m6A RNA methylation influences cancer stem cell pluripotency and cell differentiation
In recent years, a new dimension of intratumor heterogeneity and a hitherto-unappreciated subclass of neoplastic cells within tumors, termed cancer stem cells (CSCs), have aroused interests of researchers [
40]. CSCs, typically rare within tumors, may prove to regenerate all facets of a tumor as a result of their stem cell-like capacity to self-renew, survive, and become dormant in protective microenvironments, representing a reservoir of self-sustaining cells that give rise to many types of cancers [
41,
42].
Hematopoietic stem cells (HSCs) have well-defined developmental trajectories, and it is plausible to monitor and quantify their differentiation, providing an ideal model system in which to explore differentiation states. Abnormal or blocked differentiation is a common feature of myeloid hematological malignancies.
According to the recent research conducted by Ly P Vu and colleagues, leukemia cells show an elevated abundance of METTL3 as compared to normal hematopoietic cells. Utilizing miCLIP coupled with ribosome profiling, it is elucidated that METTL3 augments m
6A levels of its target genes including myelocytomatosis (MYC), B cell lymphoma 2 (BCL2), and phosphatase and tensin homolog (PTEN) genes in the human acute myeloid leukemia (AML) MOLM-13 cell line, thereby promoting the translation of these mRNAs. These data suggest that AML cells regulate their translational state through m
6A RNA methylation of specific transcripts to retain pluripotency properties and inhibit cell differentiation. Depletion of METTL3 in human myeloid leukemia cell lines induces cell differentiation and apoptosis and delays leukemia progression in recipient mice in vivo [
43].
Consistent with METTL3, METTL14 is also highly expressed in AML cells carrying t(11q23), t(15;17), or t(8;21) and is downregulated during myeloid differentiation. Mechanistically, METTL14 exerts its oncogenic role by regulating its mRNA targets (e.g., myelocytomatosis (MYB) and MYC) through m
6A modification, while the protein itself is negatively regulated by spleen focus-forming virus proviral integration oncogene (SPI1), collectively forming a SPI1/METTL14/MYB, MYC, et al. signaling axis in myelopoiesis and leukemogenesis. This highlights the critical roles of METTL14 and m
6A modification in normal and malignant hematopoiesis [
44].
RBM15, another component of the m
6A writer complex, is linked to myeloid leukemia as well where AML initiation is mediated by a chromosomal translocation t (1;22) of RBM15 (also called OTT1) with the myelin and lymphocyte (MAL) gene [
45]. Bansal and colleagues also suggested a role for m
6A RNA methylation in myeloid leukemia that WTAP expression was elevated in cells derived from 32% of patients with AML and knockdown of WTAP resulted in reduced proliferation, increased differentiation, and increased apoptosis in a leukemia cell line [
46].
However, upregulated m
6A RNA methylation expression can result in anti-proliferation effects in some circumstances. Su and colleagues have elaborated that R-2-hydroxyglutarate (R-2HG) exhibits broad and variable anti-proliferation effects in leukemia and glioma since it increases global m
6A RNA modification in the sensitive cells via suppressing FTO. The R-2HG/FTO/m
6A axis regulates MYC and CCAAT/enhancer-binding protein alpha (CEBPA) gene expression and downstream pathways [
47].
On the other hand, decreased m
6A RNA methylation levels may exert oncogenic functions in some certain types of AML. For example, FTO, an m
6A demethylase, is highly expressed in AML with t(11q23)/MLL rearrangements, t(15;17)/PML-RARA, FLT3-ITD, and/or NPM1 mutations and functions as an oncogene that promotes leukemic oncogene-mediated cell transformation and inhibits all-trans-retinoic acid (ATRA)-mediated leukemia cell differentiation. The oncogenic role of FTO is exerted through regulating expression of its target genes such as a suppressor of cytokine signaling box-2 (ASB2) and the retinoic acid receptor alpha (RARA) by reducing m
6A RNA methylation levels in these mRNA transcripts. RNA stability assays have proposed that FTO-induced repression of ASB2 and RARA expression is at least in part due to the decreased stability of ASB2 and RARA mRNA transcripts upon FTO-mediated decrease of m
6A RNA methylation levels. However, the reader(s) that targets m
6A modification and impairs mRNA stability remains to be identified, which indicates an additional reading process controlling the stability of FTO target transcripts. Studies have [
48‐
51] demonstrated the anti-leukemic effects of ASB2 and RARA, suggesting that the FTO/ASB2 or RARA axis likely plays a critical role in the pathogenesis of AML [
52].
In breast cancer, Zhang and colleagues proposed that the exposure of breast cancer cells to hypoxia could stimulate hypoxia-inducible factor (HIF)-1α- and HIF-2α-dependent expression of ALKBH5, which induced m
6A demethylation and stabilization of NANOG mRNA. Intratumoral hypoxia is a critical feature of the tumor microenvironment caused by dysregulated cell proliferation in combination with abnormal blood vessel formation and function, which drives cancer progression [
53‐
55]. NANOG, as a pluripotency factor, is required for primary tumor formation and metastasis since they play a pivotal role in the maintenance and specification of cancer stem cells. This explains the correlation between decreased m
6A RNA methylation level and promotion of breast cancer stem cell (BCSC) phenotype [
56]. The researchers have also reported that the exposure of breast cancer cells to hypoxia induces zinc finger protein 217 (ZNF217)-dependent inhibition of m
6A methylation of mRNAs encoding NANOG and Kruppel-like factor 4 (KLF4), which is another pluripotency factor that mediates BCSC speciation [
57].
When it comes to glioblastoma (GBM), a study carried out by Cui et al. demonstrates that reduced mRNA m
6A level is critical for maintaining glioblastoma stem-like cell (GSC) growth, self-renewal, and tumor development as downregulation of METTL3 or METTL14 expression reduces mRNA m
6A levels of theirs target gene A disintegrin and metallopeptidase domain 19 (ADAM19) and promotes ADAM19 expression. ADAM19 is a metalloproteinase disintegrin gene that exhibits elevated expression in glioblastoma cells and promotes glioblastoma cell growth and invasiveness [
58‐
60].
Similarly, Zhang and colleagues have identified that m
6A demethylase ALKBH5 is highly expressed in GSCs and demethylates forkhead box protein M1 (FOXM1) nascent transcripts [
61]. The nuclear RNA-binding protein HuR, which reportedly regulates both pre-mRNA splicing and expression [
62,
63], has been shown to bind with RNAs with no m
6A modification and exert stabilizing effects on its bound RNAs [
24]. Recruiting HuR to the unmethylated 3′UTR makes FOXM1 nascent transcripts more stable and upregulates its expression. Accumulating evidence has shown that the transcription factor FOXM1 functions as a key cell-cycle molecule required for G1/S and G2/M transition and M-phase progression [
64] and is overexpressed in GBM, playing a pivotal role in regulating GSC proliferation, self-renewal, and tumorigenicity [
65‐
67]. Taken together, lower m
6A level mediated by ALKBH5 in GBM helps to promote tumor progression.
The topic that m6A RNA methylation can act on cancer cell migration and tumor metastasis has now blossomed into a full-fledged field of research.
In HCC, especially in metastatic HCC, a decreased tendency of m
6A modifications is observed and METTL14 is addressed to be the main factor involved in aberrant m
6A modification [
71]. It has been demonstrated by Alarcon and colleagues that m
6A modification can mark pri-miRNAs for processing by recognizing DiGeorge critical region 8 (DGCR8) in a manner dependent on METTL3/m
6A, highlighting the important role of m
6A modification in RNA processing, including mRNAs and pri-miRNAs [
32]. Similarly, METTL14 manipulates pri-miRNA processing by regulating the recognition and binding of DGCR8 to pri-miRNAs in an m
6A-dependent manner and thus METTL14 depletion in HCC results in pri-miR126 processing arrest and reduces the expression of mature miR126 who has been identified as a metastasis suppressor, leading to advanced metastasis capability.
Besides the genetic background of cancer cells, alteration in microenvironment has emerged as a vital layer of regulating cancer metastasis. In colorectal carcinoma (CRC), serine proteases are important component in microenvironment and can selectively activate protease-activated receptor 2 (PAR2) through proteolysis of the receptor [
72]. The research conducted by Yang and colleagues has unraveled that PAR2 activation decreases the level of miR-125b through NOP2/Sun RNA methyltransferase family, member 2 (NSun2)-mediated pre-miR-125b2 methylation in CRC. NSun2 is discovered to methylate precursor of miR-125b, interferes with its processing, and reduces the level of mature miR-125b [
73]. The downregulation of miR-125b augments the expression of its target gene GRB2-associated-binding protein 2 (Gab2), thereby dramatically promoting cancer cell migration, which provides a novel epigenetic mechanism by which m
6A modification on miRNAs promotes cancer metastasis [
74].
In human pancreatic cancer (PC), the research conducted by Chen et al. showed that YTHDF2 was upregulated at both mRNA and protein levels and orchestrated proliferation and epithelial–mesenchymal transition (EMT) dichotomy [
75]. Two of the main characteristics of tumor growth are uncontrolled proliferation and abnormal cell migration [
76,
77]. However, cells are usually not supposed to respond to gene alterations by proliferating or migrating both at the same time, which is called migration–proliferation dichotomy [
78]. YTHDF2 functions as the main regulator in this phenomenon who can promote the ability of proliferation via Akt/GSK3b/CyclinD1 pathway, while it can suppress the migration, invasion, and adhesion ability by inhibiting EMT probably via downregulation of yes-associated protein (YAP) gene. There exist two m
6A RNA methylation sites in YAP mRNA transcript with one site in coding DNA sequences (CDS) and the other site in exon [
35]. Therefore, it seems reasonable to come to the hypothesis that YTHDF2 might bind to m
6A sites of YAP mRNA to decrease the stability of mRNA, while the direct link between YTHDF2 and YAP remains to be clarified.
m6A RNA methylation may contribute to tumor immunity
As elucidated by Li et al., m
6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathway. This prompts the very first attempt at studying m
6A RNA methylation as key regulators of T cell differentiation, suggesting their involvement in tumor–immune system communication and importance in tumor growth and spread [
79].
In other circumstances, toll-like receptors (TLRs) establish the first line of defense against besieging pathogens as the most conserved molecules of the innate immune system, which recognize pathogen-associated molecular patterns to facilitate an immune response. RNAs with m
6A modification are found incapable of activating TLR3, and those with 5
mC and/or m
6A do not activate TLR7 or TLR8, leading to non-recognition of pathogens carrying these RNA modifications by TLR receptors (e.g., a viral nucleic acid). The methylation in m
6A interferes with Watson–Crick base pairing; thus, its presence destabilizes RNA duplexes, which may explain why RNAs containing m
6A modification are incapable of stimulating TLR3 [
80]. Dendritic cells (DCs) exposed to such modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. These undetected viral components may then stimulate a pathway involved in cancer development [
81‐
83].