Seminars in Cancer Biology 86 (2022) 18–31 Contents lists available at ScienceDirect Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer The IGF2BP family of RNA binding proteins links epitranscriptomics to cancer Deepthi Ramesh-Kumar, Sonia Guil * Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Catalonia 08916, Spain A R T I C L E I N F O A B S T R A C T Keywords: RNA binding proteins that act at the post-transcriptional level display a richness of mechanisms to modulate the IGF2BP proteins transcriptional output and respond to changing cellular conditions. The family of IGF2BP proteins recognize M6A-RNA mRNAs modified by methylation and lengthen their lifecycle in the context of stable ribonucleoprotein particles Epitranscriptomics to promote cancer progression. They are emerging as key ‘reader’ proteins in the epitranscriptomic field, driving Noncoding RNA Cancer the fate of bound substrates under physiological and disease conditions. Recent developments in the field include the recognition that noncoding substrates play crucial roles in mediating the pro-growth features of IGF2BP family, not only as regulated targets, but also as modulators of IGF2BP function themselves. In this review, we summarize the regulatory roles of IGF2BP proteins and link their molecular role as m6A modification readers to the cellular phenotype, thus providing a comprehensive insight into IGF2BP function. during development and in cancer, generally correlating with an aggressive malignant phenotype. They orchestrate the composition of 1. Introduction RNA processing effector complexes by conferring sequence specificity and target mRNAs that are directly involved in the secretory pathway, The insulin-like growth factor 2 (IGF2) mRNA binding protein family the insulin/IGF pathway, the metabolic control of multiple tissues, or of proteins (IMPs/IGF2BPs, also labeled with different names derived the ubiquitin-dependent protein degradation pathway, among other from the different target mRNAs and contexts where they were identi­ cellular axes. The evolution, structure and RNA binding affinity of this fied (VICKZ, ZBP1, CRD-BP, KOC or Vg1RBP/Vera in Xenopus)), is family of proteins have been excellently reviewed earlier [7,8], and so involved in a variety of cellular processes, including development, we overview these topics briefly here. This review’s primary focus is on growth, and stemness. In addition, mutations and overexpression of the current knowledge about the ability of IGF2BPs to sense RNA these proteins are associated with the appearance of a high number of modifications and their consequences in downstream regulatory mech­ human tumors. IGF2BP1 was the first family member to be characterized anisms and cell biology in general. We discuss the role of IGF2BPs as as a protein that stabilized c-myc mRNA in vitro through binding to the m6A readers and how it might contribute to key roles of the proteins in coding region stability determinant (CRD) [1]. After that, IGF2BP1 was tumorigenic settings. Remarkably, recent studies have highlighted their also found to control ACTB mRNA localization through binding to a intense crosstalk with noncoding transcripts, and details of prominent “zipcode” element in its 3′ UTR [2], thereby ensuring localized trans­ examples of regulation are provided. Overall, the involvement of lation of ß-actin at the cell periphery and neuronal outgrowth in a IGF2BPs as fine-tuners of RNA metabolism is far from being completely IGF2BP1 phosphorylation-dependent manner [3]. IGF2BP2 was first understood, as the variety of targets and mechanisms of regulation identified as binding to the 5′ UTR of the IGF2 mRNA [4], and thereby increase. promoting IGF2 translation by internal ribosomal entry [5]. The human IGF2BP3 was initially identified in a screen for highly expressed genes in 2. The IGF2BP protein family structure and binding affinity pancreatic cancer [6]. Despite some specific RNA binding abilities and distinct affinities for particular targets, the canonical function for all IGF2BP1, 2 and 3 are the three members of a highly conserved RNA- three paralogs is the association to the 3′ untranslated region (3′ UTR) of binding protein (RBP) family that regulates a spectrum of processes in a number of target mRNAs to influence their localization, stability and RNA lifecycle, including localization, translation control, stability, and translation. These RNA-binding proteins (RBPs) are highly expressed * Corresponding author. E-mail address: sguil@carrerasresearch.org (S. Guil). https://doi.org/10.1016/j.semcancer.2022.05.009 Received 28 February 2022; Received in revised form 10 May 2022; Accepted 17 May 2022 Available online 25 May 2022 1044-579X/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 Nomenclature Complex Catalytic Subunit miRNA microRNA ABCF1 Binding Cassette Subfamily F Member 1 mRNP Messenger ribonucleoprotein ALKBH Alpha-ketoglutarate-dependent dioxygenase PABPC1 Poly(A) Binding Protein Cytoplasmic 1 ATF4 Activating Transcription Factor 4 PDLIM7 PDZ and LIM domain protein 7 CBX8 Chromobox protein homolog 8 PEG10 Paternally Expressed 10 eIF3h Eukaryotic translation initiation factor 3 subunit h RBP RNA binding protein eIF4E Eukaryotic translation initiation factor 4E RNP Ribonucleoprotein FEN1 Flap endonuclease-1 RPSAP52 Ribosomal Protein SA Pseudogene 52 FOXK1 Forkhead Box K1 RRM RNA recognition motif FTO FTO Alpha-Ketoglutarate Dependent Dioxygenase SG Stress Granule GHET1 Gastric Carcinoma Proliferation Enhancing Transcript 1 SNP Single Nucleotide Polymorphisms HCG11 HLA Complex Group 11 SOX2 SRY (Sex determining region Y)-box 2 HMGA2 High Mobility Group AT-Hook 2 tRNA Transfer RNA hnRNP Heterogeneous nuclear ribonucleoprotein WTAP WT1 Associated Protein IGF2BP Insulin-like growth factor-2 mRNA-binding proteins YBX1 Y-Box Binding Protein 1 IRES Internal Ribosomal Entry Site YTHDC1–2 YTH Domain Containing Protein 1–2 lncRNA Long noncoding RNA YTHDF1–3 YTH Domain N6-Methyladenosine RNA Binding Protein m6A N6-methyladenosine 1–3 MATR3 Matrin 3 YTHDF3 YTH N6-Methyladenosine RNA Binding Protein 3 METTL3 Methyltransferase 3, N6-Adenosine-Methyltransferase metabolism. IGF2BPs play a pivotal role in post-transcriptional regula­ tion of RNAs in the context of ribonucleoprotein complexes (RNPs), which contain target mRNAs together with RNA-binding proteins, and may also include noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). Although primarily observed in the cytoplasm, the presence of a nuclear export signal sug­ gests that they associate with some of their target RNAs in the nucleus, and a minor fraction of the protein is certainly detected in the nucleus [3,9,10]. In the cytoplasm, IGF2BPs form granule-like mRNP structures often located in the perinuclear region that determine mRNA fate. Since IGF2BP-containing RNP may include exon-junction complex factors but Fig. 1. Structure of IGF2BP family of proteins. The three IGF2BP paralogs are not eIF4E or eIF4G [11,12], these IGF2BP-RNP targets were initially highly similar, with two N-terminal RRMs and four C-terminal hnRNPK Ho­ linked to non-translating cytoplasmic mRNPs, suggesting that IGF2BPs mology (KH) domains, which are characterized by a conserved αβ-topology that predominantly associate with mRNAs that enter the cytoplasm and have can be structurally and functionally included in two di-domains (KH1–2 and not yet undergone their first round of translation, thereby preventing KH3–4) [195]. Association to single-stranded RNA is enhanced by the KH do­ their decay. Further research has provided a more complete picture, and mains [14], whereas the RRMs are thought to stabilize the RNA-protein in­ although the exact molecular mechanism by which IGF2BPs recognize teractions [19]. In addition, KH domains are necessary for recognition of the and regulate expression of their targets remains to be fully elucidated, m6A mark [24] and recruitment to stress granules [20]. Created with Bio­ their role has been broadened to include the regulation of mRNA sta­ Render.com. bility, translation and localization (this latter aspect specially relevant in neuronal cells, where IGF2BPs are located at the edges of neurites in formation of the protein-RNA complexes with high affinity [19]. When developing neurons) [13–15]. considering the binding affinities of each protein, it is not surprising that The three IGF2BP proteins’ structures are notably similar in order the complex array of RNA binding domains present in the IGF2BP and spacing of domains including a tandem of two RNA-recognition paralogs might dictate a not completely overlapping preference for RNA motifs (RRMs) and two tandems of two KH domains in the N- and C- binding motifs by each individual protein. Studies to address the binding terminal regions respectively [7] (Fig. 1). The overall amino acid affinity of each paralog have used both individual domains [21–23] as sequence identity between all three proteins is 56 %, with greater degree well as full-length proteins [24–28] for target RNA identification. This of similarity within protein domains. IGF2BP1 and IGF2BP3 display the fact, together with the variety of techniques employed (mostly cross­ closest similarity, showing 73 % amino acid sequence identity with each linking and immunoprecipitation (CLIP)-related approaches for other [16]. The three mammalian IMP paralogs belong to the family of full-length protein:RNA interaction identification in living cells versus zipcode-binding proteins, which include the Vegetal-1 mRNA binding the use of systematic evolution of ligands by exponential enrichment protein (Vg1RBP/Vera) in Xenopus laevis, and the zipcode-binding pro­ (SELEX) experiments for single domain analysis in vitro), and possibly tein 1 in chickens. In Drosophila, a protein lacking the N-terminal RRM differences in context have rendered results that only partially overlap. domain but comprising four KH-domains has been proposed to be KH domains recognize short (3–6 nucleotides) motifs through a char­ Drosophila IGF2BP (dIIMP) [17]. acteristic flexible loop, often with cooperation between domains [29], All members of the IGF2BPs are shown to bind RNA, irrespective of and most studies with full-length proteins report a consensus sequence the organism or tissue in which they are expressed [18]. In vitro studies containing CA-rich motifs for all three paralogs (reviewed in [8]). In have proved that the KH-domains are principally responsible for addition, a PAR-CLIP approach reported a common UGGAC motif [24]. RNA-binding, whereas RRM-domains would contribute to the stabili­ By contrast, the study of truncated proteins has revealed a preference for zation of the IGF2BP-RNA complexes with half-life greater than 2 h [19, bipartite motifs separated by a linker region of 10–25 nucleotides, 20]. This reveals that IGF2BPs play a vital role in promoting the similar to the zipcode element originally identified in the binding of 19 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 IGF2BP1 to ACTINB mRNA [2]. In this latter scenario, and despite detailed mechanism of the molecular function of IGF2BP2 in response to specific preferences of each protein as revealed by SELEX approaches, glucose remains incomplete. More recently, Imp2-/- mice have been the KH3 domain would bind to CA-rich motifs with weak nucleotide shown to display reduced levels of the CCL2 chemokine (a direct mRNA discrimination, whereas KH4 would bind to nearby motifs containing a target of IGF2BP2), which results in poor monocyte recruitment and central GG [22,23,30]. Compared to the fairly well-characterized KH3 Th17 cell polarization, protecting the animals from autoimmune neu­ and 4, the role of the KH1–2 and RRM1–2 domains in RNA recognition is roinflammation [41]. unclear [8]. Finally, the relevance of protein-protein interactions in The impact of IGF2BPs dysregulation on cancer is illustrated by the refining IGF2BPs affinity for specific RNA targets in varying cellular fact that they are among the most upregulated RBPs across the TCGA contexts is also incompletely understood [22,25], as is the role that [42]. For example, high levels of these proteins (and especially of formation of homo-and hetero-oligomers might have in protein function IGF2BP3, possibly through its ability to regulate CD44 [43]) associate [19]. with poorer prognosis in high-stage and high-grade ovarian carcinomas Of note, transcriptome-wide studies aiming at determining RNA and lung cancers [44–46]. Similarly, their upregulation associates with preferences for IGF2BPs have helped uncover targets that mediate key worse outcomes of breast cancer [47], colorectal carcinoma [48], functions of individual family members in different cellular environ­ esophageal adenocarcinoma [49], glioma [50], hepatocellular carci­ ments, including carcinogenesis. For example, integrin ITGB5 and BCL2 noma [51,52], pancreatic cancer [53–56], acute myelocyticleukemia mRNAs were identified in eCLIP experiments for IGF2BP1 and found to (AML) [57], Ewing sarcoma [58] and head and neck squamous cell mediate the pro-survival and adhesion features linked to IGF2BP1 in carcinoma [59]. Most of these studies are correlative in nature, and human pluripotent stem cells [26]. iCLIP-seq experiments for IGF2BP3 despite a large body of in vitro evidence generated over almost two uncovered the oncogenes MYC and CDK6 as direct targets mediating decades indicating that IGF2BP proteins enhance the tumorigenic IGF2BP3-associated proliferation of hematopoietic progenitors and phenotype [7,60,61], in vivo results were scarce [62], and only in recent survival of B-ALL cells [27]. In pancreatic ductal adenocarcinoma cells, years a body of data regarding their causal roles in cancer etiology in iCLIP-seq approaches revealed a potential synergistic function of vivo has been produced. For example, it has been shown in mouse ge­ IGF2BP3 with RISC complex to regulate targets involved in netic models that IGF2BP1 overexpression enhances Kras expression to cancer-related pathways, including focal adhesions and cell migration synergistically contribute to the induction of lung adenocarcinoma, [28]. whereas its downregulation diminishes the formation of metastasis [46]. Altogether, further work will be required to fully understand the In melanoma models, high levels of IGF2BP1 increases metastasis [63], specifics of RNA binding for each IGF2BP1–3, since their ability to whereas its depletion decreases melanoma progression [63] and sensi­ regulate a different pool of target RNAs is the key feature that governs tizes melanoma cells to chemotherapy [64]. In mouse xenograft models their related but distinct contributions to physiopathology. of colorectal cancer, IGF2BP1 overexpression promotes tumor growth and cell dissemination into the blood by decreasing the expression of 3. The physiological role of IGF2BPs and their dysregulation in E-cadherin and other epithelial markers while promoting clonogenicity, cancer suggesting a role in the early stages of CRC metastasis. By contrast, its knockdown in intestine cells reduces the number of tumors in a mouse Evidence thus far indicates that the IGF2BPs are essential players of model of intestinal tumorigenesis [65]. cell growth, stem cell maintenance, and differentiation during devel­ In a notable example, a recent study found that IGF2BP3 is overex­ opment. Their critical roles are highlighted by the fact that their dys­ pressed in MLL-translocated leukemia, being critical to the pathologic regulation may lead to diseases such as diabetes [31], insulin resistance proliferative phenotype. Upon IGF2BP3 depletion, there is a delay in [32], obesity [33] or neurological disorders (reviewed in [34]). All leukemia development, and the survival of mice with MLL-Af4-driven members of the IGF2BP family are highly expressed in early develop­ leukemia is significantly increased in a mechanism that involves the ment and carcinogenesis, the latter often related with poor prognosis, Ras signaling pathway [66]. In a related study, IGF2BP3 was seen spe­ tumor progression and tumor cell hierarchy establishment [17]. cifically overexpressed in mixed lineage leukemia-rearranged IGF2BP1 and 3 are expressed at very low levels in adult tissues and are (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL). IGF2BP3 bona fide oncofetal proteins, whereas IGF2BP2 expression is maintained overexpressing mice led to increased numbers of highly proliferating in most normal tissues into adulthood and is involved in metabolic progenitor cells in the bone marrow, and skewing toward B cell/myeloid processes (reviewed in [31,35]). Indeed, several loss-of-function and development, in a process involving the stabilization of the IGF2BP3 gain-of-function models have been used to investigate the physiological targets MYC and CDK6 mRNAs [27]. Thus, understanding the mecha­ role of IGF2BPs and show their association with the regulation of cell nistic details of target regulation is key to understanding IGF2BPs metabolism. For example, Igf2bp1-/- mice are 40 % smaller, with small function. For instance, IGF2BP1 plays a crucial role in shielding MYC, sized organs caused by hypoplasia, complete loss of villi and modifica­ PTEN and MDR1 mRNAs from endonucleolytic attack and BTRC mRNA tion of the composition of the intestinal extracellular matrix [36]. This from miRNA-mediated degradation in the cytoplasm [61,67–69]. phenotype is similar to that seen in the transcription factor Indeed, most mRNA targets of IGF2BP1 that are regulated by miRNAs HMGA2-deficient mice, a major regulator of IGF2BP2 [37]. In the sur­ and shielded from silencing by IGF2BP binding participate in the viving adult mice, the histological changes become gradually normal­ aggressive tumor cell phenotype, giving IGF2BP1 the most conserved ized, indicating Igf2bp1-/- mice show delayed organ maturation. oncogenic role within the family [70]. Another interesting aspect that Interestingly, human genome-wide association studies (GWAS) pro­ mediates oncogenesis is the ability of IGF2BP1 to regulate the cargo of posed IGF2BP2 as a type-2 diabetes associated gene, emerging as a direct extracellular vesicles and promote melanoma metastasis [63]. A list of mammalian target of rapamycin (mTOR) substrate that coordinates mRNA targets for each specific IGF2BP protein, together with the sug­ cellular function and nutrient metabolism [38]. Collectively, the data gested mechanism of regulation and the impact of the regulation on collected from Igf2bp2-/- mice studies show that IGF2BP2 is involved in physiopathology can be found in Table 1. the metabolism of glucose, protein, lipid and energy by controlling the An additional layer of complexity was added by the realization that translation of UCP1 mRNA, among other mitochondrial components IGF2BPs are a distinct family of methyl-RNA binding proteins that [33]. Of note, single nucleotide polymorphisms (SNPs) located in the protect m6A-modified mRNAs from decay, promote mRNA stability and/ second intron of IGF2BP2gene are associated with impaired insulin or target mRNAs for storage (e.g., MYC) [24]. Given the impact of the secretion. IGF2BP2 deficient mice displayed better metabolic traits, m6A mark on RNA metabolism and the involvement in the response to increased insulin sensitivity, improved glucose tolerance and a longer changing cellular conditions and specifically the cancer context, we will life-span compared to the control animals [39,40]. However, the revise in the following sections some key aspects of this modification 20 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 Table 1 mRNA targets of IGF2BPs and impact of the regulation on physiopathology. IGF2BP Target Cis- Regulation of target mRNA Outcome of the regulation References protein mRNA element IGF2BP1 MDR1 CDS Inhibition of CRD-dependent mRNA decay Antagonizes let-7-mediated sensitivity to chemotherapy in [68,182] ovarian cancer KRAS 3´-UTR, Inhibition of mRNA decay Promotes colon cancer cell proliferation, growth and survival [183] CDS BTRC CDS Inhibition of mRNA degradation High levels of ßTrCP1 enhance ß-catenin/Tcf signalling, activate [61,67] transcription factor NF-κB and suppress apoptosis in colorectal cancer cells HCV 5´-UTR, Enhancement of translation not assessed [184] 3´-UTR PPP1R9B 3´-UTR mRNA transport. Required for distal dendritic RNA transport [30] CD-44 3´-UTR Transcript stabilization Promotes formation of invadopodia in HeLa cells [43] ACTB 3´-UTR Inhibition of premature mRNA translation and Ensures localized translation and regulates growth cone [3,13,14,69, enhanced mRNA transport guidance. 185,186] MYC CDS Inhibition of CRD-dependent mRNA decay Enhances tumor cell survival and proliferation [1187] PTEN CDS Inhibition of CRD-dependent mRNA decay Enhances the velocity and the directionality of tumor cell [69] migration by sustaining cell polarization MAPK4 3´-UTR Inhibition of mRNA translation Enhances the velocity and the directionality of tumor cell [69] migration by sustaining cell polarization IGF2 5´-UTR Inhibition of mRNA translation Not assessed [4] CTNNB1 3´-UTR Inhibition of mRNA decay Not assessed [188] IGF2BP2 IGF2 5´-UTR Enhancement of cap-independent translation Enhanced IGF2 expression promotes tumor cell growth [5,60] following IGF2BP2 phosphorylation by mTOR HMGA1 3′ UTR mRNA stabilization following IGF2BP2 Increased proliferation of cancer cell lines [60] phosphorylation by mTOR PINCH-2 3′ -UTR mRNA destabilization Regulates adhesion structures and stabilizes microtubules in [144] skeletal myoblasts UCP1 UTRs Inhibition of mRNA translation Limits longevity and modulates nutrient and energy metabolism [33] in mouse models MURF3 5 -UTR and ′ Enhancement of translation Regulates adhesion structures and stabilizes microtubules in [144] CDS skeletal myoblasts GLUT1 3´-UTR mRNA stabilization Promotes aerobic glycolysis and proliferation of pancreatic [54] ductal adenocarcinoma cells in vitro and in vivo IGF2BP3 IGF2 5´-UTR Enhancement of translation Enhances tumor cell proliferation and survival in leukemia and [189–191] glioblastoma models CD44 3´-UTR mRNA destabilization Promotes formation of invadopodia in HeLa cells [43] ABCF1 n.a ABCF1 mRNA acts as a decoy, thereby High levels of ABCF1 counteract the oncogenic potential of [58] preventing the binding of IGF2BP3 to its IGF2BP3 in Ewing sarcoma oncogenic targets Fig. 2. Reversible m6A modification. The dynamic homeostasis and reversible process of modification of m6A is regulated by writers such as regulatory complex assisting METTL3-METTL14 core complex, known as MAC(m6A-METTL complex), catalysing Adenosine(A) to N6-methyladenosine(m6A) transformation, using S- adenosylmethionine(SAM) as the methyl donor; and its demethylation is catalysed by erasers FTO, ALKBH5 and ALKBH3, with co-substrates such as a-ketoglutaric acid (α-KG) and molecular oxygen(O2) and co-factor ferrous iron(Fe II). Created with BioRender.com. 21 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 and its protein partners, to focus subsequently on IGF2BPs binding to other proteins lacking this domain have also been shown to bind pref­ (m6A)-mRNAs in tumorigenesis. erentially to m6A-modified RNAs, including the IGF2BP1–3 family of proteins [24]. The selectivity for m6A-containing RNAs by proteins 4. The m6A mark without YTH domain is not completely understood but could be medi­ ated by i) other domains capable of directly interacting with m6A mark The methylation of position N6 of adenosine (N6 -Methyladenosine, (this would be the case of the KH domains in IGF2BPs, possibly helped or m6A), is one of the most abundant internal modifications in eukary­ by flanking regions within the protein [24,96]) and/or ii) by binding to otic mRNAs [71], being found in approximately three to five sites in surrounding or overlapping RNA motifs that become more exposed and each mRNA molecule [72,73]. M6A mark on RNA is accessible to the RBPs following local structural changes facilitated by post-transcriptionally catalyzed by a heterotrimeric methyltransferase the presence of the m6A modification (as has been seen, for example, for complex present in nuclear speckles that includes the catalytic METTL3 HNRNPC [97] or HNRNPG [98,99]. In this latter case, m6A acts as a and the allosteric activator METTL14 enzymes, and the WTAP RNA molecular ‘switch’ that may have a widespread effect on the binding of binding protein [74–76], whereas the RNA binding proteins RBM15 and additional proteins to m6A-modified RNAs. In some cases, the specific RBM15B can bind to METTL3 and WTAP and direct the complex to binding mechanism remains unclear: for example, HNRNPA2B1 protein specific RNA sites containing consensus motifs for methylation [77,78]. was initially proposed to be a direct m6A reader and interact through In mammals, mRNA methylation occurs within the consensus sequence one of its RRMs to motifs that overlap with the m6A consensus site, RRACH (R = G or A; H = A, C or U), though only a portion of these thereby regulating alternative splicing and microRNA processing in the putative methylation sites contain m6A [79–81]. An alternative nucleus [90]. However, subsequent studies suggested that HNRNPA2B1 methylation pathway involves the splicing-related METTL16, which action is helped by a “m6A switch” type of mechanism, rather than methylates U6 snRNA as well as some pre-mRNAs and ncRNAs [82,83]. functioning as a direct “reader” of m6A modification [100]. It is possible The m6A mark is reversible in nature: demethylating enzymes described that the IGF2BPs apparent direct affinity for m6A is mediated by a to date (the fat mass and obesity-associated protein FTO [84] and the similar mechanism, given the fact that the KH3 and 4 domains in alpha-ketoglutarate-dependent dioxygenases ALKBH5 [81] and IGF2BPs, necessary for m6A binding, are actually not sufficient for the ALKBH3 [85] respond to stress signals and changing cellular conditions, interaction, and that the IGF2BPs binding sites (despite the diversity of and catalyze demethylation in an Fe (II) and α-ketoglutaric motifs found in different studies) may overlap with the m6A consensus acid-dependent manner, which suggests the epigenetic feature of this sequence DRACH (D, A/G/U; R, A/G; H, A/C/U) [79,80]. Other RNA modification (Fig. 2). non-YTH-containing m6A protein readers may remain to be uncovered, Even though initially the m6A mark was associated with promotion since some untargeted quantitative proteomics approaches have re­ of mRNA degradation [71], subsequent studies have enriched this view ported a variety of potential m6A-RNA binding proteins, including RBPs by revealing that the location of this modification on the mRNA, with RRMs (such as HNRNPs or CPSF6) or with KH domains (such as together with the interplay between writer, eraser and reader complexes FUBPS) [101,102]. In some cases, confirmation of the interaction has and the subcellular location of these binding proteins ultimately deter­ been reported by direct approaches (e.g., the KH domain-containing mine the impact in the lifecycle of target RNAs, revealing the multi­ protein FMR1 and m6A-RNA, where, again, sequence elements on the faceted influence of methylation on mRNA metabolism [86]. Altogether, RNA surrounding the m6A mark seem decisive in enhancing in addition to regulating the stability of the transcript, the presence of RNA-protein affinity [102]). The number of proteins whose binding to m6A on RNA may also impact on its splicing, transport, localization and m6A-containing or neighboring regions in a m6A-dependent manner is translation [81,87–89]. Noncoding RNAs can also be m6A-modified; likely to grow in the next years, as more transcriptomic-wide compari­ primary microRNA processing is modulated by m6A presence [90], and sons between m6A RNA sets and individual Clip-seq studies in different long noncoding RNAs (lncRNAs), which are key regulators of epigenetic pathophysiological settings are developed. Thus, as future studies clarify mechanisms and of gene expression [91,92], can also be modified by the specifics of m6A-RNA recognition by non-YTH containing proteins m6A methylation, with consequences for their function. In one of the such as IGF2BPs, it is likely that some mechanistic commonalities will first described examples, the lncRNA XIST, essential for X-chromosome appear. inactivation in female cells, is highly methylated with dozens of m6A sites, which are necessary for its function as a transcriptional repressor 6. M6A and stress [77]. The distribution of m6A in tissue-specific sites has also been inves­ There are a number of mechanisms through which stress signals tigated, and the results revealed that this modification is most abun­ globally reduce cap-dependent translation, a measure that helps dantly found in the heart, brain and kidney. Furthermore, the conserve energy and nutrients while the cell promotes the expression of distribution of m6A is richer in the adult than in the fetal brain [80]. specific genes to cope with stress by using alternative mechanisms of N6-Methyladenosine plays a critical role in the development of an or­ translation initiation. This is especially relevant in the tumoral context, ganism, and changes in the levels of m6A have an impact on many life where cells face adverse conditions such as hypoxia or starvation, and processes, including tissue development, stem cell self-renewal and where the use of internal ribosomal entry sites (IRES) in stress adaptive differentiation [76,93]. M6A can also control dynamic responses to mRNAs is greatly enhanced [103,104]. In addition, under stress, most extracellular stimuli, including the heat shock response [89], and mRNAs stalled for translation are temporarily sequestered into cytosolic circadian clock [94]. aggregates called stress granules (SGs), (reviewed in [105]), from which they will be eventually released and re-associated with polysomes once 5. IGF2BPs, direct readers of m6A-RNAs? normal conditions resume. This is in contrast to RNAs recruited to the stress-induced P-bodies, which are targeted for degradation [106,107]. M6A is most frequently found in the last exons near stop codons, One key trigger of SG formation is the phosphorylation of eIF2α that allowing for 3′ UTR regulation [80,95] and thus tightly tying the modi­ happens upon several different types of stress such as oxidative, geno­ fication to the fate and function of RNAs. Changes in the mentioned toxic or hypoxic conditions, and metabolic and proteotoxic stresses “writers” and “erasers” of the modification affect the metabolism of the (such as endoplasmic reticulum stress) [108]. Some key proteins present target RNA, but so do the changes in those RNA binding proteins with in SG are small ribosomal subunits, miRNAs and Argonaute proteins, high affinity for m6A-containing transcripts (the so-called “readers”). and a number of regulators of mRNA stability and translation, including The YT521-B homology (YTH) domain-containing proteins YTHDC1–2 the RNA binding proteins TIA-1, TIAR, RCK/p54, Y-box binding protein and YTHDF1–3 are the canonical readers of the m6A mark, [71,79], but 1 (YB-1) and IGF2BPs [20,109]. SG play crucial roles in cancer biology, 22 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 by orchestrating stress signaling and providing the tumor with cell playing accessory roles). Albeit performed in a limited number of cell fitness and resistance to chemotherapy, and many SG components are types, this study clarified the global picture of IGF2BP proteins affinity overexpressed in human tumors [110,111]. Also, targeting SG formation for their RNA targets; RNA-protein cross-linking experiments revealed induces apoptosis of tumoral cells (Takahashi et al., 2013) and inhibits that most target transcripts contain the UGGAC consensus sequence, metastasis [113]. which contains the GGAC m6A core motif, and that > 80 % of the targets It is becoming increasingly clear that the reshaping of the cellular contain at least one m6A site. As is generally typical for m6A distribution, RNA methylome enhances the shift towards cap-independent translation these sites are enriched in 3′ UTRs and near stop codons, and, intrigu­ of certain transcripts and adds to the strategies included in the stress ingly, near 10 % of the targets correspond to non-protein-coding tran­ response pathways, so that the physiological roles of m6A are linked in scripts (see below). Crucially, binding to bona fide targets (including many cases to the response to changing cellular conditions, including MYC mRNA) is impaired upon METTL14 knockdown, suggesting that stress stimuli. For example, during the response to heat shock, the the modification is the primary determinant for the interaction with generally m6A-depleted 5′ untranslated regions (5′ UTRs) are preferen­ IGF2BPs. Additionally, the same study showed that m6A-RNA-IGF2BP tially methylated as a result of stress-induced nuclear localization of the binding enhances the stability and translation of m6A-bearing mRNA ‘reader’ YTHDF2, which protects the region from the m6A demethylat­ targets by recruiting the mRNA stabilizers HuR, MATR3, and PABPC1. ing action of FTO [89]. ALKBH5 also responds to hypoxia and targets the Colocalization with HuR seems to occur in P-bodies, whereas upon heat m6A-modified NANOG mRNA in breast cancer cells [114]. Of note, the shock, IGF2BPs appear in stress granules and shift to non-ribosomal increase in 5′ UTR methylation induces a widespread shift towards fractions in polysome profiles, underscoring their role as regulators of alternative, cap-independent translation initiation events, among others dynamic RNA metabolism to generally prevent degradation upon of the heat-shock protein Hsp70 mRNA [88,89] and the activating adverse cellular conditions [24] (Fig. 3). Even though evidence for transcription factor 4 (ATF4, a key effector of the integrated stress colocalization of m6A-RNAs and IGF2BP proteins in stress granules is response) [115], which depends on a direct interaction between eIF3 still missing, the proposed mechanism would be in contrast to the gen­ and m6A for ribosome recruitment. This cap-independent enhancement eral role of YTHDF2 as an m6A-RNA destabilizer, and may be a conse­ of translation may explain why certain circular RNAs may produce quence of different sequence affinity (ENCODE PAR-CLIP data indicates peptides in a m6A-dependent manner (see below). little overlap between IGF2BPs and YTHDF2-binding sites [24]) and/or location of binding sites (YTHDF2-binding sites being more abundant in 7. IGF2BPs as m6A readers in normal and stress conditions? CDS than 3′ UTRs [71]). Given the abundance of growth-related targets, it is not surprising that knockdown of IGF2BPs and specifically KH3 and One important recent development in IGF2BPs studies is their pref­ 4 mutants decreased proliferation and colony formation [24], although erential binding for m6A-modified RNAs: by using m6A-modified single- elucidating to what extent this is dependent on the roles as m6A readers stranded RNA as a bait, protein pull-down assays indicated that all three requires further studies. IGF2BP proteins were retrieved with a 3–4-fold higher affinity than with The role of IGF2BP in ensuring the stability of stress granule- the compared unmethylated control, being among the top enriched recruited mRNAs has been known for several years. For example, in proteins in the m6A pull-downs [24]. This is mediated by the proteins’ response to oxidative stress or heat shock, IGF2BP1 relocalizes to SG, KH domains (specially domains 3 and 4, with KH domains 1 and 2 where it remains bound to its target mRNAs (including MYC and IGF2 Fig. 3. Mechanism and function of IGF2BPs as m6A readers. The modification of m6A is regulated by writers, readers and erasers. Writers such as METTL3, METTL14, RBM15, ZCH13, WTAP and KAAI429 regulate m6A methylation. FTO, ALKBH5 and ALKBH3 are erasers, which are identified to induce m6A demethy­ lation. Together with the YTHDF family, IGF2BPs are reported to be the main m6A readers, and their functions are related to promoting mRNA stability, degradation, export and storage. In the cytoplasm, IGF2BPs protect RNA targets in the context of mRNPs under both physiological and stress conditions, under which these proteins and associated transcripts accumulate in stress granules for subsequent sorting. Created with BioRender.com. 23 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 mRNAs) and prevents their transfer to P bodies and subsequent decay, Table 2 thereby determining the fate of target mRNAs during cellular stress [69, Reported m6A-mRNA targets of IGF2BPs and impact of the interaction on the 109]. The association of IGF2BPs with SG is stable, in contrast to the oncogenic phenotype. RBPs that are essential for SG assembly, which are highly mobile [116]. IGF2BP m6A- Tumor type Regulation References It was noted that the KH domains facilitate the recruitment of IGF2BP1 protein target and 2 to the SG, whereas they play little role in the case of IGF2BP3 IGF2BP1 PEG10 Endometrial Stabilizes PEG10 mRNA [119] paralog [20]. Of note, while in non-stressed cells, IGF2BP may exert cancer and enhances different types of regulation on transcripts (at the stability or the expression MYC Colorectal, Increases MYC mRNA [121] translation level), the proteins adopt a general stabilizing function in ovarian, and stability and expression stress. Further studies have shown that the pivotal role of localization to other cancer SG to protect translationally-stalled mRNA targets from degradation types might be an oversimplified picture [117,118]: in fact, depletion of SRF several Enhances SRF- [96] IGF2BP1 may be the key event that destabilizes mRNA substrates, dependent transcriptional activity, irrespective of SG integrity. Likewise, impairing SG formation does not resulting in tumor cell affect the stress-induced stabilization of target mRNAs by IGF2BPs in growth and invasion intact mRNPs [116]. Rather, the maintenance of stable mRNP complexes CBX8 Colon cancer EnhancesCBX8mRNA [120] seems key to preventing stress-induced mRNA degradation. Bearing this stability and expression, which regulates in mind, and given the growing number of studies pointing to the un­ stemness and equivocal relevance of m6A modification for target recognition and chemosensitivity in function of IGF2BPs in tumorigenesis, future research shall elucidate the colon cancer specific role that modifications on the RNA targets play in the response E2F1–3 Across multiple Promotes E2F-driven [122] of IGF2BPs to stress. tumor types G1/S transition and cell growth IGF2BP2 FEN1 Hepatocellular EnhancesFEN1 mRNA [52] 8. M6A-mRNA targets mediate tumorigenic functions of IGF2BPs carcinoma stability, promoting liver cancer growth Aiming to understand the underlying mechanism(s) for IGF2BPs MALAT1 Thyroid cancer Elevates MYC [142] expression, which oncogenic features based on their ability to recognize m6A modification, consequently some recent studies have confirmed that the IGF2BP family of proteins accelerates the recognizes mRNA in a highly m6A-dependent manner, causing upregu­ proliferation, invasion lation in oncogenic expression or downregulation in tumor suppressor and migration of TC gene expression. For instance, PEG10 was shown to be bound by cells SOX2 Colorectal EnhancesSOX2 mRNA [48] IGF2BP1 via m6A modification. The polyadenylate-binding protein I cancer stability and promotes (PABPC1) is then recruited by IGF2BP1 to enhance the stability of CRC stemness PEG10 mRNA, upregulating PEG10 expression and promoting endo­ GLUT1 Colorectal Activation of glycolysis [192] metrial cancer cell proliferation [119]. In colon cancer, it is demon­ cancer and CRC progression IGF2BP3 VEGF Colon cancer Represses angiogenesis [123] strated that IGF2BP1 promotes expression of the Polycomb protein CBX8 CCND1 Colon cancer Represses S phase and [123] also in a m6A-dependent manner. This results in the regulation of proliferation of colon stemness and the suppression of chemosensitivity through induction of cancer the CBX8 target LRG5 [120]. Through a novel mechanism, the lncRNA originally annotated as LINC00266–1 produces a peptide that acts as a regulatory subunit of IGF2BP1 and enhances its binding affinity for its progression downstream of the elevated levels of METTL3 by binding to target m6A-MYC mRNA, promoting tumorigenesis by increasing the and stabilizing the m6A-modified HDGF mRNA, which is involved in level and stability of MYC transcripts [121]. IGF2BP1 also binds and increased angiogenesis and glycolysis [124]. A list of reported promotes the serum response factor SRF mRNA expression in a m6A-mRNA targets for each IGF2BP protein, and the relevance of the m6A-dependent manner by preventing miRNA-mediated down­ interaction for oncogenesis can be found in Table 2. regulation, enhancing tumor cell growth and invasion. Additionally, the It is expected that the number of bona fide m6A-modified mRNA functional link between the two genes and their oncogenic role in cancer targets of the IGF2BP family will increase in the following years, as the cells is further reinforced by the observation that IGF2BP1 stabilizes the preferential role as m6A reader proteins is further established. There is mRNAs of key downstream targets of SRF such as FOXK1 and PDLIM7 an additional aspect in the substrates of the three paralogs that deserves [96]. Perhaps the most compelling evidence linking the role of IGF2BP1 attention, namely the fact that noncoding transcripts (including m6A- as an m6A reader with an impact on oncogenesis is the finding that marked ncRNAs) are in a more complex dialogue with IGF2BPs than IGF2BP1 not only stabilizes cell cycle regulators like the transcription mRNAs, both as regulated targets and also as regulatory cofactors of RBP factors E2F transcripts in a m6A-dependent manner, but also stabilizes function. E2F-driven oncogenic targets post-transcriptionally, robustly promoting cell cycle progression and proliferation across many solid cancers [122]. 9. The emerging cross-regulation between IGF2BPs and (m6A)- In a similar manner, the METTL3/IGF2BP2 axis is shown to promote ncRNAs tumorigenic features by stabilizing the Flap endonuclease-1 (FEN1) and SRY (sex determining region Y)-box 2 (SOX2) mRNAs in hepatocellular In addition to the fairly well-characterized mRNA targets, an addi­ carcinoma and colorectal cancer, respectively [48,52]. The role of tional important aspect of the function and regulation of IGF2BPs in IGF2BP3 as m6A reader has been studied in colon cancer, were IGF2BP3 healthy and diseased tissues is provided by the noncoding tran­ expression is a predictor of progression and poor survival. IGF2BP3 scriptome. It is increasingly clear that long noncoding RNAs are key binds to Cyclin D1 and VEGF mRNAs, regulating both their stability and regulatory players in diverse cellular processes, including growth, dif­ expression via m6A modification. Through this mechanism, IGF2BP3 is ferentiation, apoptosis and cancer progression and metastasis [125, associated with DNA replication, and IGF2BP3 knockdown consequently 126]. Like m6A-mRNAs, m6A-marked noncoding transcripts are also reduced the percentage of S phase in cell cycle, cell proliferation and bound and stabilized by IGF2BP proteins [56]. Conversely, many studies angiogenesis [123]. In gastric cancer, IGF2BP3 contributes to tumor point to the role of ncRNAs as regulators of IGF2BPs function, and in 24 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 addition to the miRNAs that have been reported to target IGF2BP binding to more than one RNA through their multiple RNA binding transcripts (among which, importantly, the tumor suppressor miRNA domains, IGF2BPs may coordinate the function and fate of more than let-7) [127–130], a growing number of longer transcripts are known to one target, and/or be themselves regulated by one RNA moiety to in­ interplay with IGF2BPs. Mechanistically, many studies have focused on fluence the protein ability to bind to other RNA substrates. Similarly, a the ability of lncRNAs to recruit, organize, or modulate the activity of highly conserved lncRNA, THOR, was seen to enhance the stability of RBPs, indicating that RNA is also able to regulate RBPs (recently IGF2BP targets through direct binding to IGF2BP1. Notably, this accel­ reviewed in [131]. Therefore, similar to other RBPs, regulatory in­ erates the onset of melanoma in human, mouse and zebrafish models teractions that occur between IGF2BPs and RNA are bidirectional, [133]. Other examples of lncRNAs for which similar mechanisms have particularly in the case of lncRNAs (Fig. 4). been suggested include GHET1 in gastric carcinoma [134], HCG11 in hepatocellular carcinoma [135], LIN28B-AS1 in lung adenocarcinoma [136], and linc01021 in colorectal cancer [137]. 9.1. LncRNAs that enhance IGF2BP function A number of works have shown that binding to lncRNAs enhances 9.2. LncRNAs that hinder IGF2BP function the physical association between IGF2BPs and mRNA targets, through mechanisms that are generally not sufficiently understood but that may By contrast, interaction with other lncRNAs have been reported to imply the formation of ternary complexes. For example, a study from our impair IGF2BP function; such is the case for FGF13-AS1 in breast cancer group showed that RPSAP52 is a processed pseudogene that influences [138], the liver-specific LINC01093 in hepatocellular carcinoma [139], IGF2BP2 function through direct RNA—protein interaction and the or 91 H in colorectal cancer [140]: these lncRNAs display antitumoral enhancement of target mRNA recruitment to polysomes. In addition, effects via a potential competition with IGF2BP oncogenic targets for RPSAP52 impacts positively on the oncofetal HMGA2 gene expression, binding to the proteins. In view of the alternative regulatory modes, with both regulatory mechanisms converging on the proliferative further work will be necessary to understand the specifics of the in­ HMGA2/IGF2BP2/RAS axis to promote the growth of tumoral cells teractions that support the cooperative or competitive nature of lncRNA [132]. Although the spatial details of the interaction remain to be binding to IGF2BPs. For instance, it will be important to elucidate elucidated, one interesting possibility suggested by this study is that, by whether the affinity for the different RNA binding domains within Fig. 4. (m6A)-ncRNAs regulate IGF2BPs. A number of long noncoding RNAs and circRNAs modulate IGF2BP proteins, by impairing their function (through competition for the binding to the RNA targets or by decreasing protein levels), or conversely, by enhancing target recognition (through the establishment of ternary complexes). As a consequence, noncoding RNAs may promote or hinder the pro-tumoral features of the proteins, respectively. Specific examples are shown and listed in Table 3. Figure created with BioRender.com. 25 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 IGF2BPs is driving the ability to form ternary complexes with more than Table 3 one RNA as substrate, or how the presence of m6A sites influences the lncRNAs and circRNAs binding partners of IGF2BPs. regulatory ability of lncRNAs. ncRNA IGF2BP Regulation References protein 9.3. Other mechanisms of IGF2BP regulation by lncRNAs THOR IGF2BP1 Upregulates IGF2BP1 binding to [193] mRNA targets and enhances cell Besides impairing or enhancing target binding, lncRNAs may regu­ proliferation, migration and invasion late IGF2BPs via additional mechanisms. For instance, LINRIS is a GHET1 IGF2BP1 Enhances the gastric carcinoma cell [134] proliferation and interaction between GATA3-controlled lncRNA upregulated in colorectal cancer that binds MYC mRNA and IGF2BP1, enhancing directly to and inhibits the ubiquitination and thus the degradation of its stability and expression IGF2BP2 protein in colorectal cancer [141]. In thyroid cancer, the HCG11 IGF2BP1 Interacts with IGF2BP1 and promotes [135] highly abundant MALAT1 lncRNA is able to upregulate IGF2BP2 cell proliferation and invasion in HCC cells expression by acting as a decoy for miR-204 and preventing LIN28B- IGF2BP1 Upregulates IGF2BP1-dependent [194] miRNA-mediated silencing of IGF2BP2. As a consequence, m6A-modi­ AS1 mRNA stability and promotes HCC fied MYC mRNA is stabilized and expression of the gene upregulated, cell progression enhancing the pro-growth phenotype [142]. LINC01093 IGF2BP1 Affects expression of GLI1and [139] downstream molecules in HCC progression 9.4. LncRNAs regulated by IGF2BPs RPSAP52 IGF2BP2 Enhances target mRNA recruitment to [174] polysomes On the other hand, although with only a few examples so far, LINRIS IGF2BP2 Prevents IGF2BP2 ubiquitinationin [141] lncRNAs may be themselves regulated by IGF2BPs: the liver-specific CRC MALAT1 IGF2BP2 Promotes MALAT1 stability, [142] lncRNA HULC was shown to be destabilized by IGF2BP1 via recruit­ downstream target ATG12 expression ment of the CCR4-NOT deadenylase complex, in what was an unusual and NSCLC proliferation example of a IGF2BP protein inducing RNA decay [143]. Whether this is circNDUFB2 IGF2BP1–3 Enhances the interaction between [161] linked to the noncoding nature of the target is uncertain, since few ex­ TRIM25 and IGF2BPs, promoting amples of enhancement of mRNA decay have been reported for IGF2BP protein degradation circPTPRA IGF2BP1 Inhibits tumor progression by [162] proteins [43,144], and further studies on the composition and subcel­ blocking the recognition of mRNA lular location of the IGF2BP RNP complexes in the different contexts will tragets by IGF2BP1 be necessary to fully understand the regulatory mechanism taking place. circNSUN2 IGF2BP2 Enhances binding and stability of [151] Additionally, HULC is highly upregulated in liver cancer, where it in­ HMGA2 mRNA and promotes CRC metastasis progression duces tumor growth, but no clear correlation could be found between IGF2BP1 and the lncRNA levels in tumors, pointing to additional layers of control being present. In another recent study, IGF2BP2 was seen to circRNA expression (at least in some cell types). By contrast, m6A act as a reader of the m6A-modified ZFAS1 lncRNA and stabilize the mRNAs encoded by the parent genes of m6A circRNAs have shorter transcript. In turn, ZFAS1 enhances the activity of the Obg-like ATPase 1 half-lives among all m6A mRNAs [148]. The presence of m6A sites on (OLA1), increasing ATP hydrolysis and the Warburg effect during circRNAs has been linked to several aspects of circRNA metabolism, colorectal cancer proliferation and progression. In addition, this study including the back splicing that generates circRNAs [150], the export to shows that IGF2BP2 is highly expressed in CRC cell lines and tumor the cytoplasm [151], the stability [152], and even the translation of tissues, positively correlating with the higher levels of ZFAS1 lncRNA circRNAs. Albeit generally considered noncoding RNAs, in some cases and m6A mark [145]. circRNAs are translated through cap-independent mechanisms Table 3 lists the best characterized examples to date of lncRNAs [153–156], and m6A modifications may influence this process: interacting with IGF2BPs. m6A-modified regions may be used as internal ribosomal entry site However, for many of the reported examples, it is currently unknown (IRES) and promote translation initiation of circRNAs in a eIF4G2- and whether these mechanisms also rely on modifications of the ncRNA YTHDF3-dependent manner, which underscores the complex lives of molecules involved. Given recent evidence, though, one can foresee that this special class of transcripts. Importantly, this process is inhibited by m6A modification on the lncRNA dictates the affinity for IGF2BP pro­ the m6A demethylase FTO protein, and increased by heat-shock, once teins. This is illustrated by the case of circRNAs (covalently closed RNA more potentially linking m6A-circRNA functions with the stress response loops that generally derive from precursor mRNA back-splicing and [157]. Given the poor association of circRNAs with polysomes [158], the display regulatory functions on other genes expression [146,147]), prevalence of this phenomenon remains to be assessed, but many which are also reported to bind to IGF2BPs and are suggested to influ­ m6A-containing circRNAs were proposed to undergo similar ence IGF2BPs function through a variety of mechanisms, in many cases cap-independent translation [157,159], and the presence of modified mediated by the presence of the m6A mark. m6A sites has been seen to enhance translation [160], while in other instances, circRNA translation seems to be unaffected by m6A modifi­ 10. IGF2BPs and (m6A)-circRNAs cation [159]. Future research will clarify the mechanisms by which m6A regulates Recent literature points to circular RNAs as being prone to modifi­ circRNA metabolism, and the impact of this regulation on disease pro­ cation and interacting extensively with IGF2BPs. M6A modification is gression, but their link with IGF2BP function is increasingly clear. For widely present on circRNAs, with patterns that differ from those in the example, a report on the circRNA circNDUFB2 describes its ability to exons on linear mRNA they are related to. These patterns are cell-type downregulate IGF2BPs via scaffolding the interaction with the E3 specific, being specially present in human embryonic stem cells ubiquitin ligase TRIM25, which induces protein degradation and con­ (hESCs) and enriched in circRNAs derived from long single exons [148]. tributes to inhibition of proliferation in non-small cell lung cancer. Of In mRNAs, m6A is most commonly found on final exons, whereas note, RNA-protein interaction is enhanced by the m6A modification of circularization of the last exon of genes is rare [149], suggesting distinct circNDUFB2 [161]. In another study, circPTPRA impairs the ability of patterns of modification. M6A-circRNAs are not especially unstable, but IGF2BP1 to bind to m6A-transcripts (including MYC mRNA) by inter­ rather, high levels of modification correlate with high circRNA expres­ acting through the KH3 and 4 domains. This block in IGF2BP action sion whereas, intriguingly, low levels of m6A associate with lower 26 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 reduces the tumorigenic features of bladder cancer cells and xenografts mark, these family is emerging as one key player in m6A-dependent gene models [162]. In this case, binding to circRNA does not affect protein regulation. However, despite an increase in the number of studies that stability or levels, and the impact on downstream targets seems to focus on this new angle, the contribution of m6A reading activity to the depend on the ability of the circRNA to outcompete the mRNA substrates well-established oncogenic properties of IGF2BP family is still unclear. for the binding to IGF2BP1. A similar example was seen in melanoma, This is especially relevant for the ncRNA targets, which are emerging where the circRNA CDR1as is able to bind to IGF2BP3 via several as key molecules that can be both regulated by and regulators of CA-rich sequences. Epigenetic silencing of CDR1as during tumor pro­ IGF2BPs activity; it remains to be elucidated to what extent modifica­ gression changes the levels of a subset of IGF2BP3-bound mRNA targets tions on these types of substrates influence the binding and the regula­ involved in cell invasion, presumably by unblocking IGF2BP function tory mechanisms taking place. When considering this type of regulation, [163]. This is in contrast to what has been observed in the case of the difference in concentration between ncRNAs and IGF2BPs poses another circular RNA molecule: the modified m6A-circNSUN2 enhances difficulties. There are a few examples of highly abundant small ncRNAs IGF2BP2 function by interacting with this protein and HMGA2 mRNA, bound by IGF2BPs: for instance, the nuclear RNA 7SK is stabilized by forming a ternary complex that stabilizes the mRNA and promotes IGF2BP3 (which is able to undergo nuclear localization [171]), in a colorectal carcinoma metastasis progression [164]. The host gene of process that blocks P-TEFb activation and thus restricts megakaryocyte circNSUN2, NSUN2, is itself a 5-methylcytosine (m5C) methyltransferase development [170]. Similarly, IGF2BP1 was seen to bind to the short Y3 with a potential role in cancer and with tRNA molecules as main sub­ RNA and enhance the cytoplasmic localization of the Ro60 ribonucleo­ strates [165], suggesting a complex crosstalk between the different protein [172,173]. Regarding lncRNAs, it is easily conceivable that the epitranscriptomic modifications, and foreseeing further exciting de­ extremely abundant MALAT1 may compete for miRNA binding and velopments in the research around IGF2BPs, ncRNAs and epitran­ thereby upregulate IGF2BP2 expression [142], but how the generally scriptomics in cancer. lowly expressed lncRNAs may influence the very abundant IGF2BPs in cancer cells remains unclear. Even though an accurate comparison of the 11. IGF2BPs as a therapeutic target in cancer relative levels of lncRNA targets and IGF2BPs is generally missing, studies that have addressed this aspect in a few cancer cells show a Given the ability of IGF2BPs to bind a wide range of prooncogenic difference of several orders of magnitude between total lncRNA and mRNAs, and the growing evidence pointing to causal roles in the IGF2BP protein levels [174]. This argues against the ability of the different aspects of tumorigenesis, their pharmaceutical inhibition could lncRNAs to enhance or compete for IGF2BPs function in an effective have an important therapeutic potential. High-throughput screenings manner, even though other important variables that do not correlate that use fluorescence polarization to reveal small molecules capable of with the measure of the average expression in a population of cells may interfering with IGF2BP1 binding to key target mRNAs were developed be key in facilitating the regulation by ncRNAs. For instance, RNAs may [166], and a first small compound that inhibits the IGF2BP1-MYC mRNA be enriched or depleted neay key RBPs [175]. Also, if ternary complexes interaction was reported a few years ago. The drug reduced proliferation with more than one RNA bound by a given IGF2BP protein are formed, of ovarian and melanoma cell lines in vitro [167]. More recently, a relative levels between the target RNAs, as well as the number of binding similar screening has produced a candidate compound that specifically sites, the respective protein-RNA binding affinities and stability of the binds the hydrophobic surface at the junction of KH3 and KH4 domains interactions could be decisive in the biological outcome. Also, in IGF2BP1. Of note, the interaction hinders binding to KRAS mRNA liquid-liquid phase separation or local environments could justify the (and possibly to other key targets) and results in diminished cellular different stoichiometric abundance of ncRNA, mRNAs and IGF2BPs, migration and clonogenicity of colon and lung cancer cells in vitro, with presenting a scenario where mRNP function could be compartmental­ no apparent effect on cell proliferation [168]. Another similar screening ized and represent sites of local high concentration of otherwise aimed at targeting IGF2BP2-RNA interactions has been developed with generally lowly present ncRNAs [176]. However, this is still speculative the use of generic IGF2BP2 binding motifs. In this case, candidate and more work is warranted to uncover mechanistic commonalities and compounds inhibit cell proliferation in 2D and 3D cultures of human solidly confirm the occurrence of the proposed regulation by lncRNAs. cells, and reduce tumor growth in zebrafish embryo xenograft models Recent technical developments that allow the spatial study of the tran­ [169]. These preliminary findings suggest that at least IGF2BP1 and 2 scriptome in subcellular and membrane-less compartments will likely are druggable targets to reduce tumor cell proliferation, but further shed valuable light to understand regulation by lncRNAs [177]. studies are required to understand the different impact of each candidate Recently, the presence of multiple m6A on mRNAs has been shown to drug on cell phenotypes, and to assess the selectivity and efficacy of enhance liquid-liquid phase separation together with their bound pro­ these compounds in vivo. teins YTHDF1–3, suggesting that the diverse ways by which the cells By contrast, to date, no direct inhibitor of IGF2BP3 has been re­ interpret this modification are dictated by liquid-liquid phase separation ported, but indirect strategies have been tested. For example, indirect principles [178–180]. For example, relocalization of mRNAs and RBPs pharmacological inhibition of IGF2BP3 via the bromodomain and to P bodies or stress granules upon stress may be driven not by the mere extraterminal domain (BET) inhibitors has been described in megakar­ increase in the levels of m6A per se, but rather, by the ability of the yocytes [170] and in B-ALL [27]. Similarly, in Ewing sarcoma, where modification to scaffold the partition of the complexes in discrete high levels of IGF2BP3 are associated with worse patient outcome, the intracellular condensates. Further work will clarify whether m6A use of the BET inhibitor JQ1 in cell lines decreases IGF2BP3 expression modification influences SG formation in general or just the fate of a and the levels of its key targets ABCF1, MMP9 or CD44, with a parallel particular subset of mRNAs within that structure. It is also unclear reduction in cellular growth [58]. whether this feature of m6A is key in enhancing the presence of IGF2BPs Altogether, the existence of these inhibitors indicates potential in granules under stress conditions, but other m6A binding proteins have accessibility of IGF2BP proteins to exogenous manipulations, and war­ been reported to accumulate in stress granules linked to their “reader” rants further exploration of their targeting by pharmacological com­ ability [181]. Altogether, the detailed connection between target pounds to counteract their oncogenic actions. recognition and subcellular localization depending on post-transcriptional modification of the mRNAs/ncRNAs targets and 12. Final remarks how this determines RNA fate is still poorly understood, and future breakthroughs in the field will undoubtedly clarify our understanding of Around 7000 m6A-modified RNAs are thought to be present in the the role of IGF2BPs in cancer biology. cell. Since IGF2BPs target thousands of mRNAs, with large overlaps between the three IGF2BP1–3 proteins, and most of them bear the m6A 27 D. Ramesh-Kumar and S. Guil Seminars in Cancer Biology 86 (2022) 18–31 Acknowledgements [25] M. Hafner, et al., Transcriptome-wide Identification of RNA-binding protein and microRNA target sites by PAR-CLIP, Cell vol. 141 (1) (2010) 129–141. [26] A.E. Conway, et al., Enhanced CLIP uncovers IMP Protein-RNA targets in human We thank the CERCA program (Generalitat de Catalunya) and the pluripotent stem cells important for cell adhesion and survival, Cell Rep. vol. 15 Josep Carreras Foundation for institutional support. We are grateful to (3) (2016) 666–679. Tara Srinivas for critical reading of the manuscript, and apologize to [27] J.K. Palanichamy, et al., RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation, J. Clin. Invest vol. those authors whose work is not included. 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