Over the past few decades, it has been established that genetic dysregulation underlies various human diseases. For example, genetic errors arising from gene amplification, deletion, mutation, or chromosomal translocation have been associated with numerous cancers (1-3). Meanwhile, increasing evidence suggests that epigenetic modifications of chromatin structure also affect tumor formation and cancer development via abnormal regulation of gene expression (4). Furthermore, the impairment of epigenetic regulation of oncogenes and tumor suppressor genes has been linked to several signaling pathways that lead to cancer development (5-7). Similarly, several recent studies have suggested an important role for RNA modifications, termed the “epitranscriptome”, representing a new layer of post-transcriptional gene regulation (8, 9). Although several studies have investigated signaling pathways and transcriptional regulation in cancer, relatively little is known about the post-transcriptional regulation of cancer. Therefore, a better understanding of the gene regulatory mechanisms controlling tumorigenesis and cancer development will facilitate their therapeutic exploitation (10, 11).
To date, more than 170 chemical RNA modifications have been identified, including
Similar to DNA methylation in epigenetics, three classes of RNA-binding proteins, broadly classified as writers, readers, and erasers, mediate the regulation of RNA modification (Fig. 1) (14). Writer proteins install the modification, while eraser proteins remove the modification, and reader proteins recognize the modification and regulate the metabolism of the target RNA. The discovery of both writer and eraser proteins indicates that many RNA modifications are likely reversible. Many recent studies have suggested that abnormally regulated RNA modification may lead to tumorigenesis and cancer development (2, 15, 16).
In this review, we focus on six internal RNA modifications most closely linked to tumorigenesis and discuss the RNA species in which they have been identified, their molecular mechanisms, and evidence of their involvement in cancer (Fig. 1).
Several studies have investigated the association between m6A modification and cancer. These studies have revealed that the levels of m6A mRNA modification in cancer cells are generally elevated and closely correlated with the development of several cancers (36-39). In colorectal cancer (CRC) and gastric cancer, for example, a high degree of m6A modification is associated with mRNA stability (40, 41). m6A reader proteins, IGF2BP1, IGF2BP2, and IGF2BP3, recognize m6A modifications in oncogene mRNAs, preventing mRNA degradation and ultimately promoting cancer development (40, 41). In bladder cancer (BLC) and lung cancer, m6A modification increases the translation efficiency of oncogenes without affect-ing mRNA abundance (34, 35, 42). Integrin alpha-6 (
Cancer development has also been reported to be influenced by m6A modification of ncRNA (15, 44). MicroRNA (miRNA) m6A modification can alter the abundance of miRNA in cells, which in turn regulates the stability of the target mRNA associated with tumorigenesis in BLC and hepatocellular carcinoma (HCC) (15, 44). Specifically, METTL3 has been shown to interact with the microprocessor protein, DGCR8, affecting primary-miRNA (pri-miRNA) processing in BLC (44). A study showed that the knockdown of METTL3 in BLC induces the accumulation of
However, it has been reported that a high level of m6A modifications may also be inhibitory in the same types of cancer, depending on the target mRNAs. Recently, low levels of m6A modifications were identified in CRC and BLC (45-47). Mechanistically, the increase in m6A modification induced by the overexpression of METTL14 is thought to lead to rapid YTHDF2-mediated mRNA degradation of the essential developmental transcription factor, SRY-related high-mobility-group box 4 (
The development of several cancers is also influenced by m6A modification of ncRNA. In CRC, an oncogenic long non-coding RNA (lncRNA), the X inactive-specific transcript (
Pseudouridine (also known as 5-ribosyluracil or ψ) was first discovered in the early 1950s (48). Initially identified in tRNA and rRNA, pseudouridine has also been found in mRNA, lncRNA, and small nuclear RNA (snRNA) (49, 50). Pseudouridine is the most abundant RNA modification occurring in tRNAs and rRNAs (50). In tRNAs, pseudouridine is generally localized to the anticodon stem-loop in the D stem, and to the nucleotide position 55 in the T loop, and thus contributes to the stabilization of the tertiary structure of tRNA (51). Pseudouridines are generated post-transcriptionally via C5-ribosyl isomerization of one or a few target uridines catalyzed by pseudouridine synthases (PUSs) (52). PUSs employ two me-chanisms of pseudouridine modification: guide RNA-dependent H/ACA box small nucleolar RNA (snoRNA) and guide RNA-independent pseudouridylation. In guide RNA-dependent pseudo-uridylation, the H/ACA box snoRNA forms a complex with dyskerin pseudouridine synthase 1 (DKC1), which recognizes specific sequences for pseudouridylation on target RNAs, including rRNA, snRNA, and snoRNA (53). In contrast, in guide RNA-independent pseudouridylation, modification of the target RNA is directly catalyzed by stand-alone PUSs (54). PUS enzymes are classified into six families: TruA, TruB, TruD, RluA, RsuA, and Pus10 (54). PUS1, which belongs to the TruA family, was originally thought to pseudouridylate tRNA alone; however, recent studies have identified that PUS1 also pseudouridylates rRNA, snRNA, and mRNA (49, 50). In addition, PUS4 and PUS7 were found to target mRNAs for pseudouridylation in HEK293T cells (50).
Unlike other known RNA modifications, neither reader nor eraser proteins for pseudouridine have been identified to date. Moreover, the functional role of pseudouridylation in mRNA remains unclear.
Upregulation of pseudouridine has been shown to be associated with the progression of various cancers, including pro-state cancer (PC), BrC, and HCC (55-58). The nucleolar protein DKC1 plays an important role in two separate cell proliferation pathways: the pseudouridylation of rRNAs, which is necessary for their processing, and the stabilization of the telomerase RNA component that is necessary for telomerase activity (55). Similarly, DKC1 expression is generally upregulated in PC (56, 59). The knockdown of DKC1 by siRNA has been shown to inhibit the proliferation of the e22Rv1, LNCaP, PC3, and Du145 in PC cell lines; however, the knockdown had no significant effects on apoptosis or senescence (56). Moreover, HCC patients with high DKC1 expression have been found to exhibit shorter overall survival rates when compared to those with low DKC1 expression (57). Moreover, elevated DKC1 expression has been shown to positively correlate with MYC oncogene expression, which triggers the expression of target genes to induce cell proliferation and cell survival. In addition, DKC1 expression was shown to induce MKI67 expression, which is considered a marker for cell proliferation (57, 58).
However, pseudouridine modification has also been shown to exert negative effects on the development of several types of cancer. Downregulated pseudouridine has been associated with BrC and HCC development (60, 61). The impairment of DKC1 protein can lead to the inactivation of p53, a well-known anti-tumor development factor that induces cell cycle arrest or apoptosis, due to abnormal
Similar to m6A modification, m1A modification is reversible. Demethylation of m1A modification is mediated by ALKBH1 and ALKBH3 (65, 66). Interestingly, the m6A reader proteins, YTHDF2 and YTHDF3, have also been implicated in the recognition of m1A mRNA modifications (16, 69). Reminiscent of m6A modification, m1A-modified mRNA undergoes rapid degradation upon binding with YTHDF2 or YTHDF3 (16, 69).
To date, several studies have reported a positive correlation between m1A tRNA modification and cancer development. Elevated expression levels of both TRMT6/61A and initiator methionine tRNA (tRNAiMet) have been detected in highly aggressive GBM compared with grade II/III gliomas (67). Depletion of the TRMT6/61A complex suppresses proliferation and promotes cell death in C6 glioma cells, which can be rescued in part by tRNAiMet overexpression (67). Conversely, the ectopic expression of TRMT6/61A has been shown to upregulate the translation of oncogenic mRNAs, leading to increased colonization of C6 glioma cells (67). Similarly, ALKBH1-mediated demethylation of m1A modified tRNA attenuates translation initiation and elongation in HeLa cells, thereby reducing cell proliferation (66).
Intriguingly, another m1A demethylase, ALKBH3, has been shown to induce an opposite effect to that of ALKBH1 in m1A tRNA modification (70). ALKBH3-mediated m1A tRNA demethylation increases the susceptibility of tRNA to angiogenin cleavage and generates tRNA-derived small RNAs in various cancer cells during cancer cell proliferation, migration, and invasion (70).
A rather unique RNA modification is mediated via 2’-
Many studies suggest that FBL expression is abnormally high in various cancers, including PC and BrC (78-80). One report suggested that a p53 mutation fails to suppress transcription of the
A few studies have investigated the effects of m5C on tumorigenesis. It has been identified that NSUN2 protein is a downstream target of MYC that methylates RNA polymerase III transcripts (85). Elevated expression of NSUN2 has been shown to mediate MYC-induced cell proliferation and growth in squamous cell carcinoma and BLC (85, 89). Moreover,
While few studies have reported a correlation between m5C modification and cancer, it will be informative to consider the effects of m5C on the regulation of translation, given that m5C modifications occur mainly in 5’ or 3’ UTRs.
A positive association between internal m7G and cancer has yet to be identified. However, internal m7G modification of primary miRNA transcripts has been shown to have negative effects on lung cancer and colon cancer development. Specifically, the
Given the plethora of signaling pathways converging upon gene expression regulatory pathways to satisfy the increased anabolic demands in cancer, a better understanding of the gene regulatory mechanisms controlling tumorigenesis will facilitate their therapeutic exploitation (5, 6, 11). The field of epitranscriptomics has attracted the attention of various biological investigators in recent years; however, the molecular players and mechanisms underlying epitranscriptome regulation remain to be elucidated. In particular, several studies on the relationship between RNA modification and cancer have been published, many of which either lack data regarding the detailed mechanisms involved or often report contrasting results. For instance, pseudouridine, m1A, Nm, and internal m7G modifications have been found in mRNAs across the gene body; however, their roles in mRNA metabolism and their effects on cancer progression remain unknown (49, 65, 94, 95). Instead, most published studies have merely investi-gated the effects of the enzymes catalyzing the modifications on cancers, without confirming the effects of RNA modification. With the exception of m6A and m1A, the reversibility of RNA modifications and their specific reader proteins also remain unclear. Moreover, the molecular functions and cellular consequences of m6A or pseudouridine modification often differ across studies, depending on the degree of methylation in the specific target RNAs (Fig. 1).
In addition to the extensive interest in the roles of RNA modification in cancer, the development of potential drugs to treat cancer by modulating RNA modification has also attracted attention (98). However, there are currently no inhibitors or antagonists targeting writer and reader proteins, or the RNA modifications discussed in this review (98). Instead, several inhibitors of demethylases have been suggested. The ALKBH family and FTO share a common structure required for the binding of Fe2+ as a co-factor and 2-oxoglutarate (2OG) as a co-substrate (98). Therefore, most of the known compounds, including 2OG competitors such as N-oxalylglycine and its cell-penetrating derivative dimethyl oxalylglycine, succinate, fumarate, and 2-hydroxyglutarate,or metal chelators such as hydroxamic acids and flavonoids, were designed to target either Fe2+ or 2OG binding sites (98, 99). However, these compounds are still far from being used in anticancer drugs because they nonspecifically inhibit various demethylases. Some specific demethylase inhibitors have also been discovered, such as the ALKBH3 inhibitor, 1-(5-methyl-1H-benzimidazol-2-yl)-4-benzyl-3-methyl-1H-pyrazol-5-ol (HUHS015), and FTO inhibitor, Rhein. However, the efficacy of these drugs remains doubtful because of their nonspecificity for target RNAs (98, 99). More recently, clustered regularly interspaced short palindromic repeats (CRISPR) based RNA-editing technology has been suggested for modulating target mRNA specific modifications (100). The CRISPR-associated nuclease Cas13 has been shown to cleave the targeted single-stranded RNA (100). A catalytically inactive mutant of Cas13 (dCas13) fused with m6A methyltransferases METTL3 or METTL14 has been found to bind to the target mRNA specifically directed by the guide RNA, without the cleavage of the mRNA (100). Targeting of these fusion proteins has been shown to specifically methylate adenosine within a small range of target sites, regardless of the m6A consensus sequence (100). Thus, CRISPR-based approaches can be applied not only in the modulation of m6A modifications, but also to other types of RNA modifications. Given the abnormal up/downregulation of modified RNA transcripts and their regulatory proteins in cancer, the development of this technology will shed more light on the feasibility of controlling the modification of RNA targets for cancer treatment. Taken together, the analysis presented in this review highlights the need to further elucidate the mechanisms of RNA modification-mediated regulation of gene expression to improve cancer therapy.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (NRF-2020R1C1C1009842 and NRF- 2020R1A4A1018398).
The authors have no conflicting interests.