Research progress of m⁶A methylation in prostate cancer

Abstract

N⁶-methyladenosine (m⁶A) is a ubiquitous RNA modification in mammals. This modification is “written” by methyltransferases and then “read” by m⁶A-binding proteins, followed by a series of regulation, such as alternative splicing, translation, RNA stability, and RNA translocation. At last, the modification is “erased” by demethylases. m⁶A modification is essential for normal physiological processes in mammals and is also a very important epigenetic modification in the development of cancer. In recent years, cancer-related m⁶A regulation has been widely studied, and various mechanisms of m⁶A regulation in cancer have also been recognized. In this review, we summarize the changes of m⁶A modification in prostate cancer and discuss the effect of m⁶A regulation on prostate cancer progression, aiming to profile the potential relevance between m⁶A regulation and prostate cancer development. Intensive studies on m⁶A regulation in prostate cancer may uncover the potential role of m⁶A methylation in the cancer diagnosis and cancer therapy.

INTRODUCTION

Epigenetic modification is the regulation of the expression of heritable genes without altering the DNA sequence, including DNA methylation, RNA methylation, histone modification, and chromatin remodeling. Next-generation sequencing shows that more than 50% of cancers have mutations in chromatin-related enzymes, so DNA and RNA methylation modifications are particularly important in the tumor progression.1 N⁶-methyladenosine (m⁶A) is a ubiquitous RNA modification in mammals. This modification is “written” by m6A methyltransferase (such as methyltransferase 3, N⁶-adenosine-methyltransferase complex catalytic subunit [METTL3]/METTL14/METTL16, RNA-binding motif protein [RBM] 15/15B, vir-like m6A methyltransferase associated [VIRMA], and zinc finger CCCH-type containing 3 [ZC3H3]). Then it is “read” by m6A-binding proteins (such as YTH N⁶-methyladenosine RNA-binding protein [YTHDF] 1/2/3, YTH domain containing [YTHDC] 1/2, heterogeneous nuclear ribonucleoprotein A2/B1 [HNRNPA2B1], and insulin-like growth factor 2 mRNA-binding protein [IGF2BP] 1/2/3). Finally it is removed by demethylases (such as FTO alpha-ketoglutarate-dependent dioxygenase [FTO] and alkB homolog 5, RNA demethylase [ALKBH5]). m⁶A modification can occur in various types of RNA, such as mRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA (circRNA), microRNA (miRNA), and long non-coding RNA (lncRNA), as well as regulate the alternative splicing, translation, translocation, and stability of these RNAs.2–4 Most m⁶A sites exist near stop codons and contain the conserved motif DRACH (D = G/A/U, R = G/A, H = A/U/C).5 Extensive sequencing results show that there is a high degree of genomic and phenotypic diversity in orthotopic and metastatic prostate cancer (PCa), and these differences are determined by classical genetic and epigenetic changes.6–11 The most striking feature of PCa is that it is multifocal,10 and different lesions in PCa have unique nonoverlapping mutational profiles,12–14 indicating that they have different evolution trajectories. It also suggests a potential impact of classical genetic and epigenetic changes on PCa progression. Today, the treatment options for PCa are relatively single, while in addition to androgen receptor (AR) inhibitor-related treatments, m⁶A-related genome therapy may provide new treatments for PCa in the future.

M⁶A METHYLATION DYSREGULATION PROMOTES PCA PROGRESSION

First, significant changes in m⁶A methylation-related factors may cause gene transcriptome changes and modulate cancer progression. Invasive PCa highly expressed most m⁶A methylation regulators. For example, METTL3, RBM15B, and HNRNPA2B1 were not only highly expressed in cancer tissues, but also correlated with high Gleason scores (GS). However, FTO and ALKBH5, which act as m⁶A “erasers”, were significantly downregulated in PCa tissue samples, and this was associated with copy number variations (CNVs) events. Under the combined effect of overexpression of m⁶A-related readers and writers and downregulation of erasers, most PCas show high levels of mRNA methylation, which affects the subcellular localization of proteins in tumors and results in poor survival15 (Figure 1). Another study has also identified Cbl proto-oncogene like 1 (CBLL1), FTO, YTHDC1, and HNRNPA2B1 as key m⁶A regulators in PCa based on the data derived from The Cancer Genome Atlas (TCGA, available from: https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga).16 This result was supported by an in vitro experiment, in which knockout of FTO significantly inhibited the migration and invasion of PCa cells.16 Although only a few copy number abnormalities were observed for m⁶A regulators in PCa, the effect of copy number variation on m⁶A regulator expression was much stronger than that of DNA methylation interference.17 Similar conclusions also were made in another study, in which it was also found that the overall level of m⁶A was increased in transgenic adenocarcinoma of mouse prostate (TRAMP) mice, while the level of m⁶A was significantly upregulated with tumor progression.18 Furthermore, m⁶A writer HNRNPA2B1 was found to be significantly upregulated in advanced PCa tissues (GS>7, pT3, hazard ratio [HR]>1, and tumor protein p53 [TP53] mutation).19 In addition, Cotter’s study has also shown that METTL3 expression is low in advanced metastatic PCa tissues, in which the AR level is low, while the expression of neuroendocrine-related genes is significantly increased. It has been speculated that the low level of METTL3 in these cancers may be related to enzalutamide resistance.20

Figure 1.

Due to the enhanced expression of “writer” and “reader” and the occurrence of CNVs events related to “eraser”, the total level of m⁶A in prostate cancer cells increases, which further affects the occurrence, development, and distant metastasis of prostate cancer. CNVs: copy number variations; HNRNPA2B1: heterogeneous nuclear ribonucleoprotein A2/B1; METTL3: N⁶-adenosine-methyltransferase complex catalytic subunit; RBM15B: RNA-binding motif protein 15B; YTHDF: YTH N⁶-methyladenosine RNA-binding protein; YTHDC: YTH domain containing; FTO: FTO alpha-ketoglutarate-dependent dioxygenase; ALKBH5: alkB homolog 5, RNA demethylase.

In addition, m⁶A regulation may also play a role in therapy resistance to androgen receptor inhibitor. It has been shown that when androgen-sensitive PCa cells were treated with androgen receptor pathway inhibitors (ARPI), the m⁶A-modified AR mRNA was recruited from actively translating polysomes (PSs) to RNA protein stress granules (SGs), and further m⁶A-modified AR mRNA bound to YTHDF3 in SGs. This led to the decrease of AR protein translation and expression, which in turn resulted in the resistance of PCa cells to ARPI treatment. Likely, in this process, the m⁶A-modification is involved in the adaption of androgen-sensitive PCa cells to the stress environment induced by ARPI. In contrast, androgen sensitive PCa cells were easily killed by ARPI treatment after silencing YTHDF3 compared with cells with intact YTHDF3 expression. This result provides a new mechanism underling the adaptive survival of PCa cells facing to ARPI stress.21

M⁶A-RELATED REGULATORY MOLECULES AND PCA

METTL family

METTL3 and METTL14 serve as the core subunits of the methyltransferase complex, among which METTL3 plays a major enzymatic role, and METTL14 can recognize RNA and stabilize METTL3.22 METTL3’s expression was found to be increased in PCa tissue and significantly upregulated overall m⁶A levels. It has been supposed that METTL3 writes m⁶A modification at A2696 of USP4 mRNA, which is read by YTHDF2 and induces ubiquitin-specific peptidase 4 (USP4) mRNA degradation by recruiting the RNA-binding protein heterogeneous nuclear ribonucleoprotein D [HNRNPD]. Decreased level of USP4 protein leads to increased ubiquitination of ELAV-like RNA-binding protein 1 (ELAVL1), which in turn increases Rho GDP dissociation inhibitor alpha (ARHGDIA) expression, ultimately promoting migration and invasion of PCa cell.23 METTL3 was also found to affect lymphoid enhancer-binding factor 1 (LEF1) protein levels by affecting m⁶A methylation of LEF1 mRNA, thereby downregulating the activity of wingless/integrated (Wnt) signaling to affect PCa progression.24 However, METTL3 mutated at the catalytic site of m⁶A inhibits tumor growth due to METTL3 depletion. Further results suggest that METTL3 promotes PCa growth by regulating the hedgehog signaling pathway.25 At the same time, another study has shown that MYC is a target of METTL3-mediated m⁶A modification in PCa, and its protein level will be increased with the expression of METTL3.26 In bone metastatic PCa, METTL3 regulates the expression of integrin β1 (ITGB1) through an m⁶A-HuR-dependent mechanism, and then affects the binding of ITGB1 to collagen I and tumor cell motility, thus promoting bone metastasis of PCa.27

VIRMA

As a writer that regulates m⁶A methylation modification, VIRMA is very important for the deposition of m⁶A at the 3’-untranslated region (3’UTR) of mRNA.28 In PCa, the expression of VIRMA is elevated in hormone-insensitive PCa, and knockdown of VIRMA leads to a decrease in m⁶A methylation levels as well as the malignant biological behavior of PCa.29

YTH family

This protein family includes YTHDF1/2/3 and YTHDC1/2. The ability of these molecules in binding RNA and recognizing m⁶A depends on the YTH domain, and their protein functions are inseparable from RNA metabolism. Among them, YTHDC1 is mainly located in the nucleus, while YTHDF1/2/3 and YTHDC2 are mainly located in the cytoplasm.30 YTHDF2 knockdown can significantly reduce the proliferation and migration of PC-3 and DU145.31 In clinical samples, high expression of YTHDF2 and METTL3 is often associated with poor overall survival, and a related study has shown that YTHDF2 mediates the mRNA degradation of tumor suppressors phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) and NK3 homeobox 1 (NKX3-1) in an m⁶A-dependent manner, thereby promoting the progression of PCa by regulating AKT phosphorylation.32

FTO

As a regulator of body weight and obesity, FTO also mediates methylation reversal as an eraser in m⁶A regulation. The expression level of FTO was decreased in PCa tissue associated with advanced stage and higher GS. Knockdown of FTO promoted PCa cell invasion and metastasis in vitro, whereas overexpressing FTO inhibited the malignant biological behavior of PCa.33 In addition, high expression of FTO can also partially reverse the cancer-promoting effect of MC4R on PCa.34 In addition, a study from Xia et al.35 identified chloride intracellular channel 4 (CLC4) as a functional target of FTO using molecular techniques. It was finally confirmed that FTO inhibits PCa proliferation and metastasis by reducing the degradation of CLIC4 mRNA in an m⁶A-dependent manner.

NCRNAS REGULATED BY M⁶A AFFECT THE PROGRESSION OF PCA

miRNA

Kinesin family member 3C (KIF3C), as a proto-oncogene, is involved in the occurrence and development of tumors by activating phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and transforming growth factor β (TGF-β) signaling. It was found that the expression of KIF3C was abnormally increased in PCa, and it was negatively correlated with tumor prognosis. In terms of the upregulation of KIF3C in PCa, m⁶A methylation plays a key role. It has been shown that METTL3 enhances the m⁶A level of KIF3C and promotes its mRNA stability, while miR-320d can target and inhibit METTL3 expression, thus inhibiting progression of PCa.36 Lysine-specific demethylase 5A (KDM5A), also known as histone H3K4 demethylase, was recently shown to be involved in PCa development through the miR-495/YTHDF2/m⁶A-MOB3B signaling axis. KDM5A binds to the promoter of miR-495 and inhibits its transcription, thereby affecting the binding of miR-495 to YTHDF2 and inhibiting the malignant progression of PCa cells.37

LncRNA

lncRNAs have been confirmed to be involved in the progression of various cancers, and the relationship between their functions and m⁶A regulation is rarely reported, especially in PCa. Recent studies have shown that lncRNA PCAT6 was significantly upregulated in bone metastatic PCa, and prostate cancer-associated transcript 6 (PCAT6) can interact with the KH3–4 double domain of IGF2BP2, thereby increasing the stability of IGF1R mRNA, further promoting the growth of PCa as well as the bone metastases.38 Another study analyzed bone metastatic tissue samples derived from PCa patients as well as from patient-derived xenograft (PDX) models, and they found that the m⁶A level of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) was significantly increased in bone metastatic PCa and was associated with tumor metastasis, PSA level, and patient overall survival. In vivo, m⁶A-mutated NEAT1-1 mice were associated with low rate of bone/lung metastases and high survival rate compared with NEAT1-1 WT mice.39 In addition, it has been found that the lncRNA colon cancer-associated transcript 1 (CCAT1) and CCAT2, as m⁶A targets of VIRMA, play an important role in the malignant progression of PCa, in that, high expression of VIRMA and high m⁶A levels increase the RNA stability and abundance of CCAT1/2. Finally, it was found that the increased expression of VIRMA and CCAT1/2 as combined variable is an independent predictor of a poor prognosis of PCa.29

Although the regulatory mechanism of ncRNA m⁶A in PCa needs to be further studied, the available results consistently suggest that the regulation of ncRNA m⁶A plays an important role in the progression of PCa and has the potential to be served as a new target for tumor therapy or a new predictor for tumor prognosis.

PROSPECTS OF M⁶A REGULATION IN THE DIAGNOSIS AND TREATMENT OF PCA

To date, m⁶A regulation has been extensively studied in cancer. Based on the critical role of m⁶A regulation in carcinogenesis, targeting m⁶A regulatory molecules have great potential in current cancer treatment. For example, small-molecule FTO inhibitors FB23 and FE23-2 can directly bind to FTO to inhibit acute myeloid leukemia (AML) cell proliferation.40 IGF2BP1 inhibitors have also shown anticancer effects in leukemia, melanoma, and ovarian cancer.41–43 The small-molecule ALKBH5 inhibitor ALK04, as well as inhibitors of METTL3 and METTL14, can significantly improve the efficacy of immunotherapy and immune checkpoint inhibitors (ICIs).44 In addition, the m⁶A expression profile of tumors also has great potential in distinguishing different immune signatures, which is beneficial to individualize immunotherapy for patients with cancer.45,46 For some highly aggressive cancer, the treatment that targets tumor cells alone may not achieve the desired therapeutic effect. This may be due to the tumor reprogramming, which means that tumor cells have initiated a process of transformation adapted to the tumor microenvironment. The reprogramming of tumors by the tumor microenvironment suggests that interventions in the tumor microenvironment may sometimes yield additional benefits than treating the tumor cell itself. At present, there have been a bunch of reports on the regulation of m⁶A involved in the tumor immune microenvironment and the secretion process of tumor stromal cells,47 which shows that the regulation of m⁶A has great application prospects in the intervention of the tumor microenvironment.

For PCa, according to the analysis of many clinical data, new prognostic features integrated by five key m⁶A methylation regulators (tRNA methyltransferase activator subunit 11-2 [TRMT112], nuclear RNA export factor 1 [NXF1], YTHDF1, HNRNPG, and HNRNPA2B1) can independently predict the prognosis of PCa.48 ICIs have made significant progress in the field of tumor treatment; it, however, does not include PCa because the objective response rate of ICIs for PCa is only 5%.49 How to improve the response rate of PCa to ICIs is urgent for patients with hormone-resistant PCa. Recently, it has been shown that m⁶A regulation in PCa is closely related to the tumor immune microenvironment, and three different m⁶A regulation patterns in PCa have been identified based on m⁶A regulatory molecules. It is worth noting that the three different m⁶A regulatory patterns have different proportions of C3 immune subtypes, among which METTL14 and ZC3H13 overexpressed m⁶A regulatory patterns corresponding to the best prognosis, and these two molecules are closely related to T helper type 1 (Th1), T helper type 17 (Th17), and T helper type 2 (Th2) cells. It has been further found that the patients with lower m⁶A score in cancer tissues had a significantly higher response rate to immunotherapy than those with high m⁶A score. Further study found that this may be related to the relatively low activity of TGF-β in the low m⁶A score group. Therefore, m⁶A score may be a potential biomarker for immunotherapy response in PCa.50 In addition, a study analyzed the pathological and follow-up data of 78 clinical patients in the past 3 years. They found that a KDM5A regulated the miR-495/YTHDF2/m⁶A-MOB3B signaling axis by m⁶A modification and promoted the PCa progression. It confirmed that the high expression of KDM5A is closely related to the poor prognosis of PCa patients.37 In terms of castration resistance, downregulation of ALKBH5 can increase the m⁶A modification level of E3 ubiquitin ligase, siah E3 ubiquitin protein ligase 1 (SIAH1), resulting in decreased expression of SIAH1. It promotes the production of androgen receptor splice variant 7 (AR-V7) and induces drug resistance or a series of poor prognosis in PCa patients, which makes ALKBH5/SIAH1/cleavage and polyadenylation specific factor 1 (CPSF1)/AR-V7 axis expected to be a new target for the treatment of CRPC.35 Finally, we made statistics on the reported literature (Table 1). Among the clinical samples of a total of 887 cases of PCa in 14 institutions, we found that most of the patients showed increased m⁶A “writers” expression or decreased “erasers” expression. This strong evidence will open the way for m⁶A regulation in PCa treatment in the future.

Table 1.

Summary of N⁶-methyladenosine-related articles and cases in prostate cancer

Institution	Number of cases	Molecules and prognosis (with reference)
Affiliated Cancer Hospital of Harbin Medical University (China)	78	High KDM5A mRNA and protein levels with lower OS and DFS37
Renji Hospital Affiliated to Shanghai Jiaotong University (China)	52	SIAH1 levels are highly correlated with Gleason score35
Tianjin Medical University and the Second Xiangya Hospital (China)	60	NEAT1-1 expression and m6A level were negatively correlated with survival and overall survival39
Affiliated Hospital of Guizhou Medical University (China)	80	KIF3C is highly expressed in tumor tissues and strongly associated with PCa lymphatic metastasis and seminal vesicle infiltration36
Nanjing First Hospital of Nanjing Medical University (China)	48	METTL3 and LEF1 are highly expressed in tumor tissues with poor prognosis24
People’s Hospital of Wuhan University (China)	84 PCa/32 normal	m6A modification and METTL3 level were elevated in tumor tissues with poorer OS and DFS26
Shanghai Ruijin Hospital (China)	16 pairs	METTL3 is highly expressed in tumor tissues51
Renji Hospital Affiliated to Shanghai Jiaotong University (China)	49 PCa/11 normal tissue microarrays	METTL14 is upregulated in tumor tissues with poor prognosis52
Southern Hospital of Southern Medical University (China)	30 pairs	FTO was downregulated in tumor tissues with high lymphatic metastasis risk and high Gleason score33
Zhujiang Hospital of Southern Medical University (China)	68	FTO was downregulated in tumor tissue and was associated with poorer survival53
Porto Oncology Institute (Portugal)	198 PCa in situ/24 CRPC/13 normal	VIRMA is highly expressed in tumor tissues especially in CRPC, and is associated with poor prognosis29
The First Affiliated Hospital of Guangzhou Medical University (China)	15 pairs in situ/15 pairs of bone metastases	METTL3 is highly expressed in tumor tissues and is associated with bone metastasis and poor OS27
The Second Affiliated Hospital of Hengyang Medical College (China)	30 pairs	SNHG7 and c-Myc are highly expressed in tumor tissues54
Ningbo Medical Center LiHuiLi Hospital and Zhejiang Veteran Hospital (China)	50 pairs	FTO was downregulated in tumor tissue with high distant metastasis risk and Gleason score34

PCa: prostate cancer; CRPC: castration-resistant prostate cancer; KDM5A: lysine-specific demethylase 5A; NEAT1: nuclear paraspeckle assembly transcript 1; KIF3C: kinesin family member 3C; METTL3: N⁶-adenosine-methyltransferase complex catalytic subunit; FTO: FTO alpha-ketoglutarate-dependent dioxygenase; OS: overall survival; DFS: disease-free survival; VIRMA: vir like m6A methyltransferase associated; SNHG7: small nucleolar RNA host gene 7; SIAH1: siah E3 ubiquitin protein ligase 1; m⁶A: N⁶-methyladenosine; LEF1: lymphoid enhancer-binding factor 1

CONCLUSIONS AND PERSPECTIVES

Previous studies have shown a strong link between m⁶A regulation and PCa progression or prognosis, but many knowledge gaps remain to be filled. For example, whether m⁶A regulation plays a role in remodeling tumor microenvironment of PCa, and what role does m⁶A regulation play in regulating hormone therapy sensitivity on PCa. Based on the collecting evidence, we expect more research progress emerge in the regulation of m⁶A on PCa soon.

AUTHOR CONTRIBUTIONS

YZ and SYZ contributed to writing, acquisition of data, review, and/or revision of the manuscript. YZ supervised the study. Both authors read and approved the final manuscript.

COMPETING INTERESTS

Both authors declare no competing interests.