The current landscape of m6A modification in urological cancers

Survey methodology

We used the keywords ‘m6A’, ‘N6-methyladenosine’, ‘m6A modification’, ‘m6A regulators’, ‘Urologic Neoplasms[Mesh]’, ‘Carcinoma, Renal Cell[Mesh]’, ‘Urinary Bladder Neoplasms[Mesh]’, ‘Prostatic Neoplasms[Mesh]’, ‘Carcinoma, Transitional Cell[Mesh]’, ‘Urethral Neoplasms[Mesh]’ to conduct a literature search of PubMed database. The articles which were not associated with m6A modification and urological cancers were excluded.

M6A and urogenital cancer

More and more studies focused on the relationship between m6A modification and urogenital cancer. Here we aim to summarize the variation of m6A modification and its regulators and consequent influence on tumor cells’ malignant phenotype.

Renal cell carcinoma

Renal cell carcinoma (RCC) is one of the common types of urogenital cancer. The most common types of RCC include clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), and chromophobe renal cell carcinoma (chRCC). According to GLOBOCAN data, renal tumors account for about 2.2% of estimated cases and deaths worldwide in 2020 (Sung et al., 2021). The early diagnosis and treatment of RCC is always a research hotspot. It was found that the m6A content of total RNA increased in the RCC tumor tissues (Gan et al., 2021). A study also found that the m6A level decreased in ccRCC (Xu et al., 2022b). In terms of “writers”, The expression of METTL3 (Zhu et al., 2022), WTAP (Ying et al., 2021; He et al., 2021) was significantly increased in RCC tissues. The level of METTL14 (Zhang et al., 2021a; Liu et al., 2022d) and eukaryotic initiation factor 3 (eIF3A) (Zhang et al., 2021b) in RCC was significantly decreased. The FTO expression was different in various researches, some results show it was upgraded in ccRCC tumors (Xiao et al., 2020; Shen et al., 2022). However, others find that the expression of FTO decreased (Zhuang et al., 2019; Strick et al., 2020). The other “eraser” ALKBH5 downregulated in RCC samples (Strick et al., 2020). When we talk about “readers”, the quantity of heterogeneous nuclear ribonucleoprotein C (HNRNPC), HNRNPA2B1, YTHDC1 (Von Hagen et al., 2021), YTHDF1-3 (Mu et al., 2020; Xu et al., 2022a), and IGF2BP2 (Li et al., 2022) was significantly lower than adjacent normal tissues. More detailed information of the variation of m6A regulators in RCC can be found in Table 1. PRCC is the second most common type of renal carcinoma, different types with different m6A modulators expressions. It was reported that the expression levels of HNRNPC and YTHDF1 increased, while the expression levels of Vir-like m6A methyltransferase associated (VIRMA/KIAA1429), zinc finger CCCH-type containing 13 (ZC3H13), METTL14, ALKBH5, and YTHDF2 decreased in pRCC tissues (Yang et al., 2021b). Lei et al. (2022) revealed that the transcription level of WTAP reduced in pRCC and chRCC. Different m6A RNA methylation regulators also make different contributions to RCC. Some had a potential antitumor function in RCC. METTL14 knockout significantly increased the migratory, invasive, and metastatic ability of RCC cells, and METTL14, YTHDF2 overexpression suppressed tumor growth and metastasis (Liu et al., 2022d). On the contrary, depletion of METTL3 could significantly decrease ccRCC tumor cell growth (Zhu et al., 2022). Knockdown of METTL3 (Shi et al., 2021) or WTAP (He et al., 2021) decreased RCC migration and proliferation. Cell proliferation in RCC was also suppressed by knockout of WTAP or IGF2BP (Ying et al., 2021). The function of FTO is ambiguous. Some found that FTO knockdown inhibited cell migration and proliferation (Shen et al., 2022), and FTO negatively influenced autophagy (Xu et al., 2022b). But it was also be validated that the proliferation of ccRCC cells was suppressed by higher expression of FTO (Zhuang et al., 2019). Above all, m6A RNA methylation regulators play an important role in the development of RCC. However, the changes in m6A modulators in different types of RCC are not the same. These need more research to clarify the function of m6A in renal cell carcinoma and explore its change and mechanism.

Bladder cancer

Bladder cancer is the ninth most common tumor among all types of tumors worldwide, with about 570,000 new cases per year (Sung et al., 2021). More and more studies have shown that bladder cancer was related to m6A modification. The m6A modification content in bladder cancer tissues was lower than that in normal tissues (Gu et al., 2019), but some studies also found that the m6A modification level was increased in bladder cancer samples (Liu et al., 2022a), which may be related to the different sample source and small sample size. It was found in humans and animal models that METTL3 (Cheng et al., 2019; Jin et al., 2019; Xie et al., 2020), FTO (Tao et al., 2021; Zhou et al., 2021), IGF2BP1 (Xie et al., 2021), IGF2BP3, YTHDF1 (Zhu et al., 2023), YTHDF 2, ELAV-like protein 1 (ELAVL1), HNRNPA2B1 (Deng et al., 2022) were upregulated in bladder cancer cells. In contrast, METTL14 (Gu et al., 2019; Guimarães Teixeira et al., 2022), WTAP (Liu et al., 2021a), YTHDC1, YTHDF3, ZC3H13 (Deng et al., 2022) were reduced in bladder cancer samples. However, it was also reported that METTL14 mRNA transcription level was significantly increased (Liu et al., 2022a). The change of the expression level of m6A regulators could affect cell function. Stable knockdown of METTL3 (Cheng et al., 2019; Yang et al., 2019; Han et al., 2019; Xie et al., 2020; Wang et al., 2021a) and IGF2BP1 (Xie et al., 2021) effectively reduced cell cycle process, cell proliferation, migration, and invasion ability, while inducing apoptosis and carcinogenic transformation. Knockout of METTL14 enhanced invasion ability and tumor proliferation ability (Gu et al., 2019). Using METTL14 knockout cells resulted in smaller tumor size and fewer tumor surrounding blood vessels (Guimarães Teixeira et al., 2022). However, there are also other studies showing that ectopic expression of METTL14 increased tumor cell survival ability, proliferation and migration ability (Liu et al., 2022a). After knocking down METTL14, cell apoptosis increased, migration and proliferation decreased instead (Guimarães Teixeira et al., 2022). FTO was closely related to the self-renewal of bladder tumor stem cells (Gao et al., 2020). FTO inhibited the growth of bladder cancer cells by G1 phase arrest and inhibits their migration ability (Sun et al., 2022). But at the same time other studies demonstrated that, after FTO overexpression, cells in G0/G1 phase were less while those in S phase increased, cell proliferation ability migration and invasion ability all increased (Zhou et al., 2021). Depletion of YTHDF2 reduced Epithelial-mesenchymal transition (EMT) pathway-related proteins and matrix metalloproteinase family proteins expression so that reduced cell migration rate (Xie et al., 2020). Different m6A regulators showed different functions in bladder cancer. There are many references found the correlation of bladder cancer and writers and erasers. Further research related to readers is still needed to clarify the role of each m6A regulator.

Prostate cancer

Prostate cancer, as one of the six most common tumors in the world (Soerjomataram & Bray, 2021), has also been proven to be closely related to m6A. In prostate cancer, m6A modification level was upregulated, and the expression levels of METTL3 (Chen et al., 2021b), METTL14 (Barros-Silva et al., 2020), VIRMA (Barros-Silva et al., 2020), RNA binding motif protein 15 (RBM15), HNRNPC, HNRNPA2B1, YTHDC2, YTHDF1 (Liu et al., 2022c) and YTHDF2 (Li et al., 2020) increased. While FTO (Zou et al., 2022; Zhu, Li & Xu, 2021), ALKBH5 (Wu et al., 2021a), ZC3H13 (Zhang et al., 2021c) and IGF2BP2 (Liu et al., 2022c) decreased. The expression of METTL3 and METTL14 also significantly increased in castration-resistant prostate cancer (Li et al., 2023b). A special case is IGFBP3, which had a lower expression level in prostate cancer tissues, but a higher expression level in patients with advanced stage (Quan, Zhang & Ping, 2022). Then the rule found out and summarized from the experiment is that prostate cancer was positively related to methyltransferases and negatively related to demethylases. Knockdown of METTL3 reduced the proliferation, migration, invasion and DNA synthesis ability of prostate cancer cells, and inhibited tumor formation and reduced lung metastasis (Chen et al., 2021b; Mao et al., 2022). This ability was produced by catalyzing m6A modification (Yuan et al., 2020). Overexpression of METTL14 positively regulated the number of cells in S phase and negatively regulated the number of cells in G2 phase to improve cell proliferation ability (Wang et al., 2022). Upregulated FTO (Zhu, Li & Xu, 2021; Zou et al., 2022) or knockdown of VIRMA (Barros-Silva et al., 2020) inhibited cell proliferation and invasion ability. But there are also studies showing that knockdown of FTO reduced the migration and invasion ability of prostate cancer cells (Su, Wang & Li, 2021). Overexpression of YTHDF2 can also promote cell proliferation and migration ability (Li et al., 2020).

To sum up, urogenital cancer had no specific relation with the increase or decrease of m6A modification. There are still some researches demonstrating opposite results. However, the change of m6A modification and regulators’ expression could alter the biological behaviors of tumor cells. The mechanism of these changes needs further research in our work from now on.

Mechanism

Regulating RNA stability

M6A modification has various functions in cells. One of the essential assignments is to regulate mRNA expression levels and affect the stability of different RNAs. This feature may also be one of the mechanisms by which m6A regulators play a role in the urinary system. The writer and eraser regulate mRNA stability by regulating m6A modification levels, while the reader recognizes m6A modified regions, interacts with mRNA, and affects its decay, regulating its stability. The m6A modification catalyzed by METTL3 in SETD7, KLF4 (Xie et al., 2020), LHPP and NKX3–1 (Li et al., 2020) was recognized by YTHDF2 to promote mRNA degradation. It was also reported that the decay of METTL14-methylated bromodomain PHD finger transcription factor (BPTF) (Zhang et al., 2021a) and THBS1 (Wang et al., 2022) accelerated. Eraser FTO promoted the demethylation of PPARg coactivator-1 α (PGC-1 α) (Zhuang et al., 2019) and CLIC4 (Zou et al., 2022) and enhanced mRNA stability. But there are also other researches demonstrated that the m6A modification mediated by METTL3, METTL14 (Zheng et al., 2022) and WTAP (Ying et al., 2021) maintained the stability of mRNA. IGF2BPs stabilized their target genes by the read of m6A modified sites (Gu et al., 2021; Ying et al., 2021; Li et al., 2022; Huo et al., 2022). Meanwhile, m6A regulators modulated the interaction between transcripts and other proteins to affect the stability of mRNA. METTL3 knockdown regulated the interaction between Rho GDP dissociation inhibitor α (ARHGDIA) and ELAV-like RNA-binding protein 1 (ELAV1) and the interaction between ELAV1 and ubiquitin-specific protease 4 (USP4) to impact its function (Chen et al., 2021b). But the mechanism behind this phenomenon is not known. We summarized the mRNA and its functional role which related to m6A modification in Table 2. Others kind of RNA also modulated mRNA stability via m6A modification. A Circular RNA circPTPRA bound with IGF2BP1, blocking its recognition of downstream m6A modified mRNA and reducing target mRNA stability (Xie et al., 2021; Zheng et al., 2023). Conversely, LncRNA TRAF3IP2 antisense RNA 1 (TRAF3IP2-AS1) accelerated the poly ADP-ribose polymerase (PARP1) mRNA decay by stimulating m6A mRNA modification (Yang et al., 2021a). Taken together, although the function of m6A modification in various mRNA stability was different, there is no doubt that regulating RNA stability is one of the essential functions of m6A modification.

Regulating gene expression

The mRNAs with different degrees of m6A methylation have different effects on tumor occurrence and development. M6A regulators and other enzymes regulated gene expression by changing the m6A methylation level of mRNA, thereby influencing cell function. In RCC, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulated hypoxia-inducible factor (HIF)-2 α mRNA methylation dependent on METTL3, thereby increasing HIF-2 α protein levels and promoting glycolysis (Green et al., 2019). CUB-domain containing protein 1 (CDCP1) is a kind of transmembrane glycoprotein which associated with metabolic pathways (Khan et al., 2021). METTL3 and YHDF1 bound to CDCP1, promoting CDCP1 mRNA modification and translation (Yang et al., 2019). METTL3 promoted m6A modification in PCAT6 and increased PCAT6 expression in an IGF2BP2-dependent manner (Lang et al., 2021). M6A modification, which induced be METTL14, inhibited the expression of oncogene nuclear-enriched abundant transcript 1_1 (NEAT1_1) in RCC through YTHDF2-dependent RNA degradation (Liu et al., 2022d). SOD2, a superoxide dismutases, was positively correlated with WTAP expression and WTAP affected the expression of SOD2 by regulating SOD2’s m6A methylation level (Liu et al., 2021a). In bladder cancer, the upregulation of circSLC38A1 was associated with m6A modification, circSLC38A1 promoted tumor invasion and metastasis (Li et al., 2023a). M6A modification could also regulate the distribution of Circular RNA in cells. CircMET was exported to the cytoplasm by YTHDC1 through m6A modification. When circMET was distributed in the cytoplasm, cell proliferation and growth were inhibited (Yang et al., 2022).

In a word, m6A regulators modulated downstream gene expression to regulate cell metabolism, and the mechanism behind was concluded in Fig. 1. It is a worthy research task how to control tumor occurrence and development in this way.

Modulating the immune system

It is increasingly recognized that m6A RNA methylation regulators are closely related to the human immune system. M6A score was used to quantify m6A modification patterns, which could predict the immune phenotype of RCC patients (Li et al., 2021b). The tumour microenvironment (TME) is one of the important factors that influence the progress and oncogenesis. The infiltrating immune cell formed a stimulating or inhibiting network to transform TME (Li et al., 2021c). People judged the degree of immune cell infiltration in cancer tissues by detecting m6A modification level and the expression level of m6A regulators. The expression level of m6A RNA methylation regulators affected the immune infiltration of urogenital tumors. The level of m6A modification was related to T cell infiltration degree (Liang et al., 2022). In RCC, a study grouped according to 23 m6A regulators found that different groups had different degrees of immune infiltration (Deng et al., 2022). High EIF3A expression upregulated the overall immune infiltration level (Zhang et al., 2021b). METTL14 inhibited Tregs infiltration in ccRCC by inhibiting CCL5 (Xu et al., 2021a). The expression of IGF2BP3 was demonstrated to be closely related to the infiltration of immune cells in PRCC (Chen et al., 2021a), which was mainly related to Th2 cell infiltration enrichment and affected PD-1 treatment effect (Zhong et al., 2021). In bladder cancer, cluster analysis based on the expression level of m6A regulators found that nearly all of immune cells in the m6ACluster B group decreased. Patients in the m6ACluster B group had a better prognosis than others (Ye et al., 2021). Also, the B group in another research was closely related to immune activation (Zhu et al., 2021).

M6A RNA methylation regulators were closely related to immune cells. It was validated that m6A regulators partook in the differentiation and recruitment process of immune cells (Deng et al., 2022). Changes in METTL3 expression were closely associated to the down-regulation of CD4 + T cells, neutrophils, and dendritic cells infiltration level in bladder cancer (Wang et al., 2021b), while positively correlated with naive B cells, CD4+ memory resting T cells, and M1 macrophages number and negatively correlated with regulatory T cells and M2 macrophages number in prostate cancer (Liu et al., 2022c). The expression of YTHDF2 was positively correlated with B cells, CD8+T cells, CD4+T cells, macrophages, neutrophils, and dendritic cells. It was be validated that this correlation influenced treatment outcomes (Su et al., 2021). FMR1 expression was significantly negatively associated with M2 macrophages, while decreased M0 macrophages in urothelial carcinoma tissues upon HNRNPA2B1 overexpression (Kong et al., 2022). In ccRCC, Chen et al. (2020b) confirmed that a higher m6A risk score leaded to a higher abundance of regulatory T cells (Tregs) and follicular helper T cells (Tfhs). On the contrary, in bladder cancer, CD56dim natural killer cells, central memory CD4T cells, eosinophils, mast cells, monocytes, and17-type helper T cell numbers were lower in high-m6Ascore group, while activated CD4 + T cell, memory B cell, natural killer T cell, neutrophil, 2-type helper T cell etc., and mainly related to immunosuppression-related cell numbers were less in the low-m6Ascore group (Deng et al., 2022). However, another study of bladder cancer demonstrated that the m6A score was positively correlated with CD4 T immune cell, CD8 T immune cell, and dendritic immune cell number, while negatively correlated with PD-L1 expression level (Zhu et al., 2021). At the same time, studies also demonstrated that the number of each group’s immune cells was different through cluster analysis (Liu et al., 2021b).

After a hundred years of development, immunotherapy has also become one of the important treatment options for tumor patients. Anti-PD-1 immunotherapy as an emerging first-line treatment option has been used for treatment of advanced ccRCC (Atkins & Tannir, 2018) and it is also a promising treatment option for patients with advanced bladder cancer (Inman et al., 2017). Several studies indicated that IGF2BP3 was positively associated with the expression of PD-L1 in immune cells (Cui et al., 2022), while FTO was just the opposite (Deng et al., 2022). Kong et al. (2022) found that in bladder cancer, the expression of HNRNPA2B1, FMR1, IGF2BP1, IGF2BP3, and YTHDF2 were significantly upregulated in the anti-PD-1 immunotherapy response group, while FTO expression was significantly downregulated. They also constructed a nomogram to predict the responsiveness of patients to atezolizumab monotherapy (Kong et al., 2022). But there were also other studies showing that FTO expression level was higher in bladder cancer patients who did not respond to immunotherapy (Deng et al., 2022). In addition, patients with low m6A score had a better response to immunotherapy. The influence of m6A modification and m6A regulators on the immune system provides possibilities for discovering new treatment methods, selecting suitable treatment methods for different patients, and performing precision medicine in the future.

Clinical value