, and chromatin immunoprecipitation (ChIP) assays of NF-κB occupancy on 5′ regulatory region of the AR gene enhanced AR expression in LNCaP cells and promoted cell proliferation.^128,129 NF-kappaB2/p52 also promoted AR nuclear translocation and activation by interacting with its NH(2)-terminal domain and enhanced the recruitment of coactivators such as p300 to the promoters of AR-regulated genes.^130,131 Besides regulation of AR, NF-κB promoted splicing factor heterogeneous nuclear RNA-binding protein A1 (hnRNPA1) production and led to the generation of AR splice variants such as AR3 which may confer resistance to ADT¹³² (Figure 4(b)). Overall, data implicated that interrupting NF-κB combined with ADT may be an effective strategy to reduce castration-resistant PCa cell growth.^68,133

MAPK

Chronic stimulation of the Ras-mitogen-activated protein kinase/extracellular signal–regulated protein kinase (MEK-ERK) signaling pathway can sustain AR-induced gene transcription and the growth of PCa cells, and MEK/ERK inhibitor U0126 impeded R1881-stimulated ARE luciferase reporter gene activity and the expression of prostate-specific antigen.¹³⁴ U0126 also inhibited androgen-induced PSA promoter luciferase gene activities and androgen-independent growth of LNCaP cells via the inhibition of AR phosphorylation at Ser81.¹³⁵ MEK inhibitor PD 98059 was found to reduce the induction of AR expression by Capsaicin, and studies suggested that MAPK pathway was positively related with AR expression.^136,137 Study based on two castrate-resistant cell lines, R1 and Rv1, which were derived from two independent CWR22 relapsed tumors, found that ERK phosphorylation increased levels of low-molecular-weight (LMW) form of AR by promoting proteolysis of the AR, and LMW-AR molecules would be able to translocate into the nucleus in an androgen-independent manner, bind to DNA, and activate or repress gene transcription.²⁴ Raf/MEK/ERK signal can downregulate full-length as well as LBD-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b.¹³⁸ Thus, MAPK may confer to castration resistance. Conversely, whether DHT activates the ERK pathway in PCa is controversial, and Recchia et al.¹³⁹ suggested that DHT exerted no effect on ERK phosphorylation and DHT induced rapid bona fide p38MAPK phosphorylation in LNCaP cells. Pang et al.¹⁴⁰ suggested that AR was able to phosphorylate ERK-1/2 (Thr202 and Tyr 204) in cultured genital skin fibroblasts independent of DNA binding (Figure 4(c)). In mice prostate, AR promoted ERK signaling pathway via downregulating U19/EAF2,¹⁴¹ which is involved in tumorigenesis.

TGFβ/Smad

AR interacted with TGFβ/Smad signal had been put forward for a long period. Martikainen et al.¹⁴² demonstrated that TGFβ1 can induce the death of androgen-dependent rat prostatic glandular cells in 1990s, and it is consistent with the fact that both the receptor and the mRNA for TGFβ1 increase rapidly in the ventral prostate after castration which led to cancer cell death. TGFβ1 induces nuclear to cytoplasmic translocation of AR and inhibits androgen response in prostate smooth muscle cells,¹⁴³ but it was unknown whether TGFβ1 inhibits cancer cell proliferation mainly through inhibiting AR translocation or other mechanisms. Nevertheless, Smad3 specifically represses transcriptional activation mediated by AR on androgen-induced gene promoters;¹⁴⁴ TGFβ1 reduced the E2F transcriptional activity activated by DHT-induced AR activation, and luciferase reporter gene assays showed that AR signaling was inhibited in TGFβ receptor (TGFβR2) stably transfected LNCaP cells (express AR and lack TGFβR2 expression).¹⁴⁵ However, Smad3 may act as an important coregulator for the AR signal pathway and play a positive role for TGFβ in androgen-promoted PCa growth,¹⁴⁶ and the action of Smad3 to AR activity may depend on Smad4 expression.¹⁴⁷ In turn, luciferase reporter assays showed that TGFβ/Smad signal was inhibited in PC3 transfected with AR (PC3 lacks AR expression, but has a functioning TGFβ1/Smad signaling cascade).¹⁴⁵ The ability of androgens to stimulate growth of rat prostatic epithelial cells involved the downregulation of the production of TGFβ1, TGFβ2, and TGFβ3.¹⁴⁸ DHT modulates transcriptional activity of Smad3 and TGFβ receptor type II and hence cell sensitivity to TGFβ1, leading to suppression of the inhibition of TGFβ to Bcl-xL, cyclin Ds, and activated caspase-3.^149–152 Electrophoretic mobility shift assays indicated that the AR-LBD directly inhibited the association of Smad3 to the Smad-binding element¹⁵³ (Figure 4(d)). It was noted that DHT and AR may have distinct effect on cells, and downregulation of AR signaling in PCa cells activated the TGFβ/Smad signaling and promoted the PCa metastasis,^154,155 and recent study suggested that ADT enhanced the expression of AR and AR3 concomitant with upregulation of the expression of epithelial–mesenchymal transition (EMT) and stem cell marker genes.¹⁵⁶ More importantly, both positive and nAREs were found functional in TGFβ1 promoter, and AR signal may positively or negatively regulate TGFβ1 expression in response to various signals or under different environmental conditions.^157,158

Conclusion

AR and its mutation, amplification, and splice variants play a critical role in PCa progression, increasing evidences highlight that AR splice variants particularly AR-V7 could be used as a potential predictive biomarker and a therapeutic target.¹² The action of AR-V7 relies on its nuclear translocation independent of ligand which is essential for AR-FL. Nonetheless, research also underlines miRNA, lncRNA in addition to protein in modifying AR dimerization, phosphorylation, and its expression. Such alterations of AR lead to AR nuclear translocation or functional change and subsequently positively or negatively regulate the growth and invasion ability of cancer cell. Apart from the genomic action of AR, non-genomic action of AR that is the action of AR in cytoplasm^9,159 should be considered in target therapy.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Dissecting the roles of the androgen receptor in prostate cancer from molecular perspectives

Abstract

Keywords

Introduction

Genetic alterations of AR: mutations and variants

Nuclear translocation of AR

Modifications of the AR action

AR dimerization

AR phosphorylation

AR coregulators

Regulation of AR expression

AR and cell signals

PI3K/Akt

NF-κB

MAPK

TGFβ/Smad

Conclusion

Footnotes

Declaration of conflicting interests

Funding

References