Epigenetic (de)regulation in Prostate Cancer

Abstract

Prostate cancer (PCa) is a heterogenous disease exhibiting both genetic and epigenetic deregulations. Epigenetic alterations are defined as changes not based on DNA sequence, which include those of DNA methylation, histone modification and chromatin remodeling. Androgen receptor (AR) is the main driver for PCa and androgen deprivation therapy (ADT) remains a backbone treatment for patients with PCa; however, ADT resistance almost inevitably occurs and advanced diseases develop termed castration resistant PCa (CRPC), due to both genetic and epigenetic changes. Due to the reversible nature of epigenetic modifications, inhibitors targeting epigenetic factors have become promising anti-cancer agents. In this chapter, we focus on recent studies about the dysregulation of epigenetic regulators crucially involved in the initiation, development and progression of PCa and discuss the potential use of inhibitors targeting epigenetic modifiers for treatment of advanced PCa.

1. Introduction

Prostate cancer (PCa) is the second most commonly diagnosed cancer in the men and one of the leading causes of cancer-related death (Sung et al., 2021). According to Globocan estimates, approximately 1.4 million of new PCa cases were diagnosed worldwide in 2020 (Sung et al., 2021). Androgen receptor (AR), a nuclear hormone receptor and transcription factor that mediates signaling of testosterone and 5-α-dihydrotestosterone (DHT), not only is essential for normal prostate development but also remains a main driver in PCa (Watson et al., 2015). Accordingly, androgen deprivation therapy (ADT) has been a backbone of treatment for PCa patients (Huggins and Hodges, 1972). However, most PCa patients eventually become refractory to ADT and, almost inevitably, the disease develops to castration-resistant prostate cancer (CRPC) (Watson et al., 2015). Patients with CRPC often develop metastases (termed metastatic CRPC or mCRPC), mainly in the skeleton, and become incurable due to a lack of effective therapy (Tsuzuki et al., 2016). CRPC exhibits a diverse disease presentation with variable outcomes, including neuroendocrine PCa (NEPC) (Conteduca et al., 2019). NEPC is relatively rare at initial diagnosis of PCa but its incidence increases dramatically following ADT (thus termed therapy induced-NEPC or t-NEPC) and ranges from ~17–30% based on different clinical studies (Patel et al., 2019a). The available therapy for CRPC is very limited, therefore, it remains an urgent task to dissect precise mechanisms responsible for CRPC progression and develop new methods to overcome therapy resistance.

Genetic and epigenetic alterations, as well as changes in tumor microenvironment (TME), collectively contribute to malignant transformation of cancer (You and Jones, 2012). Whole genome sequencing (WGS) studies in patient samples have identified numerous genomic alterations in primary and advanced PCa, which include the loss of tumor suppressor genes such as PTEN, RB and TP53, amplification of oncogenes such as AR and MYC, formation of structural variants such as TMPRSS2-ERG, and mutations of genes important for various signaling pathways (Quigley et al., 2018, Armenia et al., 2018, Robinson et al., 2015, Cancer Genome Atlas Research, 2015, Grasso et al., 2012, Tomlins et al., 2005, Taylor et al., 2010). On the other hand, epigenetics, here by a relatively loose definition, refers to biological processes that regulate gene expression and function without altering DNA sequences (Sharma et al., 2010). Genetic information or DNA of mammalian cells is organized in the form of chromatin and the building unit of chromatin is termed as nucleosomal core particle, which consists of a histone octamer (two copies of each of the four core histones H2A, H2B, H3 and H4) surrounded by double-stranded DNA (Felsenfeld and Groudine, 2003). Epigenetic regulations include DNA methylation, histone modification, chromatin remodeling, amongst other mechanisms. DNA methylation mainly occurs in CpG dinucleotides in which a methyl group is added to the 5’-carbon position of cytosine by a family of DNA methyltransferases (DNMTs) (Greenberg and Bourc’his, 2019). Histone modification refers to covalent post-translational modification (PTM) of histone proteins. The cellular machineries that mediate the epigenetic regulation can be grouped as writers, erasers and readers (Zhao et al., 2021). The writer refers to an enzyme that deposits a specific histone modification or DNA methylation. The common writer enzymes include histone acetyltransferases (HATs), histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs). Histone modifications and DNA methylation are reversible and can be removed by erasers. Common erasers include histone deacetylases (HDACs), histone demethylases (HDMs) and TET family proteins. Readers refer to a set of specialized proteins that recognize and bind specific histone modification or DNA methylation and convey epigenetic information to downstream effectors (Zhao et al., 2021). The change of chromatin accessibility is mediated by chromatin remodeling complexes such as the SWI/SNF complex (Clapier et al., 2017). DNA methylation, histone modifications and chromatin remodeling are important epigenetic mechanisms to mediate gene expression, and their deregulations act independently and/or cooperatively in the process of PCa initiation and progression and during the development of drug resistance (Kumaraswamy et al., 2021, Kukkonen et al., 2021, Natesan et al., 2019, Ruggero et al., 2018). In this book chapter, we will use prostate cancer as an example to highlight the recent discoveries on how to deregulation in DNA methylation and histone modification modifiers and chromatin remodelers contributes to the cancer initiation and progression, as well as potential targeting strategies, as summarized in Figure 1.

Figure 1. Overview of key epigenetic regulators and targeting drugs in PCa.

This schematic illustration only covers a set of selectively focused epigenetic modifications including DNA methylation (top), histone methylation and acetylation (middle), and chromatin remodeling (bottom) involved in PCa. Certain key writers, erasers and readers, as well as the respective targeting compounds, are listed. *, the compound used in the ongoing clinical trials in PCa as shown in Table 1. Many other histone modifications, epigenetic modifiers and targeting drugs, which might have equally important functions in PCa, are not listed. The figure is created with BioRender.com.

2. DNA Methylation in prostate cancer

Patterns of cytosine methylation within CpG dinucleotide sequences are important for regulating gene expression, DNA damage repair, DNA recombination and DNA replication (Chin et al., 2011). Cytosine methylation is de novo ‘written’ by DNMT3A and DNMT3B and maintained by DNMT1, which transfer a methyl group from S-adenosylmethionine, a methyl donor, to the 5’-carbon of the cytosine ring to form 5-methylcytosine (5mC) (Greenberg and Bourc’his, 2019). 5mC can be ‘erased’ through sequential oxidation by Ten-eleven translocation (TET) family proteins, with the assistance of base excision repair (BER) pathway, first to 5-hydroxymethyl cytosine (5hmC), then 5-formyl cytosine (5fC) and finally 5-carboxyl cytosine (5caC) (Ikeda and Kinoshita, 2009). CpG islands refer to the CpG-rich regions of ~200 bp to several kilobases in length, usually located near the promoters of highly expressed genes (Illingworth et al., 2008). CpG islands are often subject to hyper-methylation in human tumors including PCa, compared to normal tissues (Nowacka-Zawisza and Wisnik, 2017). Hypermethylated CpG islands are often associated with target gene silencing (Greenberg and Bourc’his, 2019).

Mechanistically, 5mC can regulate gene expression by recruiting readers such as methyl-CpG binding proteins (MBP1, MBP2, MBP3), Kaiso, and methyl CpG-binding protein-2 (MeCP2) (Buck-Koehntop and Defossez, 2013, Wu et al., 2015, Jones et al., 1998, Nan et al., 1998). Once bound to promoter-associated 5mC, methyl-CpG-binding domain (MBD) family proteins can further recruit corepressors such as HDACs, thereby mediating repression of target gene transcription (Du et al., 2015). Meanwhile, DNA methylation can profoundly modulate binding of various DNA-binding proteins such as transcription factors (TFs) and thus directly influences gene activity (Zhu et al., 2016). In this case, 5mC may either inhibit or promote TF binding (Ren et al., 2018, Zhu et al., 2016).

In 1987, Bedford and van Helden reported that global DNA methylation was significantly lower in metastatic PCa compared to benign prostatic hyperplasia (BPH) (Bedford and van Helden, 1987). This is the first study to demonstrate a correlation between global hypomethylation and metastatic potential of PCa. In general, promoter hypomethylation and gene body hypermethylation are positively correlated with gene expression (Jones, 2012). In PCa, DNA hypomethylation has been linked to higher expression of genes and usually occurs later in PCa progression and contributes to tumor metastasis (Yegnasubramanian et al., 2004). Over the last decade, several studies have compared genome-wide DNA methylation patterns in benign prostate tissues and various stages of PCa (Kirby et al., 2017, Bhasin et al., 2015, Geybels et al., 2015, Cancer Genome Atlas Research, 2015, Zelic et al., 2015). Very recently, Zhao et al. used whole genome bisulfide sequencing (WGBS) and further confirmed that advanced PCa is overall less methylated than primary PCa, which is predominantly less methylated than benign prostate (Zhao et al., 2020). Numerous oncogenic factors involved in various signaling pathways are reported to be hypomethylated in PCa including Urokinase plasminogen activator (uPA) (Pakneshan et al., 2003), Heparanase (HSPE) (Hulett et al., 1999), Cytochrome P450 1B1 (CYP1B1) (Tokizane et al., 2005), and wingless-related MMTV integration site 5A (WNT5A) (Wang et al., 2007). In the aforementioned study, Zhao et al. demonstrated promoter hypomethylation at AR and key androgen-responsive genes including KLK3 and NKX3–1 in advanced PCa compared to benign prostate samples (Zhao et al., 2020). Global DNA hypomethylation has also been proposed to be correlated with genetic instability by promoting formation of more open chromatin (Ehrlich, 2002). Therefore, DNA hypomethylation potentially contributes to PCa development and progression through altering transcriptomics and/or genome stability.

Conversely, DNA hypermethylation is one of the most commonly observed phenomena and best characterized epigenetic alterations in PCa. A plethora of genome-wide DNA methylation analyses have been performed which suggest that differential methylation among distal and genic regions is one of key drivers of PCa tumorigenesis and progression (Wang et al., 2016b). For example, Zhao et al. reported that about 22% of CRPC tumors exhibit a hypermethylation subtype termed a CpG methylator phenotype (CMP), which is enriched with somatic mutations of DNMT3B, TET2, BRAF and IDH1 (Zhao et al., 2020). In contrast to promoter hypomethylation, hypermethylation of gene promoters often coincides with transcriptional repression of target genes, most of which are tumor suppressor genes (TSGs) such as adenomatosis polyposis coli (APC) (Richiardi et al., 2009), retinoic acid receptor β (RARβ) (Jeronimo et al., 2004) and RAS-associated domain family 1 (RASSF1) (Liu et al., 2002, Kuzmin et al., 2002), DNA damage repair related genes such as Glutathione S-Transferase Pi 1 (GSTP1) (Mahon et al., 2014, Lee et al., 1994) and O-6-methylguanine-DNA methyltransferase (MGMT) (Gerson, 2004), hormonal response related genes such as AR (Jarrard et al., 1998), ER-α (Sasaki et al., 2002) and ER-β (Lau et al., 2000), as well as genes involved in cell cycle control, apoptosis and cell adhesion such as cyclin dependent kinase inhibitor 2A (CDKN2A) (Herman et al., 1995), Target of Methylation-induced Silencing 1 (TMS1) (Das et al., 2006) and E-cadherin (CDH1) (Graff et al., 1995, Li et al., 2001).

Measurement of global or targeted DNA methylation can be used as biomarkers to help the risk stratification of PCa patients. For instance, ConfirmMDx, an epigenetic test that evaluates the methylation status of three genes, GSTP1, APC and RASSF1, can distinguish true negative prostate biopsies from occult PCa, therefore avoiding unnecessary repeat prostate biopsies of patients (Partin et al., 2014). Likewise, the methylation panel of GSTP1, together with those of GAS6 and HAPLN3, was identified by Patel et al. to separate PCa and benign prostate tissues in a sensitive and specific way (Patel et al., 2019b).

While 5mC is commonly associated with transcriptional repression, further conversion to 5hmC is related to transcriptional activation (Xu et al., 2011). The laboratory of Dr. Felix Feng has recently profiled 5hmC among 145 PCa samples representing different disease stages (including 52 localized PCa and 93 mCRPC) and integrated datasets of WGS, WGBS and RNA-seq from the same samples (Sjostrom et al., 2022). In this study, 5hmC in mCRPC was shown to mark the activation of major cancer driver genes (such as AR and EZH2) at both gene-regulatory sites and their downstream binding sites (Sjostrom et al., 2022), consistent with the transcriptional changes during disease progression captured by RNA-seq (Bolis et al., 2021). Since metastatic PCa is notoriously heterogenous and hard to biopsy, circulating tumor DNA (ctDNA) obtained from liquid biopsy provides an alternative approach for understanding the underlying tumor biology (Vandekerkhove et al., 2019, Wu et al., 2020). Indeed, 5hmC level in ctDNA represents the individual advanced tumors, making it a potential liquid biomarker for mCRPC (Sjostrom et al., 2022, Bolis et al., 2021). A very exciting work done by Chen et al. performed plasma DNA methylome analysis from patients with localized (60 samples) and metastatic PCa (175 samples) using the cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) technology (Chen et al., 2022, Shen et al., 2018). They observed global hypermethylation in metastatic samples, together with hypomethylation in the pericentromeric regions (Chen et al., 2022). This study indicated that the cell-free DNA methylome may accurately distinguish different disease stages, highlighting a potential use in PCa diagnosis and prognosis (Chen et al., 2022).

Deregulation of DNMT occurs commonly in cancer and is associated with tumorigenesis (Zhang and Xu, 2017). DNMT1 is often overexpressed in localized and metastatic PCa when compared to BPH (Valdez et al., 2013, Zhang et al., 2015). Increased expression of DNMT3A and DNMT3B led to hypermethylation at a substantial subset of CpG sites in PCa (Kobayashi et al., 2011). TET2 binds AR and TET2 loss is correlated with PCa progression (Nickerson et al., 2017). To target aberrant DNA hypermethylation, DNMT inhibitors such as Azacytidine and Decitabine have been developed and tested in various types of cancers including PCa (Cheng et al., 2019). Currently, there are two ongoing clinical trials using Decitabine/Cedazuridine together with Enzalutamide (NCT05037500), or Guadecitabine (SGI-110) together with Perbrolizumab (NCT02998567), for the treatment of CRPC patients (see Table 1).

Table I.

Selective ongoing clinical trials of epigenetic drugs for the treatment of PCa (Source: https://clinicaltrials.gov/)

Trial ID	Drug name	Phase	Conditions	Status
DNMT inhibitors
NCT05037500	Decitabine/Cedazuridine	I/II	mCRPC (+Enza)	Recruiting
NCT02998567	Guadecitabine(SGI-110)	I	CRPC (+Pembrolizumab)	Active
LSD1/KMD1A inhibitors
NCT04628988	CC-90011	I	mCRPC(+Abi/Pred)	Recruiting
EZH2 inhibitors
NCT03460977	PF-06821497	I	mCRPC (alone or with SOC)	Recruiting
NCT04179864	Tazemetostat	I/II	mCRPC (+Enza or Abi/Pred)	Recruiting
NCT04846478	Tazemetostat	I	mCRPC(+Talazoparib)	Recruiting
NCT04104776	CPI-0209	II	mCPRC	Recruiting
NCT03480646	CPI-1205	I/II	mCRPC (+Enza or Abi/Pred)	Active
NCT04388852	DS3201(Valemetostat)	I	CRPC(+Ipilimumab)	Recruiting
p300/CBP inhibitor
NCT03568656	CCS1477	I/II	mCRPC(+Enza or Abi or Daro)	Recruiting
HDAC inhibitors
NCT04703920	Belinostat	I	mCRPC(+Talazoparib)	Recruiting
BET protein inhibitors
NCT05252390	Nuv-868	I/II	mCRPC(alone or +Enza or Talazoparib)	Recruiting
NCT04471974	ZEN-3694	II	mCRPC(+Enza and Pembrolizumab	Recruiting
NCT04986423	ZEN-3694	II	CRPC(+Enza)	Recruiting

Abbreviation in the table: mCRPC, Metastatic castration-resistant prostate cancer; Enza, Enzalutamide; Abi, Abiraterone acetate; SOC, Standard of Care; Pred, Prednisone/Prednisolone; Daro, Darolutamide

3. Histone methylation in prostate cancer.

Histones are a group of highly conserved, highly basic proteins that are responsible for chromatin organization and compaction by folding DNA into the nucleus (Marino-Ramirez et al., 2005). There are four core histones, namely H2A, H2B, H3 and H4. Amino acid residues of histones, especially those in the unstructured N-terminal tails, are known to be potential sites for various types of post-translational modifications (PTMs), such as methylation, acetylation, phosphorylation and ubiquitination (Allis and Jenuwein, 2016). Histone methylation and acetylation have been relatively intensively studied in cancer and hence, we will focus the next sections on the recent findings showing the involvement of these modifications in PCa.

Histone methylation and acetylation are established, respectively, by histone lysine methyltransferases (KMTs) and histone acetyltransferases (HATs), and can be removed, respectively, by histone lysine demethylases (KDMs) and histone deacetylases (HDACs). Histone methylation is critically involved in a wide variety of biological processes ranging from transcriptional regulation to heterochromatin formation (Greer and Shi, 2012). Lysine (Lys or K) and arginine (Arg or R) residues serve as the most common sites of methylation. Here we mainly discuss about lysine methylation. The histone H3 lysine 4, 9, 27, 36 and 79 (H3K4, H3K9, H3K27, H3K36 and H3K79) and histone H4 lysine 20 (H4K20) are among the most extensively studied sites of histone lysine methylation. Lysine can be mono-, di- or tri-methylated (Kme1, Kme2, or Kme3) on its ε-amine group (Greer and Shi, 2012). The degree of histone lysine methylation is controlled by the relative activities of site-specific methyltransferases and demethylases.

3.1. H3K4 methylation

Highly methylated H3K4, such as H3K4me3 and H3K4me2, is localized to the promoter-proximal regions of actively transcribed genes, with H3K4me3 exhibiting a more punctuate pattern than H3K4me2 (Ng et al., 2003, Santos-Rosa et al., 2002). H3K4me3 and H3K4me2 are associated with active transcription. H3K4me1, on the other hand, is a marker of gene enhancer (Creyghton et al., 2010, Rada-Iglesias et al., 2011). While the poised enhancers are marked by a presence of both H3K4me1 and H3K27me3 and a lack of H3K27 acetylation (H3K27ac), those active ones carry both H3K4me1 and H3K27ac and a lack of H3K27me3 (Creyghton et al., 2010, Rada-Iglesias et al., 2011). In mammalian cells, methylation of H3K4 is catalyzed by the KMT2/MLL family methyltransferases: KMT2A/MLL1, KMT2D/MLL2, KMT2C/MLL3, KMT2B/MLL4, KMT2F/SETD1A and KMT2G/SETD1B. Another SMYD family of H3K4 methyltransferases include SMYD1, SMYD2, and SMYD3. On the contrary, H3K4 demethylation is primarily catalyzed by KDM1A/LSD1, KDM1B/LSD2, KDM5A/JARID1A, KDM5B/JARID1B, KDM5C/JARID1C and KDM5D/JARID1D (Zhao et al., 2021).

The H3K4 methyltransferase KMT2A/MLL1 can function as a co-activator for AR in CRPC (Malik et al., 2015, Grasso et al., 2012). Here, AR directly interacts with the MLL complex via the menin subunit, and the inhibition of menin-MLL complex suppresses AR signaling (Malik et al., 2015). Notably, a phase I clinical trial with revumenib (SNDX-5613), a potent and selective oral inhibitor of the menin–MLL interaction, demonstrated strong efficacy in MLL-rearranged or NPM1-mutated acute leukemia (Issa et al., 2023). It would be worthwhile to test this inhibitor in advanced PCa. KMT2D/MLL2 is mutated in 8.6% of PCa cases (Grasso et al., 2012). KMT2D/MLL2 promotes prostate tumor growth through epigenetically activating LIFR and KLF4, which are involved in PI3K/AKT and epithelial-mesenchymal transition (EMT) pathways (Lv et al., 2018). KMT2F/SETD1A promotes tumor cell proliferation in metastatic CRPC by regulating FOXM1 transcription (Yang et al., 2020). Upregulation of SMYD3 expression occurs frequently in multiple cancer types including PCa (Huang and Xu, 2017). SMYD3 was shown to promote prostate tumorigenesis through upregulation of AR expression (Liu et al., 2013).

LSD1 (also known as KDM1A) is the first identified histone demethylase (Shi et al., 2004) and also represents one of the most widely studied KDMs in cancers including PCa (Rotili and Mai, 2011). LSD1 is overexpressed in patients with advanced PCa compared to normal tissues (Crea et al., 2012). LSD1 was reported to interact with AR and activate AR signaling through demethylating histone H3K9 methylation (Wissmann et al., 2007, Metzger et al., 2005, He et al., 2018). Cai et al. demonstrated that LSD1 can play dual roles in PCa (Cai et al., 2014). In the presence of high concentration of androgen, LSD1 acts as a co-repressor of AR expression by inducing demethylation of H3K4me1 and H3K4me2 at AR intronic region, and additionally, AR recruits LSD1 for mediating silencing of AR-repressed gene targets (Cai et al., 2011). However, in CRPC cells where androgen level is generally low, the expression of AR and AR-repressed genes is increased due to an impaired recruitment of LSD1, which in turn promotes CRPC cell proliferation (Cai et al., 2011). LSD1-mediated epigenetic reprogramming also drives PCa progression through regulating CENPE expression (He et al., 2018, Liang et al., 2017). However, it remains to be elucidated as of the molecular determinants responsible for LSD1 specificity towards H3K4 versus H3K9 demethylation under different biological contexts. In addition to its role in demethylating histone substrates (He et al., 2018), LSD1 also demethylates a number of non-histone substrates such as p53 and FOXA1. LSD1-mediated repression of p53 functionality was reported to be relevant during oncogenesis (Huang et al., 2007). More recently, LSD1 was shown to demethylate FOXA1, an oncogenic TF and functional partner of AR in PCa, at its residue K270, which positively regulates FOXA1’s binding to chromatin targets and then enhances AR’s transcriptional activity (Gao et al., 2020). LSD1 blockade, alone or in synergy with AR antagonist treatment, dramatically decreased PCa growth in vivo (Gao et al., 2020). LSD1 also has demethylase-independent functions to promote survival of the AR-independent prostate cancer (Sehrawat et al., 2018).

A number of LSD1 inhibitors, which include CC-90011 (Kanouni et al., 2020), SP-2577 (Soldi et al., 2020), ORY-1001 (Maes et al., 2018), TCP (Yang et al., 2007) and GSK-2879552 (Mohammad et al., 2015), have been reported and are under clinical evaluation for the treatment of cancers, either alone or in combination with other drugs (Fang et al., 2019, Cheng et al., 2019). An ongoing clinical trial (NCT04628988) is testing the LSD1 inhibitor CC-90011 together with ADT in mCRPC (Table 1).

The KDM5 family of Jumonji domain-containing KDMs are H3K4-specific and also frequently altered in PCa (Vieira et al., 2014). For instance, KDM5A is upregulated in PCa (Vieira et al., 2014). KDM5B was reported to be frequently upregulated in PCa tissues and associate with AR to regulate its transcriptional activity (Xiang et al., 2007b). KDM5B is also a key regulator of PI3K/AKT signaling in PCa through directly binding the PIK3CA promoter (Li et al., 2020). Loss of KDM5B results in a significant reduction of P110α and PIP3 levels and inhibition of the proliferation of PCa cells (Li et al., 2020). KDM5C overexpression in PCa was found to be associated with a reduced biochemical recurrence-free survival in patients after prostatectomy (Stein et al., 2014). KDM5C is highly expressed in metastatic PCa and promotes tumor growth and metastasis (Lemster et al., 2022). In contrast, KMD5D, a male-specific protein, was shown to suppress the invasion of PCa cells via demethylating H3K4me3 on the promoters of invasion-associated genes or recruiting co-repressor ZMYND8 (Li et al., 2016, Padeken et al., 2022). Furthermore, KDM5D is associated with altered docetaxel sensitivity in PCa through modulating the AR signaling (Komura et al., 2016). Loss of KDM5D expression results in aggressive PCa and confers a poorer prognosis in PCa patients (Komura et al., 2018). Taken together, these findings underscore the roles of KDMs in PCa initiation, progression and metastasis, indicating that KDMs might represent attractive therapeutic targets.

The reader proteins for H3K4 methylation also play a role in PCa progression. For example, an in vivo open reading frame (ORF) screening identified PYGO2, a H3K4me2 and H3K4me3 reader, as a driver for metastatic PCa (Lu et al., 2018). While overexpression of PYGO2 promotes PCa tumor growth and invasion, depletion of PYGO2 has the opposite effects in vitro and in vivo (Lu et al., 2018). Upregulation of PYGO2 is associated with higher Gleason score and metastasis in PCa patients, further indicating PYGO2 as a potential therapeutic target in advanced PCa (Lu et al., 2018). A recent follow-up study showed that targeting PYGO2 through genetic or pharmacological inhibition enhanced cytotoxic T cell responses and sensitized PCa to immunotherapy (Zhu et al., 2023).

3.2. H3K9 methylation

H3K9 methylation is deposited by the KMT1 writer enzymes (including KMT1A/SUV39H1, KMT1B/SUV39H2, KMT1C/G9a/EHMT2, KMT1D/GLP/EHMT1, KMT1E/SETDB1 and KMT1F/SETDB2), recognized by readers such as HP1α and MPP8, and eliminated by erasers such as the KDM3 (KDM3A-C/JHDM2A-C) and KDM4 (KDM4A-E/JMJD2A-E) family enzymes (Padeken et al., 2022). H3K9me2 or H3K9me3 is a gene silencing-related histone PTM and often found in high levels at constitutive heterochromatin regions (Barski et al., 2007). H3K9me3 mediates transcriptional silencing of various transposable elements (TEs) and regular genes as well. Malfunction of H3K9 methylation-associated regulators and modifier enzymes can profoundly affect either the level or the genomic distribution of H3K9 methylation, thus resulting in de-regulation of gene expression and/or genome instability during the course of pathogenesis (Padeken et al., 2022). H3K9 methylation writers, readers and erasers were reported to influence PCa development and progression by a variety of different mechanisms.

KMT1B/SUV39H2 is the writer responsible for H3K9me1/2 deposition and its overexpression was reported in PCa (Vieira et al., 2014). Through yeast two hybrid screening, SUV39H2 was identified as a coactivator of AR (Askew et al., 2017). Similarly, the H3K9me3 writer KMT1E/SETDB1 was also upregulated in PCa (Strepkos et al., 2021, Sun et al., 2014). Knockdown of SETDB1 induced the G0/G1 cell cycle arrest and inhibited PCa cell proliferation and migration (Sun et al., 2014). Dutta et al. discovered a NKX3.1-G9a-UTY transcriptional axis, which is essential for prostate differentiation (Dutta et al., 2016). More studies, however, are required to decipher the role of this axis in PCa. Notably, H3K9 methylation was found to be essential to drive antiandrogen resistance in advanced PCa (Baratchian et al., 2022). ADT induced de-repression of retroelements (REs) leading to a phenomenon termed viral mimicry, which activates the immune-related signaling pathways and thus inhibits tumor growth (Baratchian et al., 2022). Overexpression of H3K9me3 writers (for example, GLP or G9a) conferred drug resistance through mediating repression of antiandrogen-induced activation of REs, whereas inhibition of these writers and readers (such as CBX5, also known as HP1α) restored RE expression and abolished antiandrogen resistance (Baratchian et al., 2022). Therefore, a combined treatment with antiandrogen and inhibitors targeting H3K9me3 regulators serves as a promising therapeutic strategy for treating CRPC, based on the linkage between H3K9me3-mediated RE silencing and tumor microenvironment.

The H3K9me3 reader HP1α showed elevated expression in NEPCs, which was found to be associated with poor prognosis (Ci et al., 2018). HP1α promoted neuroendocrine trans-differentiation and silencing of HP1α inhibited NEPC cell proliferation (Ci et al., 2018). Another H3K9me3 reader MPP8 was reported to repress E-cadherin through binding to H3K9 methylation and promote EMT phenotypes in PC3 cells (Sun et al., 2015).

H3K9 methylation is removed by the KDM3 and KDM4 family of erasers, which are often highly expressed in PCa (Bjorkman et al., 2012). KDM3A (also known as JMJD1A) regulates activities of AR and c-Myc and promotes prostate cancer progression (Fan et al., 2016). Acetylation of KDM3A by P300 blocks its degradation and enhances AR activity in CRPC (Xu et al., 2020). In addition, KDM3A promotes alternative splicing of AR to generate the constitutively active AR spliced variant 7 (AR-V7), the formation of which represents one of the main ADT resistance mechanisms (Fan et al., 2018). Collectively, multifaceted roles of KDM3A in regulating the AR activation, c-Myc activity and AR-V7 alternative splicing suggested it to be a promising therapeutic target for PCa. KDM3B (also known as JMJD1B) was identified as a key regulator of cell proliferation in CRPC cells by a focused shRNA screening (Sarac et al., 2020). KDM3C has been shown to have a synthetic lethal relationship with AR and the AR-negative PCa cells are sensitive to KDM3C inhibition (Yoshihama et al., 2021). Knockdown of PHF8, another H3K9 demethylase, showed that it plays a role in PCa cell proliferation, migration and invasion (Bjorkman et al., 2012).

KDM4A and KDM4D form complexes with ligand-bound AR and function as AR coactivators (Shin and Janknecht, 2007). KDM4A (also known as JMJD2A) is a coactivator of E2F1 and regulates PCa metabolism through transcriptional regulation of pyruvate dehydrogenase kinase (PDK) (Wang et al., 2016a). Overexpression of KDM4A is positively correlated with Gleason score and metastasis in human PCa (Kim et al., 2016). Kim et al. further identified a KDM4A-ETV1-YAP1 axis that operates to promote PCa initiation (Kim et al., 2016). KDM4B interacts with AR and regulates its stability and activity (Coffey et al., 2013). In addition, KDM4B promotes alternative splicing to generate AR-V7, thus contributing to the development of CRPC (Duan et al., 2019). Furthermore, KDM4B promotes CRPC cell proliferation through physically interacting with c-Myc to activate a set of metabolic genes such as LDHA (Wu et al., 2021). Overexpression of KDM4B may act promote the recruitment of AR to the enhancer of c-Myc gene and induces its expression, which causes anti-androgen resistance (Tang et al., 2020). Together, targeting KDM4B may theoretically repress the nodes of AR-FL, AR-V7 and c-Myc, making it an attractive therapeutic strategy in PCa. In fact, small-molecule inhibitors targeting KDM4 were shown to suppress PCa cell proliferation (Duan et al., 2015, Chu et al., 2014).

In summary, H3K9 methylation and its various regulators profoundly affect PCa cell proliferation and survival, as well as PCa-TME interaction, through different molecular mechanisms. Targeting the H3K9 methylation-related modifiers and regulators provide promising clinical strategies to fight against PCa, which awaits additional studies.

3.3. H3K27 methylation

Trimethylation of histone H3 lysine 27 (H3K27me3) is another histone PTM closely associated with transcriptional repression (Di Croce and Helin, 2013). H3K27me3 deposition is carried out by Polycomb Repressive Complex 2 (PRC2), a multi-subunit methyltransferase complex that contains Enhancer of zeste homolog 2 (EZH2) as the catalytic subunit. PRC2 core complex is composed of EZH2 or related EZH1, EED, SUZ12 and RbAP46/48 (Guo et al., 2021). To gain a comprehensive view of PRC2 function in development and cancer, please refer to recent comprehensive reviews (Kim and Kingston, 2022, Piunti and Shilatifard, 2021, Blackledge and Klose, 2021).

EZH2 mRNA and protein levels were found to be progressively increased in metastatic PCa samples compared with benign prostate tissues (Varambally et al., 2002). In a clinical PCa cohort, higher expression of EZH2 was found to be correlated with a worse prognosis (Varambally et al., 2002). EZH2 overexpression promotes the proliferative and invasive capacity of PCa cells (Varambally et al., 2002, Varambally et al., 2008). Loss of EZH2-targeting microRNAs such as micro-RNA-101, which negatively regulates EZH2 at a posttranscriptional level, contributes to overexpression of EZH2 during PCa progression (Varambally et al., 2008).

As an oncogenic driver of PCa, overexpression of EZH2 inhibits the expression of TSGs through deposition of H3K27me3, which then promotes tumorigenesis (Jain and Di Croce, 2016). In addition to this canonical role of EZH2 in H3K27me3 deposition and gene silencing, EZH2 also carries non-canonical oncogenic functions independent of PRC2 and H3K27me3 (Wang and Wang, 2020). For instance, EZH2 can methylate STAT3 to promote its activity in glioblastoma stem-like cells (Kim et al., 2013). EZH2 methylates elongin A to regulate target gene transcription in embryonic stem cells (Ardehali et al., 2017). Besides its canonical function to repress TSGs in PCa, EZH2 was reported to be involved in transcriptional activation of AR target genes, and PI3K/AKT phosphorylation of EZH2 at serine 21 was proposed to promote such a gene-activation effect by EZH2 (Xu et al., 2012). EZH2 was further demonstrated to directly bind the AR gene promoter to activate its transcription, thereby potentiating AR signaling in PCa (Kim et al., 2018). A more recent study from the same group showed that EZH2 can methylate FOXA1 at the K295 residue to enhance FOXA1’s stability, thereby regulating the cell cycle-related genes in PCa cells (Park et al., 2021a). Furthermore, Yi et al. demonstrated that EZH2 increases rRNA 2’-O methylation and regulates translation through its direct interaction with fibrillarin in PCa (Yi et al., 2021). EZH2 also plays a significant role in regulating lineage plasticity, drug resistance and antitumor immunity (Morel et al., 2021, Abida et al., 2019, Bai et al., 2019, Xiao et al., 2018, Davies et al., 2021, Berger et al., 2019, Puca et al., 2018, Zhang et al., 2018, Dardenne et al., 2016, Clermont et al., 2015, Ku et al., 2017).

Because of the aforementioned important roles of EZH2, many EZH2 inhibitors, such as DZNeP, GSK126, UNC1999, EPZ-6438, PF-06821497, CPI-1205, CPI-0209 and DS3201, have been developed and tested in preclinical and clinical settings (Park et al., 2021b, Li et al., 2021, Duan et al., 2020). Tazemetostat (EPZ-6438) is the first FDA-approved EZH2 inhibitor for the treatment of epithelioid sarcoma and follicular lymphoma (Hoy, 2020). In the domain of mCRPC, there are a couple of ongoing clinical trials, either with EZH2 inhibitors alone or together with antiandrogen treatment, PARP inhibitor or immune checkpoint blockade (ICB) therapy (NCT03460977, NCT04846478, NCT04179864, NCT03480646, NCT04104776, NCT04388852; Table 1). Despite much effort, there seems a lack of efficacy of EZH2 inhibitors in solid tumors, which could be explained, at least partially, by the enzymatically independent functions of EZH2.

Interestingly, a gene-activation-related function EZH2 has been linked to an intrinsic transactivation activity harbored within a cryptic transactivation domain (TAD) of EZH2 (Jiao et al., 2020, Wang et al., 2022b). Here, EZH2 TAD was found to directly interact with p300 and c-Myc, which then act to mediate gene activation (Jiao et al., 2020, Wang et al., 2022b). To more thoroughly inhibit multifaceted activities of EZH2 in cancers, a Proteolysis-Targeting Chimera (PROTAC)-based degrader specific to EZH2 was recently developed for blocking both H3K27me3-dependent and H3K27me3-independent tumor-promoting functions of EZH2 (Wang et al., 2022b, Wang et al., 2022a). Wang et al. further reported that, in advanced PCa, EZH2’s TAD associates with both AR and AR-V7 to recruit the transactivation-related machineries at target sites that lack binding of PRC2 and H3K27me3 (Wang et al., 2022a). These non-canonical target sites of EZH2 and AR/AR-V7 were found to be enriched for the clinically relevant oncogenes, notably CDK2 and MYBL2 (also known as B-Myb) (Wang et al., 2022a). EZH2 TAD facilitates EZH2’s recruitment to these non-canonical target oncogenes, stimulating their activation to enhance CRPC growth in vitro and in vivo (Wang et al., 2022a). And when compared to the matched EZH2 catalytic inhibitor, the EZH2 degrader MS177 showed superior antitumor efficacy, presumably through on-target depletion of both EZH2’s canonical (EZH2:PRC2) and non-canonical (EZH2TAD:AR/AR-V7:co-activators) complexes in PCa (Wang et al., 2022a). EZH2-targeting PROTACs emerge as a promising therapeutic method for treating the aggressive cancers (Wang et al., 2022b, Wang et al., 2022a).

H3K27me demethylases include KDM6A (or UTX), KDM6B (or JMJD3), and KDM7A. KDM6A interacts with AR in advanced PCa (Grasso et al., 2012). Although KDM6A was found mutated in metastatic CRPC, its exact function in PCa progression or AR signaling remains elusive. A role for KDM6B was also implicated in PCa (Xiang et al., 2007a, Farzaneh et al., 2022). KDM7A is upregulated in PCa, which is correlated with Gleason score (Lee et al., 2018). KDM7A directly interacts with AR and regulates expression of its downstream target genes (Lee et al., 2018). Knockdown of KDM7A inhibits PCa cell proliferation in vitro and in the tumor xenografted animal model, indicating that targeting KDM7A might be a possible strategy to treat advanced PCa (Lee et al., 2018).

3.4. H3K36 methylation

H3K36 methylation is involved in a range of biological processes including transcriptional regulation, mRNA splicing, DNA replication and DNA repair, and it also cross-talks with other epigenetic modifications, such as DNA methylation and H3K27me3 (Husmann and Gozani, 2019, Li et al., 2019). H3K36me2 is enriched at the 5’ regions of actively transcribed genes and intergenic area, while H3K36me3 is mainly localized to the gene body of actively transcribed genes (Pokholok et al., 2005, Li et al., 2019, Husmann and Gozani, 2019). There exist several H3K36me2-specific writers in the human cells, which include ASH1L and the nuclear receptor binding SET domain (NSD) family proteins, NSD1, NSD2 (also known as WHSC1 and MMSET) and NSD3. In contrast, SETD2 is the sole H3K36me3 writer (Husmann and Gozani, 2019). H3K36 methylation is removed by the KDM2 (KDM2A and KDM2B) and KDM4 (KDM4A-KDM4D) subfamilies of erasers (Greer and Shi, 2012). H3K36 methylation can be recognized by reads that contain the PWWP, Chromodomain, or Tudor domain (Li et al 2019).

Deregulation of NSD family writer is known to be involved in development of human cancers including PCa (Topchu et al., 2022). For example, NSD1 and NSD2 were both shown to interact with AR, enhancing transactivation of AR signaling in PCa (Wang et al., 2001, Kang et al., 2009). The role of NSD2 in PCa has been extensively studied. NSD2 mediates constitutive activation of NF-κB signaling by proinflammatory cytokines in CRPC through direct interaction with NF-κB, which involves NSD2’s enzymatic activity (Yang et al., 2012). NSD2 is overexpressed in PCa and required for PCa tumor growth (Yang et al., 2012). In addition, NSD2 has been found to be overexpressed in metastatic PCa compared to primary tumors and associated with disease stage and biochemical recurrence (Li et al., 2017b). NSD2, together with PTEN loss, promotes PCa metastasis (Li et al., 2017b). Aytes et al. further showed NSD2 to be a conserved driver of metastatic PCa through analysis of genetically engineered mouse models (GEMM) of PCa and correlative study of human PCa patient datasets (Aytes et al., 2018). Interestingly, H3K36me2 has recently been associated with epithelial plasticity and metastasis in pancreatic ductal adenocarcinoma (PDAC) through the H3K36 to H3M36 mutation (H3K36M), which directly and globally inhibits this mark (Yuan et al., 2020b). via CRISPR-Cas9 screening, the authors discovered both NSD2 and KDM2A (the writer and eraser of H3K36me2) to be involved in regulating EMT and tumor metastasis of PDAC (Yuan et al., 2020b). NSD2 also cross-talks with other epigenetic players. For instance, EZH2 regulates the expression of NSD2 through repressing the NSD2-targeting microRNAs (Asangani et al., 2013). On the other hand, the oncogenic function of EZH2 was mediated by NSD2, which affects PCa tumor formation and metastasis (Asangani et al., 2013). NSD2 has also been shown to be involved in regulating immune infiltration in PCa (Want et al., 2021). NSD3 is amplified in lung squamous cell carcinoma (LUSC) and functions as a key regulator of tumorigenesis (Yuan et al., 2021). In breast cancer, NSD3-mediated H3K36 methylation is crucial for tumor initiation and metastasis (Jeong et al., 2021). However, the function of NSD3 in PCa remains to be identified.

SETD2 serves as the sole methyltransferase for writing H3K36me3 (Edmunds et al., 2008) and is frequently mutated in a variety of human cancers (Li et al., 2019). Via H3K36me3 deposition, SETD2 has been shown to be involved in regulation of DNA damage repair and mRNA splicing (Luco et al., 2010). Additionally, SETD2 also methylates non-histone substrates to regulate oncogenesis. For example, Yuan et al. reported that loss of SETD2 cooperates with PTEN deletion to promote PCa metastasis in an EZH2-dependent manner (Yuan et al., 2020a). Mechanistically, SETD2 methylates EZH2 at its K735 residue, an event that promotes degradation of EZH2, an oncoprotein in PCa, and thus inhibits progression of PCa (Yuan et al., 2020a). The K735R mutant of EZH2 (a SETD2 methylation deficient mutant) induces metastatic PCa in mice (Yuan et al., 2020a). These results thus provide an explanation for the known antagonism between SETD2-mediated H3K36me3 and EZH2-catalyzed H3K27me3 (Yuan et al., 2011, Schmitges et al., 2011, Yuan et al., 2020a).

PHF19, a component of PRC2 complex, promotes the binding and spreading of PRC2 complex into active chromatin region through its Tudor domain-mediated recognition of H3K36me3, thereby promoting gene silencing during development (Li et al., 2017a, Brien et al., 2012, Ballare et al., 2012, Cai et al., 2013). PHF19 also plays a role in many cancers including PCa (Jain et al., 2020, Abdelfettah et al., 2020). In vitro studies with the AR-negative PCa indicated that PHF19 regulates cell proliferation, invasiveness and angiogenesis (Jain et al., 2020). Another H3K36 methylation reader, LEDGF (or PSIP1), is overexpressed in PCa and knocking down LEDGF sensitizes chemo-resistant PCa cells to taxanes (Rios-Colon et al., 2017). The exact functions of these H3K36 methylation readers in PCa tumorigenesis and progression warrant more detailed study in future.

The H3K36 methylation erasers include the family members of KDM2 and KDM4. KDM2B has recently been shown to play a role in PCa cell motility (Zacharopoulou et al., 2020). The four KDM4 family members (namely, KDM4A, KDM4B, KDM4C and KDM4D) can act as the erasers of both H3K36me2/3 and H3K9me2/3 methylation (Klose et al., 2006, Whetstine et al., 2006), as mentioned in the previous H3K9 methylation section. KDM4 proteins can form complex with AR and promote its activity in an enzymatic dependent fashion (Coffey et al., 2013, Chu et al., 2014, Wissmann et al., 2007, Shin and Janknecht, 2007).

3.5. H3K79 methylation

H3K79 methylation, located inside the globular domain of histone H3, is associated with the actively transcribed genes (Zhao et al., 2021). Disrupter of telomeric silencing 1L (DOT1L) is the only identified writer for H3K79 methylation (Feng et al., 2002). So far, eraser of H3K79 methylation has not been reported. Very recently, Menin is identified as the first H3K79me2 reader protein (Lin et al., 2023). Menin is a well-known component of MLL complex, the H3K4 methylation writer, and also a critical regulator of AR signaling (Malik et al., 2015). The expression of Menin is higher in CRPC when compared to primary PCa and benign prostate tissues, which also correlates with poor overall survival of PCa patients (Malik et al., 2015). How exactly such a newly identified function of Menin as a H3K79me2 reader is involved in PCa development warrants further investigation. H3K79me2 and H3K79me3 marks participate in the regulation of gene transcription, cell cycle progression and DNA damage response (Nguyen and Zhang, 2011). Besides its function in the development and maintenance of MLL-rearranged (MLL-r) leukemia (Okada et al., 2005), DOT1L has also become a promising therapeutic target for treating the solid tumors (Alexandrova et al., 2022). DOT1L was identified as a novel cofactor for ERα and regulates transcription of ERα target genes in breast cancer (Nassa et al., 2019). DOT1L inhibition impairs the growth of antiestrogen-resistant breast cancer cells (Nassa et al., 2019). Vatapalli et al. found that DOT1L expression was significantly increased in PCa relative to normal prostate and correlated with Gleason score and poor clinical outcome in multiple datasets of PCa patients (Vatapalli et al., 2020). Interestingly, DOT1L blockade specifically reduces the viability of AR-positive PCa cells, including enzalutamide-resistant ones (Vatapalli et al., 2020). Mechanistically, DOT1L and AR co-bind a distal enhancer of MYC, thus promoting the MYC expression in AR-positive PCa cells; conversely, treatment with the DOT1L inhibitor promotes AR and MYC degradation via upregulation of the E3 ubiquitin ligases HECTD4 and MYCBP2, thus impairs the PCa tumor growth (Vatapalli et al., 2020). Taken together, these studies provide a foundation for rationalizing the targeting of DOT1L in endocrine therapy resistant AR-positive or ERα-positive cancers.

4. Histone acetylation in prostate cancer

Histone acetylation is closely associated with transcriptional activation and this histone PTM is achieved through the transfer of an acetyl group to the histone lysine by HATs (Struhl, 1998). Conversely, HDACs conduct deacetylation reaction by the removal of an acetyl group off lysine (Struhl, 1998). There are over 40 different histone lysine residues that have been reported to be modified by acetylation (Zhao and Garcia, 2015). Histone acetylation influences numerous physiological and pathogenic processes (Shvedunova and Akhtar, 2022). For instance, HATs and HDACs act as AR’s coactivator and corepressor, respectively, thus profoundly affecting the AR-mediated gene transcription in PCa (Lavery and Bevan, 2011). H3K27ac is a marker for active enhancers and promoters. Patterns of global AR binding and H3K27ac are reprogrammed in mCRPC compared to localized PCa (Pomerantz et al., 2020), suggesting the importance of chromatin states in governing tumor progression. In addition, H3K27ac profiling before and after AR-targeted therapy revealed that a subset of H3K27ac peaks are associated with treatment resistance (Severson et al., 2023).

Super-enhancers are defined as a cluster of enhancers formed by exceedingly high levels of binding of master transcription factors and mediator complex, also marked with strong histone acetylation such as H3K27ac (Whyte et al., 2013), which play a crucial role as oncogenic drivers in various tumor types including PCa. Activation of AR-associated enhancers is well correlated with histone acetylation in PCa (Valdes-Mora et al., 2017).

4.1. Histone acetyltransferases (HATs)

Classic HATs include p300, CREB-binding protein (CBP), TIP60, GCN5 and PCAF. These HATs are involved in AR signaling and PCa tumorigenesis, which are nicely summarized in a recent review (Jaiswal et al., 2022). Here, we focus on p300 and CBP, the two highly homologous HAT proteins, each containing two ZZ-type zinc finger (ZZ) domains and three cysteine/histidine-rich (CH) domains (CH1, CH2 and CH3), a bromodomain (BD) that recognizes acetylated residues, and a catalytic histone acetyltransferase (HAT) domain that catalyzes lysine acetylation (Dancy and Cole, 2015). p300 and CBP are well-known AR coactivators through directly acetylating AR to enhance AR’s transcriptional activity (Fu et al., 2000). They are overexpressed and involved in the progression of PCa (Comuzzi et al., 2004, Debes et al., 2003). A number of CBP/p300 inhibitors have recently been developed. For example, GNE-049, a CBP/p300 bromodomain inhibitor, inhibits the growth of CRPC in vitro and in vivo (Jin et al., 2017). A-485, a highly selective catalytic inhibitor of p300/CBP, potently downregulated the AR transcriptional output in both androgen-sensitive PCa and CRPC cells (Lasko et al., 2017). Furthermore, A-485 inhibited the growth of LUCaP-77 xenograft, a patient-derived AR-positive CRPC model (Lasko et al., 2017). In addition, A-485 treatment greatly increases the efficacy of PD-L1 blockade in a syngeneic PCa mouse model through inhibiting the secretion of exosomal PD-L1 (Liu et al., 2020). More recently, CCS1477, another CBP/p300 inhibitor targeting the bromodomain, exerts anti-tumor activity through regulating AR, AR-V7 and c-MYC nodes and it is currently in a phase I/II clinical trial NCT03568656 (Table 1) (Welti et al., 2021). In summary, these findings underscore CBP/p300 as a compelling therapeutic target, either alone or in combination with other drugs, for the treatment of advanced PCa.

4.2. Histone deacetylases (HDACs)

HDACs removes acetylation of histones and non-histone substrates (Shvedunova and Akhtar, 2022). So far, eighteen different HDACs have been identified, which comprise four major classes, namely, HDAC Class I, II, III and IV (Seto and Yoshida, 2014). Overexpression of HDACs is observed in different types of cancers, including PCa (Ropero and Esteller, 2007).

Class I HDACs include HDAC1, 2, 3 and 8, which are highly expressed in PCa compared to normal prostate tissue (Weichert et al., 2008). HDAC inhibitors block AR-mediated activation of target genes in both hormone-sensitive and refractory PCa (Welsbie et al., 2009). Similarly, HDAC3 inhibitors were shown to block the activity of AR-V7, inhibiting the growth of 22Rv1 xenografted tumors (McLeod et al., 2018). A very recent study further demonstrated that selective elimination of senescent neutrophils through HDAC inhibitors delays PCa progression in vivo (Bancaro et al., 2023). Class III HDACs, namely sirtuins (SIRT1–7), are nicotinamide adenine dinucleotide (NAD+)-dependent and different from other zinc-dependent HDAC classes (Chalkiadaki and Guarente, 2015). The function of sirtuins in PCa is complex and context dependent. For example, loss of SIRT1 results PIN formation (Powell et al., 2011); and SIRT1 inhibits PCa cell proliferation through AR deacetylation (Lavery and Bevan, 2011, Fu et al., 2006). In contrast, Huang et al. showed that lowering SIRT1 leads to inhibition of PCa growth (Huang et al., 2021).

HDAC inhibitors, which cover five different classes of compounds (namely, hydroxamic acids, aliphatic acids, cyclic peptides, benzamides and sirtuin inhibitor), have been developed and tested in clinical trials (Eckschlager et al., 2017). Suberoylanilide hydroxamic acids (SAHA) was the first FDA-approved HDAC inhibitor for the treatment of cancer in the US (Grant et al., 2007). SAHA treatment has been found to inhibit the proliferation of PCa cell lines in vitro and the progression of PCa tumors in vivo (Butler et al., 2000, Chinnaiyan et al., 2005, Lakshmikanthan et al., 2006). Although HDAC inhibitors showed efficacy for some hematological malignancy subtypes, a majority of the clinical trials involving HDAC inhibitors in PCa failed due to high toxicity and a lack of specificity (Rana et al., 2020). There are still ongoing clinical trials with HDAC inhibitor in PCa (Thompson et al., 2022). For example, the FDA-approved HDAC inhibitor Belinostat is currently tested together with PARP inhibitor Talazoparib in mCRPC (NCT04703920, Table 1). Nevertheless, different HDACs can exert either tumor-promoting or anti-tumorigenic functions (Chalkiadaki and Guarente, 2015), therefore demanding more mechanistic studies to provide guidance on how to employ class-specific HDAC inhibitors, either as a single-agent therapy or in combination with other therapies, to treat advanced PCa.

4.3. Histone acetylation readers

The bromodomain-containing family proteins recognize histone acetylation to regulate gene expression (Fujisawa and Filippakopoulos, 2017). The bromodomain and extra terminal domain (BET) subfamily of bromodomain-containing proteins include BRD2, BRD3, BRD4, and BRDT, all of which contain two tandem bromodomains (BD1 and BD2) (Perez-Salvia and Esteller, 2017, Stathis and Bertoni, 2018). BD1 and BD2 play an important role in regulating transcription via recognizing acetylated lysine residues of histones and non-histone proteins (Dhalluin et al., 1999). BRD4, the best characterized BET family protein, recruits the elongation factor p-TEFb to stimulate RNA polymerase II-dependent transcription (Jang et al., 2005). BET proteins are frequently overexpressed in various types of human cancer including PCa (Urbanucci et al., 2017, Urbanucci and Mills, 2018, Fujisawa and Filippakopoulos, 2017, Stathis and Bertoni, 2018). They play a critical role in tumorigenesis and represent a class of attractive therapeutic targets for cancer treatment (Perez-Salvia and Esteller, 2017).

Pan-BET inhibitors, such as JQ1, I-BET151, I-BET762, ABBV-075, OTX-015 and ZEN-3694, were developed to block interaction between BD domain and the acetylated residue, which were shown to affect a large variety of cellular processes and have antitumor effects in numerous preclinical and clinical models (Perez-Salvia and Esteller, 2017). In PCa, BRD4 physically interacts with the N-terminal domain of AR and promotes AR-related gene-expression programs in CRPC (Asangani et al., 2014). JQ1, an inhibitor of BET family member proteins, inhibited the binding of BRD4 to AR enhancers globally, thereby repressing the AR-mediated gene transcription (Asangani et al., 2016). Welti et al. reported that the nuclear BRD4 level increases significantly over disease progression and its higher expression at diagnosis is associated with a worse clinical outcome (Welti et al., 2018). Furthermore, they found that BET inhibitors reduce AR and AR-V7 signaling in patient-derived xenograft model of CRPC (Welti et al., 2018). Furthermore, BET inhibitors also overcome antiandrogen resistance in advanced PCa (Shah et al., 2017, Asangani et al., 2016). In addition, BRD4 seems to have AR-independent functions (Civenni et al., 2019, Coleman et al., 2019). BRD4 also recognizes the acetylated non-histone proteins such as NF-κB and TMPRSS2-ERG (Hajmirza et al., 2018, Li et al., 2018). Treatment of the BET inhibitor OTX-015 increases the efficacy of radiation therapy (RT) and overcomes radio-resistance through blocking DNA repair (Li et al., 2022b). In summary, these aforementioned studies lay a strong foundation for the future clinical exploration of BET inhibitors.

The mechanisms for resistance to BET inhibitors have been reported, some of which involve PCa-associated somatic mutation of SPOP, an E3 ligase substrate binding protein (Zhang et al., 2017, Janouskova et al., 2017, Dai et al., 2017). Due to its frequent mutation in PCa, mutant SPOP failed to induce the ubiquitination and proteasomal degradation of BET domain proteins (Zhang et al., 2017, Janouskova et al., 2017, Dai et al., 2017). This resistance mechanism then led to activation of AKT-mTORC1 signaling, which can be overcome by combination treatment with AKT inhibitors (Zhang et al., 2017).

PROTAC-based small-molecule degrader for specifically targeting BET domain proteins have also emerged (Raina et al., 2016, Zhou et al., 2018). For Instance, ARV-771, a potent pan-BET degrader, more potently suppressed the AR signaling and tumor progression in the CRPC-xenografted mouse models, compared to the original inhibitor (Raina et al., 2016). ZBC-260, another BET degrader, preferentially affects AR-positive PCa cells over AR-negative ones, and suppresses PCa growth in vitro and in vivo (Kregel et al., 2019).

Interestingly, a study based on CRISPR-Cas9-directed protein domain scanning has previously uncovered that the BD1 and BD2 domains of BRD4 have distinct functions (Shi et al., 2015). Accordingly, selective inhibitors targeting either BD1 or BD2 have recently been developed (Gilan et al., 2020, Faivre et al., 2020). While the BD1-specific inhibitor iBET-BD1 phenocopies the pan-BET inhibitors in a majority of the tested cancer cell lines, the BD2-specific inhibitor iBET-BD2 is predominantly effective in models of inflammatory and autoimmune disease (Gilan et al., 2020). The activity of another BD2-specific inhibitor, ABBV-744, is predominantly restricted to AML and AR-positive PCa (Faivre et al., 2020). ABBV-744 displaces BRD4 from AR-containing super-enhancers, inhibits the AR-dependent transcription, and reduces tumor growth in PCa xenografts (Faivre et al., 2020). There are a plethora of ongoing clinical trials with inhibitors or degraders targeting BET domain proteins (Guo et al., 2023). In mCRPC, a number of trials with the pan-BET inhibitor ZEN-3694 are currently underway (Table 1). A completed trial (NCT02711956) showed that ZEN-3694 plus Enzalutamide demonstrated acceptable tolerability and potential efficacy in mCRPC patients, and further prospective study is required (Aggarwal et al., 2020). Interestingly, a phase I/II clinical trial with NUV-868, a BD2-selective oral small-molecule BET inhibitor, is ongoing in mCRPC patients, either alone or in combination with Enzalutamide or Talazoparib (NCT05252390, Table 1).

5. Chromatin remodeling in prostate cancer

Chromatin accessibility is regulated by chromatin-remodeling complexes, which use the energy from ATP hydrolysis to slide or eject nucleosomes (Clapier et al., 2017). There are four subfamilies of chromatin-remodeling complexes, namely Chromodomain Helicase DNA-binding (CHD), Inositol Requiring 80 (INO80), Imitation Switch (ISWI) and Switch/Sucrose Non-Fermentable (SWI/SNF, also known as BRG1/BRM associated factor (BAF) complex) (Clapier et al., 2017). In this section, we mainly highlight the recent discoveries on SWI/SNF and CHD remodeling complexes in PCa.

Alteration of the SWI/SNF complex subunit occurs in about 20–25% of all cancers including PCa (Shain and Pollack, 2013, Kadoch et al., 2013). The catalytic ATPase components, SMARCA4 (BRG1) and SMARCA2 (BRM), mediate ATP hydrolysis to reposition nucleosomes at non-coding regulatory elements, thereby enabling the transcription factors to bind DNA and promote gene expression (Mittal and Roberts, 2020). The mutant SWI/SNF complex usually enhances oncogenic transcriptional programs, making it a promising therapeutic target (Centore et al., 2020). BRG1 has been reported to be overexpressed in PCa and associated with tumor progression (Cyrta et al., 2020, Muthuswami et al., 2019, Sun et al., 2007). Sandoval et al. identified a strong interaction between TMPRSS2-ERG and BAF complex in VCaP cells, a PCa cell line containing endogenous TMPRSS2-ERG fusion (Sandoval et al., 2018). They further found that this interaction drives retargeting of BAF complex globally to ETS target sites; meanwhile, the ATP-dependent catalytic activity of BAF complex is required for ERG’s chromatin association, ERG’s downstream target gene expression and ERG’s oncogenic function in PCa (Sandoval et al., 2018). The interdependency between ERG and BAF complex indicates that TMPRSS2-ERG-positive PCa could be sensitive to BAF complex inhibitors. Indeed, Xiao et al. reported that the treatment of AU-15330, a PROTAC degrader of BRG1 and BRM, dismissed the main transcription factors such as AR, FOXA1, ERG and MYC from chromatin, disrupted their super-enhancer and promoter looping, inhibited the down-stream oncogenic pathways, and induced strong inhibition of CRPC growth, either alone or in combination with enzalutamide (Xiao et al., 2022). BRD9, a component of non-canonical SWI/SNF complex (ncBAF), has recently been shown to interact with AR and regulate its activity and promote tumor progression (Alpsoy et al., 2021). In addition, the expression of BAF57 is positively correlated with Gleason score and functions as a critical regulator of AR (Link et al., 2008, Balasubramaniam et al., 2013).

Chromatin-remodeling complex often intertwines with other signaling pathways. For example, a synthetic lethal relationship was characterized between PTEN and BRG1 by the Qin laboratory (Ding et al., 2019). In this study, they found that PTEN loss stabilized BRG1 via inhibiting the AKT/GSK3b/FBXW7 pathway, which drove a tumor-promoting transcriptome through chromatin remodeling (Ding et al., 2019). Additionally, PTEN-deficient PCa is sensitive to the treatment with BRG1 antagonist, indicating a therapeutic strategy (Ding et al., 2019).

Another chromatin remodeler CHD1 is altered in 7–10% of PCa cases (Baca et al., 2013, Grasso et al., 2012). CHD1 has been reported to play significant roles in PCa in a context-dependent manner (Li et al., 2023, Zhao et al., 2017). CHD1 depletion leads to the defects of DNA double-strand break (DSB) repair via homologous recombination (HR) and thus sensitizes PCa cells to PARP inhibitors (Kari et al., 2016). Coordinate loss of MAP3K7 and CHD1 occurs in 10% to 20% of localized PCa, correlating with poor disease-free survival (Rodrigues et al., 2015). Likewise, combined loss of MAP3K7 and CHD1 increased tumor growth and decreased survival in LNCaP xenografted animal models (Rodrigues et al., 2015). In addition, CHD1 loss alters AR binding and regulates different sets of target genes to promote PCa (Augello et al., 2019). Zhang et al. identified that depletion of CHD1 leads to lineage plasticity and antiandrogen resistance through an in vivo shRNA screening (Zhang et al., 2020). In a mCRPC patient cohort, low CHD1 expression is correlated with a worse response to the second-generation antiandrogen treatment (Zhang et al., 2020).

Interestingly, both SWI/SNF complex and CHD1 have been shown to play an important role in cancer immunotherapy including PCa (Li et al., 2022a, Belk et al., 2022). Since PCa is known to be immune-cold (Topalian et al., 2012), it would be worthwhile to test whether targeting chromatin remodeling pathway makes PCa sensitive to immunotherapy.

6. Conclusions and future perspectives

This chapter provides an overview of epigenetic alterations, focusing on DNA methylation, histone methylation and acetylation, and chromatin remodeling during the development and progression of PCa. AR-mediated signaling is one of the most critical drivers for PCa initiation and progression. Various histone-modifying enzymes regulate ligand-dependent or ligand-independent transcription of AR target genes through interacting with AR. Additionally, epigenetic regulators are also critically involved in the activation of other oncogenic signaling pathways, independent of AR signaling and usually in a more advanced setting of PCa such as mCRPC. Due to the reversible nature of epigenetic modifications, targeting epigenetic regulators has become a promising therapeutic intervention. Table I lists some of the ongoing clinical trials for testing drug candidates targeting epigenetic factors in patients with mCRPC. Development of epigenetically centered therapy alone generally showed less efficacy in PCa, probably because of the heterogeneity nature of this disease. However, we remain optimistic about potential combination therapies with other drugs, such as antiandrogen, PARP inhibitor and immune therapy, for the treatment of patients with advanced PCa. We look forward to new exciting development along these lines in the years to come.