Upregulated CircRNAs With Efficacy in Preclinical Ιn Vivo Models Reveal New Targets for Therapeutic Intervention in Glioma

Abstract

There is a significant need to improve treatment efficacy for patients with glioma. Therefore, we have analyzed the literature for the role of circular RNAs (circRNAs) with respect to selected pathology-related topics of glioma. We have focused on upregulated circRNAs with efficacy in preclinical in vivo models. The identified circRNAs cover the following topics: Temozolomide (TMZ) resistance, transmembrane and secreted proteins, wingless-related integration site (WNT) signaling, cytoskeleton dynamics, cell-cell and cell-extracellular matrix interactions, as well as circRNAs with additional functions. We describe possible therapeutic tools and challenges with respect to delivery of the corresponding inhibitory agents.

Introduction

Gliomas are the most common brain tumors and comprise different pathological and molecular subtypes. Glioblastomas are the most frequent subtype (1). The latter are very aggressive, and the median survival time of patients is 12-15 months after diagnosis (2). Gliomas can be derived from astrocytes, oligodendrocyte precursor cells and neural stem cells (3). Heterogeneity of gliomas has been revealed by transcriptional profiling and several molecular glioma subtypes have been identified such as classic, mesenchymal, proneural and neural (4). Spatial and temporal heterogeneity is a hallmark of gliomas involving normoxic, hypoxic, quiescent, proliferative, vascularized and infiltrative regions (5, 6). Distinct genetic features have also been uncovered in gliomas; examples include: O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, chromosomal 1p/19q deletion, loss of neurofibromatosis type 1 (NF1) and phosphatase and tensin homolog (PTEN), epidermal growth factor receptor (EGFR) amplification and truncation (EGFRvIII), mutations in TP53, isocitrate dehydrogenases 1,2 (IDH1,2), telomerase reverse transcriptase (TERT), alpha-thalassemia/mental retardation syndrome X-linked (ATRX), cyclin-dependent kinase inhibitors 2A and 2B (CDKN2A/B), and other alterations (7). Recent investigations have uncovered networks between tumor cells and cells of the tumor microenvironment (TME) which promote tumor growth, infiltration and therapy resistance. Intratumoral networks are formed and long membranous protrusions such as tumor microtubes (TM) and tunneling tubes (TNT) promote connectivity among tumor cells (8). In addition, neuron-glioma synapses that mediate glioma proliferation and tumor-cell survival, have been identified (8). Recently, it was demonstrated that autonomous rhythmic activity in glioma networks drives brain tumor growth (9). Other cell types of the TME interacting with glioma cells have been shown to contribute to the evolution and pathogenesis of gliomas. Examples are glial cells, endothelial cells, microglia, tumor-associated macrophages, T-cells, myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs) and neutrophils (10).

The standard of care for patients with glioma involves a combination of surgery, chemotherapy, radiotherapy and Tumor Treating Fields (TTFields) therapy, as well as off-label and supportive medications such as corticosteroids and anti-convulsants (11). The DNA-methylating agent temozolomide (TMZ) is used as the standard chemotherapy agent and bevacizumab has been approved for treatment of recurrent glioma. Agents targeting gliomas with specific mutations in IDH1 or BRAF (V600E) or H3 K27M histone mutations such as vorasidenib, tovorafenib and dordaviprone have been approved (12). However, the therapeutic efficacy of all of these agents is limited. Recently, immunotherapeutic approaches such as chimeric antigen receptor- (CAR-) and natural killer (NK) cell therapy in combination with immune-checkpoint inhibitory antibodies gave rise to promising clinical results in glioma patients (13). In order to identify mediators of glioma pathogenesis and corresponding therapeutic targets, we have scrutinized the literature using PubMed for the role of circular RNAs (circRNAs) in this context. We have focused on circRNAs which are involved in TMZ-resistance, affect the level of transmembrane and secreted proteins, WNT-signaling, mediate the organization of the cytoskeleton, cell-cell and cell-extracellular matrix interactions, or induce other selected additional targets. We discuss upregulated circRNAs mediating efficacy in preclinical in vivo models of glioma; downregulated circRNAs are not in the focus of this review.

Circular RNAs

CircRNAs are covalently closed single-stranded RNAs. They are generated by circularization of mRNA transcribed from genes driven by lariats, intron pairing or RNA-binding proteins through back-splicing (14). CircRNAs are a subgroup of non-coding RNAs such as microRNAs (miRs), small-interfering RNAs (siRNAs), piwi-interacting RNAs (piRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNAs), long non-coding RNAs (lncRNAs), transfer RNAs (tRNAs) and ribosomal RNA (rRNA) (15). CircRNAs can contain exonic, intronic, as well as intergenic sequences and harbor unique junctional sequences due to the back-splicing process (16). The latter are specifically amenable for therapeutic intervention (16).

CircRNAs are transcribed from hundreds of genes of the human genome (17) and are conserved among species (18). They exert a plethora of physiological functions, and their expression is deregulated in many diseases, including cardiovascular, neurological, and inflammatory diseases, as well as cancer (18). CircRNAs are expressed in many organs including the brain (19). For example, a circRNA which modulates synaptic responses in vivo has been identified, emphasizing the physiological role of circRNAs (20).

CircRNAs exert their functions through a variety of mechanisms, such as sponging of miRs, protein binding, generation of protein-complex scaffolds, interactions with DNA and RNA, and, in rare cases, by encoding functional proteins. They act intracellularly but also can be transferred by exosomes to other types of cells and organs from the cells in which they are generated (21). CircRNAs are involved in processes such as transcription, splicing, translation, signaling, generation of mutations, DNA repair and epigenetic modifications (21). They can affect hallmarks of cancer such as proliferation, migration, invasion, metastasis, angiogenesis and modulation of antitumor immune responses. CircRNAs can exert oncogenic as well as tumor-suppressive functions (21). Their preclinical activity has been demonstrated in numerous in vitro systems as well as in xenograft, orthotopic and patient-derived in vivo cancer-related models (22-24). Therefore, reconstitution or repression of the activity of defined circRNAs might be a future option for treatment of cancer. The role of circRNAs in glioma as predictive, prognostic clinical markers and possible therapeutic targets has been summarized in several reviews (25-27). In this review we focus on the role of selected classes of upregulated circRNAs as tools for target identification and possible new modalities for therapeutic intervention in glioma patients.

CircRNAs Mediating TMZ Resistance

TMZ is the standard agent for chemotherapy of glioma. It has high oral bioavailability, alkylating and lipophilic properties, a small size, and can cross the blood-brain barrier (BBB) (28-30). TMZ can deliver a methyl group to O6 guanine, N7 guanine and N3 adenine of DNA. However, therapy resistance develops consistently, a phenomenon observed for many chemotherapeutic interventions in cancer (28-30). Herein, we present a selection of circRNAs which mediate TMZ-resistance in glioma. CircRNAs affecting TMZ-resistance in glioma are also summarized in (31).

CircWD repeat domain 62 (circWDR62) induces TMZ-resistance by upregulation of O6-methylganine-DNA-methyltransferase (MGMT). CircWDR62 (Figure 1) was increased in TMZ-resistant gliomas and correlated with poor prognosis (32). Its knockdown abolished TMZ-resistance in U343-R and U251-R glioma cells and in corresponding xenografts in nude mice. Exosome-mediated transfer of circWDR62 increased proliferation, viability, invasion, and migration of U343 cells in vitro and enhanced their tumor volume after implantation as xenografts into nude mice (32). CircWDR62 sponged miR-370-3p and upregulated methyl-removing enzyme MGMT, which transfers methyl groups from adenine and guanine bases of DNA to cysteine residues in proteins (32, 33).

Figure 1.

Circular RNAs mediating temozolomide (TMZ) resistance in preclinical glioma-related in vivo models. The first column outlines the corresponding circRNA, the second column shows involved microRNAs or interacting proteins, and the third column refers to the affected target. ALDOA: Aldolase A; ALKBH5: ALKB homolog H5; circASAP1: circ ADPribosylation factor GTPase activating protein with SH3 domain, ankyrin repeat and PH domain 1; circCABIN1: circ calcineurin-binding protein cabin-1; circGLIS3: circ GLIS family zinc finger 3; circHIPK3: circ homeodomain-interacting protein kinase 3; circKIF4A: kinesin family member 4A; circNEIL3: Nei like DNA glycosylase 3; circVPS18: circ vacuolar protein sorting-associated protein 18 homolog; circWDR62: circ WD repeat domain 62; ITGα1: integrin α1; MED31: mediator complex subunit 31; MGMT: O6 methylguanine DNA methyltransferase; miR: microRNA; N-RAS: neuroblastoma RAS viral oncogene homolog; OLFML3: Olfactomedin-like 3; RUNX1: runt-related transcription factor 1; SPI-1: transcription factor SPI-1; U2AF2: U2 small nuclear RNA auxiliary factor 2; ZIC5: transcription factor zic family member 5.

Circ vacuolar protein sorting-associated protein 18 homolog (circVPS18) induces TMZ-resistance by upregulation of runt-related transcription factor 1 (RUNX1). CircVPS18 (Figure 1) was upregulated in TMZ-resistant glioma cell lines (34). Its knockdown in TMZ-resistant glioma cell lines U251TR and LN229TR restored TMZ sensitivity, induced apoptosis, repressed migration and invasion in vitro and enhanced TMZ sensitivity of corresponding xenografts in nude mice. CircVPS18 sponged miR-370 and upregulated transcription factor RUNX1 (34). It was shown that RUNX1 can induce Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling in gliomas (35) and increase expression of multidrug-resistance associated protein 1 (MRP1) in this tumor entity (36, 37).

Circ homeodomain-interacting protein kinase 3 (circHIPK3) mediates TMZ-resistance by upregulation of transcription factor zic family member 5 (ZIC5). CircHIPK3 (Figure 1) was increased in TMZ-resistant glioma cells and found in corresponding exosomes (38). Knockdown of circHIPK3 resolved TMZ-resistance and induced apoptosis in vitro in glioma cells and in corresponding xenografts in nude mice (38). CircHIPK3 sponged miR-421 and upregulated ZIC5, a member of a transcription factor family comprising five homologues (39). In glioma, ZIC5 has been shown to be involved in promotion of proliferation and migration (40).

Circ nei like DNA glycosylase 3 (circNEIL3) upregulates transcription factor SPI-1. Inhibition of circNEIL3 enhanced sensitivity of glioma cells to TMZ in vitro and in a glioma xenograft model (41). It has been shown that circNEIL3 interacts with U2 small nuclear RNA auxiliary factor 2 (U2AF2) which binds to SPI-1 mRNA, resulting in its stabilization and improved translation (41). It has been shown that SPI-1 (42) acts as a mediator of TMZ resistance (43).

Circ0072083 mediates TMZ resistance by upregulation of methyl-adenosin demethylase ALKB homolog H5 (ALKBH5). Circ0072083 (Figure 1) was increased in TMZ-resistant glioma cells as well as in TMZ-resistant glioma patients and its expression correlated with poor prognosis (44). Its knockdown attenuated TMZ-resistance of U251TR and U87TR glioma cells in vitro and in vivo after subcutaneous implantation into nude mice. Circ0072083 was also found in exosomes derived from these cell lines and promoted TMZ-resistance in vitro. Exosomes derived from U251TR and U87TR glioma cells injected intra-tumorally (i.t.) into U251 and U87 xenografts enhanced growth and TMZ-resistance. Circ0072083 sponged miR-1252 and upregulated ALKBH5 (44, 45) which demethylated N6-methyladenosine (m6A)-modified transcription factor NANOG mRNA, increasing its stability and expression. NANOG is a homeobox transcription factor which mediates stem-cell-like properties of glioma cells (46).

Circ ADP-ribosylation factor GTPase activating protein with SH3 domain, ankyrin repeat and PH domain 1 (circASAP1) mediates TMZ-resistance by up-regulation of neuroblastoma RAS viral oncogene homolog (N-RAS). CircASAP1 was upregulated in TMZ-resistant glioma cells and recurrent glioma tissues (47). CircASAP1 increased proliferation and migration in N3T3rd and U251rd glioma cell lines in vitro. In an intracranial xenograft model, depletion of circASAP1 restored sensitivity to TMZ of N3T3rd xenografts in nude mice. CircASAP1 sponged miR-502-5p, upregulated N-RAS and activated mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (47). N-RAS is overexpressed in glioma cell lines, glioma stem cells and glioma tissues (48). It has been shown independently that N-RAS enhances TMZ-resistance in glioma cells (49).

Circ calcineurin binding protein 1 (circCABIN1) upregulates olfactomedin-like 3 (OLFML3). CircCABIN1 was identified in exosomes secreted by TMZ-resistant glioma cells and can convey TMZ-resistance to TMZ-sensitive glioma cells. si-circCABIN1 sensitized TMZ-resistant glioma cells to TMZ in vitro and in an orthotopic glioma xenograft model in mice (50). CircCABIN1 sponged miR-637, resulting in the upregulation of the secreted protein OLFML3. The latter is a member of a family of five proteins comprising 13 isoforms, is located in the extracellular matrix (ECM) and promotes angiogenesis and pericyte coverage (51). It also stimulates erythroblastic oncogene B (ErbB) signaling as well as glioma cell stemness (51). Expression of OLFML3 was linked to a poor prognosis in patients with glioma (52).

Circ0110757 and circ0043949 induce TMZ-resistance by upregulation of integrin α1 (ITGα1). Circ0110757 (Figure 1) was more highly expressed in TMZ-resistant gliomas compared to TMZ-sensitive gliomas (53). Circ0110757 induced TMZ-resistance in U87 glioma cells in vitro and TMZ-resistance of this cell line after subcutaneous implantation into nude mice (53). It sponged miR-1298-5p, upregulated ITGα1 and activated the phosphoinositide 3-kinase (PI3K)/Ser/Thr kinase AKT/B-cell lymphoma 2 (BCL2) pathway. Circ0043949 (Figure 1) was also up-regulated in TMZ-resistant gliomas, induced proliferation, migration, invasion, TMZ resistance and inhibited apoptosis in glioma cells in vitro and in nude mice by sponging miR-876-3p and up-regulating ITGα1 (54).

Circ kinesin family member 4A (circKIF4A) upregulates aldolase A (ALDOA). Circ KIF4A promoted growth and TMZ resistance in A172, SHG-44 and U251 glioma cells in vitro and insubcutaneous and intracranial glioma xenograft models (55). Circ KIF4A sponged miR-335 and upregulated the glycolytic enzyme ALDOA. Glycolytic enzymes have significant potential for the treatment of gliomas (56).

Circ GLIS family zinc finger 3 (circGLIS3) mediates TMZ-resistance by upregulation of mediator complex subunit 31 (MED31). CircGLIS3 was upregulated in TMZ-resistant glioma cells, promoted proliferation, invasion, migration, and inhibited apoptosis in vitro and its knockdown attenuated growth of TMZ-resistant glioma xenografts in nude mice (57). It sponged miR-548m and upregulated MED31, a component of the mediator large complex required for basal and regulated expression of most RNA Pol II transcribed genes (57, 58).

CircRNAs Inducing Transmembrane Receptors and Encoded Receptor Variants With Efficacy In Preclinical In Vivo Models

Circ cyclin-dependent kinase 14 (circCDK14) upregulates platelet-derived growth factor receptor α (PDGFRA). CircCDK14 (Figure 2) was overexpressed in glioma patients and correlated with poor prognosis (59). CircCDK14 accelerated proliferation, migration, invasion, and inhibited ferroptosis in U87 and U251 glioma cells in vitro, while it promoted growth of U251 xenografts after subcutaneous implantation into nude mice. CircCDK14 sequestered miR-3938 and upregulated PDGFRA (59). Platelet-derived growth factor (PDGF) is expressed as five isoforms which interact with two receptors (PDGFRA and PDGFRB) (60, 61). Activation of the PDGF/PDGFR signaling pathway is associated with cancer proliferation, metastasis, invasion, and angiogenesis through modulation of multiple downstream pathways such as PI3K and MAPK/ERK signaling (60, 61). However, targeting of the PDGF/PDGFR pathway in glioma did not result in significant clinical benefit (62).

Figure 2.

CircRNAs upregulating transmembrane receptors and encoded receptor variants with efficacy in preclinical in vivo models. The first column outlines the corresponding circRNA, the second column shows involved microRNAs or interacting proteins, and the third column refers to the affected target. Downward arrow: downregulated. circARID1A: circ AT-rich interaction domain 1A; circEGFR: circ epidermal growth factor receptor; circCDK14: circ cyclin-dependent kinase 14; circMELK: circ maternal embryonic leucine zipper kinase; circMET: circ receptor tyrosine kinase MET; circNFIX: circ nuclear factor 1 X-type; circRPPH1: circ ribonuclease P RNA component H1; circRYK: circ receptor like tyrosine kinase; circSMO: circ smoothened; circTLK1: circ ser/thr-protein kinase tousled-like 1; circXRCC5: circ X-ray repair cross complementing 5; CIC-3: vesicular chloride transporter CIC-3; EPHB2: erythropoietin-producing hepatoma B2; ERBB4: transmembrane receptor tyrosine kinase ERBB4; huR: human antigen R; IGF1R: insulin-like growth factor receptor 1; IGF2BP2: insulin growth factor 2 binding protein 2; ITGB8: integrin β8; MAGT1: magnesium transporter 1; MET404: 404-aa MET variant; NCR3LG1: natural killer cell cytotoxicity receptor 3 ligand 1; NOTCH1: neurogenic locus notch homolog protein 1; NRX3: neurexin 3; PANX1: pannexin 1; PDGFRA: platelet-derived growth factor receptor α; RPN2: ribophorin 2; rtEGFR: rolling-translated EGFR variants; SDC1: syndecan 1; SMO-193: 193-aa protein derived from smoothened; TGFBR2: transforming growth factor beta receptor 2; VLDLR: very-low-density-lipoprotein receptor.

Circ0001162 and circ0001588 upregulate transmembrane receptor tyrosine kinase ERBB4. Circ0001162 (Figure 2) was highly expressed in glioma and mediated proliferation, colony-formation, invasion and migration of A172 and LN18 glioma cells in vitro and in vivo and in corresponding xenografts implanted subcutaneously into nude mice. Circ0001162 sponged miR-936 and upregulated ERBB4 (63). Expression of circ0001588 was closely related to tumor size and WHO grade of gliomas and stimulated cell proliferation, migration, invasion, tube formation, and decreased glioma cell apoptosis in vitro and accelerated growth of glioma xenografts in nude mice (64). Circ0001588 (Figure 2) sponged miR-1281 resulting in the upregulation of ERBB4, a member of the epidermal growth factor receptor family composed of four members: HER1 (EGFR, ERBB1), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4) (65). ERBB4 is highly expressed in the brain and has a critical role in brain development (65). ERBB4 interacts with numerous ligands such as heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin and neuregulins 3 and 4, and can activate MAPK and PI3K pathways (65, 66). ERBB4 has a tumor-promoting role in glioma (67).

Circ maternal embryonic leucine zipper kinase (circMELK) upregulates receptor tyrosine kinase erythropoietin-producing hepatoma B2 (EPHB2). CircMELK (Figure 2) was upregulated in glioma tissues and enhanced glioma mesenchymal transition and maintenance in vitro in U87 and pGBM-1 cells (68). CircMELK promoted growth of pGBM-1 xenografts after subcutaneous implantation into nude mice. From a mechanistic point of view, circMELK sponged miR-593 and upregulated EPHB2 (68). EPH receptor tyrosine kinases consist of 14 members which interact with membrane-bound ligands, referred to as ephrins. They can interact with five glycosyl-phosphatidyl-inositol (GPI)-linked type A ephrins and three transmembrane type B ephrins across cell membranes and are involved in bidirectional signaling which is unique to this system (69). Ephrin signaling is involved in cell adhesion, migration and axon guidance. Tumor-suppressing as well as tumor-promoting functions of ephrin receptors have been observed (69). It has been shown that EPHB2 promotes glioma invasion by phosphorylation of paxillin (70).

Circ0006168 upregulates insulin-like growth factor receptor 1 (IGF1R). Circ0006168 (Figure 2) was increased in glioma tissues and induced cell proliferation, migration, invasion, and expression of mesenchymal markers vimentin and SNAIL in A172 and LN229 glioma cells (71). Circ0006168 promoted growth of LN229 xenografts in nude mice after subcutaneous implantation. It sponged miR-628-5p and upregulated IGF1R (71). This receptor is part of a complex signaling system which consists of soluble ligands insulin-like growth factors-1 and -2 (IGF-1 and IGF-2), cell surface transmembrane receptors IGF1R and IGF-2 receptor (IGF2R) as well as six soluble IGF binding proteins (IGFBPs1-6) (72). In glioma, several components of the IGF-signaling system are linked to poor prognosis (73). However, numerous inhibitors of this pathway evaluated in clinical studies in several types of cancer did not achieve significant clinical benefit (74).

Circ receptor tyrosine kinase MET (circMET) encodes a 404 aa MET variant (MET404). High MET404 (Figure 2) expression predicted poor prognosis in glioma patients (75). CircMET promoted proliferation, invasion, migration, angiogenesis and neurosphere formation in GSC28 and GSC456 glioma cells in vitro and in vivo after intracranial implantation into nude mice. CircMET encoded secreted MET variant MET404 which activated the c-MET receptor by interaction with its ß subunit and formed a constitutively active c-MET receptor, which does not need activation by HGF (75). It has been shown that overexpression of c-MET is associated with poor prognosis in glioma patients (76).

Circ epidermal growth factor receptor (circEGFR) encodes rolling-translated EGFR variants (rtEGFR). Glioma patients with high circEGFR (Figure 2) exhibited worse overall survival (77). CircEGFR promoted proliferation and neurosphere formation of 456 and 4121 brain-tumor initiating cells (BTICs) and SW1783 and Hs683 glioma cells in vitro and tumorigenicity after intracranial implantation into nude mice. CircEGFR encoded several EGFR variants (rtEGFR) corresponding to 35 kD, 40 kD, 55 kD and 70 kD generated by rolling translation, while programmed -1 ribosomal frameshifting (-1 PRF) induced an out of frame stop codon polymeric protein complex referred to as rtEGFR. The latter sustained EGFR membrane localization and attenuated EGFR endocytosis and degradation via direct binding to EGFR (77). EGFR signaling plays an important role in the pathogenesis of glioma (78).

CircRNAs Affecting Intracellular Transmembrane Receptors Involved in N-glycosylation

Three circRNAs involved in protein N-glycosylation were identified. This emphasizes the role of this kind of protein modification in the pathogenesis of glioma. They affect two targets which are components of the oligosaccharyl-transferase complex.

Circ nuclear factor 1 X-type (circNFIX) and circ0037655 upregulate ribophorin 2 (RPN2). CircNFIX and circ0037655 (Figure 2) were upregulated in glioma cells and tissues and expression of circNFIX correlated with poor prognosis in glioma patients (79, 80). Both circRNAs upregulate RPN2 by sponging miRs-378e or -130-5p, respectively. CircNFIX promoted glycolysis, invasion, migration of T98 and U251 glioma cells in vitro and enhanced growth of corresponding orthotopic and subcutaneous xenografts in nude mice (79). Circ0037655 increased migration and invasion of glioma cells in vitro and tumor growth in vivo in nude mice (80). RPN2, the target of both circRNAs, is a transmembrane protein located in the membrane of the rough endoplasmic reticulum (ER) and is involved in co-translational N-glycosylation of proteins coded by the Asp-X-Ser/Thr aa motif by modifying the Asp residue (81). RPN2 is a subunit of the oligosaccharyl-transferase complex which is composed of eight subunits. RPN2 transfers oligosaccharides from a dolichole-oligosaccharide donor associated with the membrane of the ER to the sequence motif as described above in the lumen of the ER. Oligosaccharyl-transferase subunit STT3 acts as the catalytic subunit in this complex (81-83). RPN2-based N-glycosylation stimulates many signaling pathways resulting in proliferation, migration, invasion and multi-drug resistance. In addition, RPN2 inhibits apoptosis of many types of cancer cells and is associated with poor prognosis of several cancer entities (81). It has been shown that RPN2 can activate STAT3 (84) and wingless-related integration site (WNT) signaling (85) in glioma.

Circ002755 upregulates magnesium transporter 1 (MAGT1). Circ002755 (Figure 2) was upregulated in glioma cells and tissues and promoted glucose consumption, cell viability, migration and invasion of glioma cells in vitro. Circ002755 increased growth of glioma xenografts in nude mice (86). It sponged miR-628-5p and upregulated MAGT1 (86). The latter functions as a Mg2+ transporter in the ER. It contains four transmembrane domains and is a bona fide component of oligosaccharyl-transferase complex which catalyzes N-linked glycosylation (87). MAGT1 has been shown to be involved in glioma progression, to up-regulate programmed death-ligand 1 (PD-L1) through phosphorylation of ERK and to mediate growth and radio-resistance of glioma cells (88, 89).

CircRNAs Affecting Nerve Cell-related Receptors

Circ X-ray repair cross complementing 5 (circXRCC5) upregulates vesicular chloride transporter CIC-3. CircXRCC5 (Figure 2) was overexpressed in the tumors of patients with glioma and correlated with shorter survival time (90). CircXRCC5 promoted proliferation and NLR family pyrin domain containing 3 (NLRP3)-mediated inflammasome activation in glioma cells and growth of glioma xenografts in nude mice. CircXRCC5 bound to insulin growth factor 2 binding protein 2 (IGF2BP2), increasing the stability of CIC-3 mRNA (90). CIC-3, also known as CLC3 or CLCN3, functions as a voltage-gated chloride channel (91), increases proliferation, invasion and migration by regulating membrane ruffling and expression of integrins in glioma cells (92). Suppression of CIC-3 inhibits proliferation of glioma cells by inhibition of nuclear factor κB (NFκB) signaling (93). Glioma patients with high CIC-3 expression have an overall shorter survival time (93).

Circ0001367 upregulates neurexin 3 (NRX3). Albeit we focus on upregulated circRNAs in this review, we discuss Circ0001367 because it regulates a synapse-related protein. Circ0001367 (Figure 2) was downregulated in glioma tissues and suppressed proliferation, migration and invasion of LN229 and T98G glioma cells in vitro and tumor growth of LN229 xenografts in an intracranial xenograft glioma model in nude mice. Circ0001367 sponged miR-431 and upregulated NRX3 (94). This transmembrane protein is primarily localized in the presynaptic membrane and is involved in synapse development and function. It interacts with various ligands such as neuroligins in the postsynaptic membrane (95). NRX3 functions in excitatory as well as in inhibitory synapses of the central nervous system (CNS). Despite these proneuronal activities, NRX3 has been shown to act as a tumor suppressor by inhibiting proliferation and invasion of glioma cells (96).

CircRNAs Affecting Cell-Cell and Cell-Extracellular Matrix Connectivity

Circ nuclear factor 1 X-type (circNFIX) upregulates neurogenic locus notch homolog protein 1 (NOTCH1). CircNFIX (Figure 2) was overexpressed in glioma tissues compared to paired normal brain tissues (97). CircNFIX promoted cell migration, proliferation and inhibited apoptosis in U87 glioma cells in vitro and in corresponding xenografts after subcutaneous implantation into nude mice. It sponged miR-34a-5p and upregulated transmembrane protein NOTCH1 (97). The NOTCH signaling pathway consists of four members (NOTCH1-4), three delta-like ligands (DLLs) and two jagged (JAG) ligands (98, 99). After ligand-mediated stimulation, an intracellular domain of the NOTCH receptors is released from the transmembrane domain through cleavage by γ-secretase and is translocated into the nucleus to mediate transcription of a set of genes (98, 99). It has been shown that NOTCH1 is upregulated in glioma, promotes EMT, metastasis (100) and represents a marker for worse prognosis in glioma patients (101). Recently, the Food and Drug Administration (FDA) has approved nirogacestat, an oral γ-secretase inhibitor for treatment of desmoid tumors (102).

Circ ser/thr-protein kinase tousled-like 1 (circTLK1) upregulates pannexin 1 (PANX1). CircTLK1 (Figure 2) was upregulated in glioma tissues compared to adjacent normal tissues and its expression correlated with higher grade tumors (103). CircTLK1 overexpression promoted glioma growth, migration and invasion, inhibited apoptosis and stimulated PANX1/MAPK/ERK expression in vitro and in xenografts in nude mice (45). CircTLK1 sponged miR-17-5p and upregulated PANX1 (103). The latter is a member of a three-member protein family that forms heptameric channels belonging to the family of gap junction proteins. These channels are permeable to molecules such as ATP and are involved in propagation of Ca²⁺ waves (104, 105). PANX1 can exert tumor-suppressive as well as oncogenic functions (104, 105). Silencing of PANX1 inhibits proliferation of U87MG glioma cells (106) and PANX1 plays a role in the release of glutamate in U87 cells (107).

Circ ribonuclease P RNA component H1 (circRPPH1) upregulates syndecan 1 (SDC1). CircRPPH1 (Figure 2) was increased in glioma cells and tissues in comparison to healthy brain tissues (108). Knockdown of circRPPH1 led to decreased cell proliferation, migration and invasion, increased cell apoptosis and inhibited growth of glioma xenografts in nude mice. CircRPPH1 sponged miRs-627-5p and -663a, resulting in upregulation of SDC1 (108). Syndecans are type I cell surface receptors which interact with several ligands such as VEGFs, FGFs, TGFβ and are involved in organization of the ECM and the cytoskeleton (109, 110). SDC1 expression is related to poor prognosis in glioma patients and stimulates migration of U251 glioma cells (111).

CircRNAs Upregulating Other Types of Transmembrane Receptors

Circ receptor-like tyrosine kinase (circRYK) upregulates very-low-density-lipoprotein receptor (VLDLR). CircRYK (Figure 2) was elevated in glioma and correlated with poor prognosis (112). CircRYK induced glioma cell epithelial-mesenchymal transition (EMT) and glioma stem cell maintenance in pGBM-1 and U87 cells in vitro and tumor growth of pGBM-1 cells in an intracranial xenograft model in nude mice (112). CircRYK sponged miR-330-5p, resulting in the upregulation of VLDLR. In addition, circRYK bound to RNA-binding protein huR, leading to stabilization of VLDLR mRNA (112). The multifunctional protein VLDLR is a member of the low-density lipoprotein receptor family (113). It is involved in cholesterol uptake and can interact with numerous ligands. VLDLR is involved in endocytosis and regulation of cellular signaling pathways (113). VLDLR has been identified as a promoter of metastasis in several types of cancer by stimulating ß-catenin signaling (114). In addition, it was shown that VLDLR stimulates proliferation, migration and invasion of glioma cells (115).

Circ AT-rich interaction domain 1A (circARID1A) upregulates transforming growth factor beta receptor 2 (TGFBR2). CircARID1A (Figure 2) was upregulated in glioma cell lines and tissues and was also found in corresponding exosomes (116). Its knockdown inhibited glioma cell migration and invasion in U87 and U118 cells in vitro and growth of U87 xenografts after intracranial implantation in nude mice. CircARID1A sponged miR-370-3p and upregulated TGFBR2 (116). TGFβ binds to type II TGFBRs, bridges type I and type II TGFBR on the cell surface, and stimulates the ser/thr kinase activity of type I receptors. This results in translocation of intracellular effector SMAD proteins into the nucleus to regulate expression of dedicated genes. TGFβ suppresses the growth of normal cells; in cancer, however, it can promote proliferation and metastasis, in a context-dependent manner (117, 118). It has been shown that inhibition of TGFBRII reduces proliferation of glioma cells (119). TGFβ has been found to be deregulated in gliomas, but inhibitors of this pathway evaluated in clinical studies did not show significant beneficial effects in corresponding patients (120).

Circ0037655 upregulates integrin β8 (ITGB8). Circ0037655 (Figure 2) was upregulated in glioma cells and corresponding tumor tissues (121). In T98G and LN229 glioma cells, circ0037655 mediated proliferation, migration, invasion, EMT and promoted growth of LN229 xenografts after subcutaneous implantation into nude mice. Circ0037655 sponged miR-1229-3p and upregulated ITGB8 (121). In mammals, integrins are composed of 18 α and 8 β subunits, which can form 24 heterodimers that bind distinct ECM proteins and their signaling can affect all hallmarks of cancer (122). It has been shown that ITGB8 promotes proliferation, invasion, angiogenesis and inhibits ferroptosis in glioma cells (123, 124).

Circ smoothened (circSMO) encodes a 193-aa protein (SMO-193). Higher expression of circSMO (Figure 2) predicted worse prognosis and poorer survival in glioma patients (125). SMO-193 mediated self-renewal of 456 and 3691 glioma stem cells (CSC) in vitro and tumorigenicity in nude mice. SMO-193 directly interacted with SMO and promoted its activation and Hedgehog (Hh) signaling. It enhanced cholesterol modification of SMO and released SMO from inhibition by patched transmembrane receptors (125, 126). Hh signaling has been uncovered as a promoter of glioma pathogenesis (127).

Circ0000215 upregulates natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1). Circ0000215 (Figure 2) promoted proliferation and metastasis and inhibited apoptosis of glioma cells, and its knockdown decreased growth of glioma xenografts in nude mice (128). Circ0000215 sponged miR-1200 and upregulated NCR3LG1, also known as B7 homolog 6 (B7-H6). Knockdown of NCR3LG1 suppressed proliferation, migration, invasion, induced apoptosis and cell-cycle arrest in U87 and U251 glioma cells (128, 129). It has been shown that NCR3LG1 enhances glioma stem cell proliferation via MYC-mRNA-cap-methyltransferase (RNMT) signaling (130). On the other hand, it has been shown that NCR3LG1 activates NK cells by interaction with NKp30 pointing to a double-edged function of NCR3LG1 in cancer biology (131).

Circular RNAs Affecting Expression of Secreted Proteins

Secreted factors such as proteins, small molecules and nucleic acids have an impact on the pathogenesis of glioma (132). In this review we focus on secreted proteins which activate glioma-related autocrine and paracrine pathways (132).

Circ0073237 upregulates hepatoma-derived growth factor (HDGF). Circ0073237 (Figure 3) was overexpressed in glioma patients and enhanced viability of glioma cells in vitro and their growth as xenografts in nude mice. Circ0073237 sponged miR-345, resulting in enhanced expression of heparin-binding protein HDGF (133). The latter is overexpressed in many malignancies, and its expression correlates with worse prognosis (133). HDGF interacts with nucleolin in the plasma membrane of cancer cells and can activate MAPK and PI3K signaling and angiogenesis in different types of cancer (133, 134). In glioma, HDGF enhances cell growth, EMT and TMZ-resistance through AKT and TGFβ signaling, supporting its function as a neurotrophic factor (135). Furthermore, HDGF has been identified as a negative prognostic factor in glioma patients (136).

Figure 3.

CircRNAs upregulating secreted proteins with efficacy in preclinical in vivo models. The first column outlines the corresponding circRNA, the second column shows involved microRNAs or interacting proteins, and the third column refers to the affected target. circCDC45: Circ cell division cycle 45; circE-Cad: circE-cadherin; circHGF: circ hepatocyte growth factor; circLGMN: circ legumain; circPOSTN: circ periostin; circZNF652: circ zinc-finger protein 652; CSF1: colonystimulating factor 1; C-E-Cad: secretory E-cadherin protein variant; C-HGF: 119-aa HGF-variant; EDN1: endothelin 1; EGFR: epidermal growth factor receptor; HDGF: hepatoma-derived growth factor; LGMN: legumain; PAI-1: plasminogen activator inhibitor-1; SERPINE1: serpin family E member 1; SPARC: secreted protein acidic and rich in cystein.

Circ cell division cycle 45 (circCDC45) upregulates colony-stimulating factor 1 (CSF1). CircCDC45 (Figure 3) was highly expressed in glioma cells and corresponding tissues (137). Downregulation of circCDC45 blocked proliferation, invasion and migration in A172 and LN229 glioma cells in vitro and in LN229-derived xenografts aftersubcutaneous implantation into nude mice. CircCDC45 sponged miR-485-5p and upregulated CSF1 (137). It was shown independently that CSF1 promotes proliferation, invasion and migration of U81 and U251 glioma cells (138) and stimulates formation of glioma-associated microglia and macrophages without modulating their polarization status (139). Due to these properties, the CSF1/CSF1R system should be scrutinized in further detail for its role as a potential target for the treatment of glioma (140).

Circ101491 upregulates endothelin 1 (EDN1). Circ101491 (Figure 3) overexpression correlated with poor prognosis in glioma patients (141). Circ101491 was also found in exosomes derived from U251 cells, promoting viability, migration and invasion of U87 and U118 glioma cells. Overexpression of circ101491 in U251 cells stimulated tumor growth aftersubcutaneousimplantation into nude mice (141). U118 cells treated with exosomes derived from U251 cells exhibited increased lung metastasis after tail vein injection into nude mice. From a mechanistic point of view, circ101491 sponged miR-125b-5p and upregulated EDN1, a 21-aa peptide with vascoconstrictive function (141, 142). EDN1 is a member of a family of three isoforms. EDN1 interacts with two G-protein coupled receptors (GPCRs), referred to as ETRA and ETRB. EDN1 is involved in signal transduction via β-arrestin 1 and 2 (143). In glioma, EDN1 has been identified as a mitogenic, angiogenic and anti-apoptotic peptide, and its receptors are highly expressed on glioma endothelial cells (144).

Circ zinc-finger protein 652 (circZNF652) upregulates serpine family E member 1 (SERPINE1). High expression of circZNF652 (Figure 3) correlated with poor prognosis in glioma patients (145). Suppression of circZNF652 in U87 and A172 glioma cells inhibited cell growth, invasion, migration and EMT in vitro and growth of A172 xenografts after subcutaneous implantation into nude mice. CircZNF652 sponged miR-486 and upregulated SERPINE1, also known as plasminogen activator inhibitor-1 (PAI-1) (145). This protein acts as an inhibitor of plasminogen activation through binding to urokinase (uPA) and tissue-type plasminogen activator (t-PA). Therefore, the upregulation of SERPINE1 in glioma might appear to be counterintuitive, but it has been shown that PAI-1 exerts additional functions, such as interacting with ECM protein vitronectin and mediating endocytosis of receptors of the low-density lipoprotein receptor family and playing a role in tumor vascularization (146-149). Monoclonal antibodies (mABs) directed against PAI-1 have been shown to interfere with tumor growth in a preclinical in vivo model (150). Furthermore, PAI-1 increases the expression of VEGF in glioma cells (151). In addition, it was shown that plasmin had an anti-metastatic effect on brain colonization of breast- and lung-cancer cells (152). This was due to cleavage of astrocytic FAS ligand (FASL), with a paracrine death signal for cancer cells and inactivation of e L1 cell adhesion molecule (L1CAM), which is expressed by metastasizing cells for spreading along brain capillaries and metastatic outgrowth (152).

Circ legumain (circLGMN) upregulates protease legumain (LGMN). CircLGMN (Figure 3) was highly expressed in glioma patients and was associated with poor prognosis (153). In U87-MG and U251-MG glioma cells, circLGMN promoted cell proliferation, colony formation and cell migration in transwell- and wound-healing assays in vitro. CircLGMN stimulated growth of intracranial U87-MG xenografts in nude mice by sponging miR-127-3p and upregulation of LGMN. The latter is a member of the family of cysteinyl proteases and acts as an asparaginyl endopeptidase (154). LGMN is expressed in tumor cells as well as in macrophages and is an important mediator of angiogenesis, invasion and metastasis due to intracellular and extracellular functions (154). In addition to its protease activity, LGMN can also exert functions as a ligase (155). It has been shown that LGMN can mediate activation of matrix metalloproteinases MMP2 and MMP9 and is able to cleave extracellular fibronectin in the context of ECM remodeling (156). In glioma, LGMN promotes polarization of tumor-associated macrophages to the immunosuppressive M2-type via activation of the glycogen synthase kinase-3β (GSK-3β)-STAT3 signaling pathway (157).

Circ periostin (CircPOSTN) upregulates secreted protein acidic and rich in cysteine (SPARC). CircPOSTN (Figure 3) was upregulated in glioma cells and tissues and its knockdown inhibited clonogenicity, migration, promoted apoptosis in glioma cells and decreased growth of glioma xenografts in nude mice. CircPOSTN sponged miR-433-3p and upregulated SPARC, a calcium-binding protein, also known as osteonectin (158). SPARC is a 40 kD acidic cysteine-rich glycoprotein which is also secreted by osteoblasts. It is a multifaceted protein located in the ECM that can bind to collagen and promote bone formation (159). SPARC increases production and activity of MMPs and is highly expressed in metastatic tumors (159). SPARC has been identified as a marker of glioma progression (160). Inhibition of SPARC in glioma cells attenuates focal adhesion kinase (FAK) and integrin-linked kinase (ILK) activity (161). Downregulation of SPARC also inhibits migration and invasion of glioma cells (162).

Circ hepatocyte growth factor (circHGF) encodes a 119-aa HGF-variant (C-HGF). CircHGF (Figure 3) was highly expressed in gliomas compared to normal adjacent tissue (163). C-HGF possesses a unique 49-aa terminal sequence formed by a ribosomal frameshift, interacts with c-MET and activates STAT3, AKT and ERK signaling (163). CircHGF promoted proliferation, invasion and migration of GBM6 and HK296 PDX glioma cells in vitro. Furthermore, circHGF mediated growth of intracranially implanted HK296 PDX in nude mice (163). It has been shown that c-MET/HGF signaling is one of the driving pathways for the pathogenesis of glioma (164).

CircE-cadherin (circE-Cad) encodes a secretory E-cadherin protein variant (C-E-Cad). CircE-Cad (Figure 3) was overexpressed in glioma and promoted glioma stem cell tumorigenicity (165). C-E-Cad mediated EGF-independent activation of EGFR signaling by directly interacting with EGFR through a unique 14-aa C-terminus. Inhibition of circE-Cad enhanced the anti-tumor activity of EGFR inhibitors in glioma models (165). Deregulation of EGFR signaling has been identified as a hallmark of glioma (78).

CircRNAs Regulating WNT Signaling

WNT signaling has an important role during early development, is conserved across species and is deregulated in many types of cancer. The interaction between WNT ligands and frizzled (FZD) receptors or other co-receptors can induce canonical or non-canonical WNT signaling (166). Canonical WNT signaling results in activation of transcriptional programs due to translocation of β-catenin into the nucleus, whereas non-canonical pathways lead to activation of cellular actin and have an impact on the plasticity of the cytoskeleton (167). WNT signaling is a key driver of glioma proliferation and progression, invasion, TMZ and radiotherapy resistance, as well as glioma stem cell maintenance and proliferation (168, 169).

Circ0082375 upregulates wingless-type MMTV integration site family 7B (WNT7B). Circ0082375 (Figure 4) was upregulated in glioma tissues and its expression correlated with poor prognosis (170). Circ0082375 promoted proliferation, invasion, migration, EMT, and glycolysis, while it inhibited apoptosis in LN229 and U251 glioma cells. Knockdown of circ0082375 hampered growth of U251 xenografts after subcutaneous implantation into nude mice. Circ0082375 sponged miR-485-5p and upregulated WNT7B. Co-culturing of LN229 and U251 cells with human umbilical vein endothelial cells (HUVECs) increased angiogenesis mediated by the expression of circ0082375 (170). WNT7B is one of 19 WNT ligands which can interact with 10 FZD receptors to initiate canonical WNT signaling (168, 169). WNT7B has been shown to promote proliferation and invasion of glioma cells (171).

Figure 4.

CircRNAs regulating wingless-related integration site (WNT) signaling with activity in preclinical in vivo models. The first column outlines the corresponding circRNA, the second column shows involved microRNAs or interacting proteins, and the third column refers to the affected target. circAHCY: Circ adenosylhomocysteinase; circEXOC6: circ exocyst complex component 6; circMMD: circ monocyte to macrophage differentiation associated; DVL1: dishevelled homolog 1; EIF4A3: eukaryotic translation initiation factor 4A3; FIR: FBP-interacting repressor; FUBP1: far upstream element binding protein 1; FZD6,7: frizzled 6,7; TCF4: transcription factor 4; WNT7B: MMTV integration site family 7B.

Circ exocyst complex component 6 (circEXOC6) upregulates frizzled 6 (FZD6). CircEXOC6 (Figure 4) expression was elevated in glioma cells and tissues. Knockdown of circEXOC6 inhibited proliferation, migration, invasion, glycolysis, and facilitated apoptosis of A172 and U251 glioma cells (172). Co-culturing of these cells with the human brain microvascular endothelial cell line HCMEC/D3 induced circEXOC6-mediated tube formation and angiogenesis. CircEXOC6 stimulated growth of A172 xenografts after subcutaneous implantation into nude mice. It sponged miR-433-3p and upregulated FZD6 (172). It has been shown independently that FZD6 promotes proliferation of glioma and correlates with poor prognosis (173).

Circ monocyte to macrophage differentiation associated (circMMD) upregulates FZD6 and segment polarity protein dishevelled homolog 1 (DVL1). Expression of circMMD (Figure 4) was elevated in glioma and its high expression indicated poor prognosis (174). In DBTRG and U251 glioma cells, circMMD promoted proliferation in vitro. Knockdown of circMMD reduced growth of DBTRG subcutaneously and intracranially implanted xenografts in nude mice (174). This was due to the downregulation of FZD6 and DVL1. FZD6 was upregulated by circMMD through sequestration of miR-15b-5p (174). DVL1 was induced by the binding of circMMD to far upstream element binding protein 1 (FUBP1) (175), resulting in the dissociation of the FUBP1/FBP-interacting repressor (FIR) complex which binds to an upstream element of the DVL1 gene. FUBP1/FIR interaction has been shown to inhibit transcription of MYC (176). DVL1 is an essential component for WNT signaling and acts through inhibition of ß-catenin degradation (177).

Circ0000177 up-regulates frizzled class receptor 7 (FZD7). Circ0000177 (Figure 4) was upregulated in glioma cell lines and tissues and was associated with poor prognosis in glioma patients (178). Circ0000177 knockdown inhibited glioma cell proliferation and invasion in vitro and as xenografts in vivo in nude mice. Circ0000177 sponged miR-638 and upregulated FZD7 (178). It has been shown that overexpression of FZD7 promotes glioma cell proliferation by upregulating the transcriptional co-activator with PDZ-binding motif (TAZ), a downstream effector of WNT signaling (179).

Circ adenosylhomocysteinase (circAHCY) upregulates transcription factor 4 (TCF4) and β-catenin. CircAHCY (Figure 4) was highly expressed in glioma cell lines, stimulated proliferation and inhibited apoptosis in A172 and LN229 glioma cells (180). Exosomal circAHCY derived from glioma cells increased proliferation of astrocytic NHA cells. CircAHCY also stimulated the growth of LN229 xenografts in nude mice. CircAHCY transcriptionally activated the β-catenin gene by sponging miR-1294 resulting in the upregulation of the MYC oncogene. Furthermore, circAHCY recruited eukaryotic translation initiation factor 4A3 (EIF4A3) to upregulate TCF4 by stabilization of its mRNA and by inducing the formation of TCF4/β-catenin complexes (180). The latter are mediators of the transcriptional program induced by WNT signaling (170, 181). Currently, many new approaches targeting β-catenin are under preclinical and clinical evaluation in cancer-related indications (182). They involve stabilized peptides, condensate modulators and degraders (182).

CircRNAs Involved in Cytoskeleton Dynamics, Microtube Formation and Function, and Cell-Cell Interactions in Gliomas

Cytoskeleton-assisted interactions result in migration, invasion and brain colonization of gliomas (183). These processes are mediated by actin filaments, intermediate filaments (IFs), microtubes (MTs) and TNTs (184-187). MTs and TNTs connect multiple glioma cells forming a syncytium transmitting rhythmic calcium signals from pacemaker cells to other glioma cells of the corresponding network (185, 186). MTs and TNTs are composed of actin, myosin IIA, tubulins, cellular organelles and vesicles and other functionally important proteins such as growth-associated protein 43 (GAP43) and connexin 43 (Cx43). MTs and TNTs also mediate therapy resistance by making cells of the syncytium less sensitive to drugs than individual cells (185, 186). Tight junctions, desmosomes, hemidesmosomes, gap junctions as well as adherens junctions and lamellopodia are the main elements facilitating the aggressiveness of glioma (184-187). Therefore, interference with their functions might lead to control of growth and local spread of gliomas (187).

Circ GLIS family zinc finger 3 (circGLIS3) stabilizes EZRIN. CircGLIS3 (Figure 5) was highly expressed in high-grade glioma and promoted migration and invasion of U87 and U251 glioma cells in vitro and in an intracranial xenograft model in nude mice with no impact on tumor volume (188). CircGLIS3 could be excreted by glioma cells through exosomes and induce angiogenesis. CircGLIS3 is bound to phosphorylated EZRIN and is elevated in glioma cells (188). EZRIN cross-links the actin cytoskeleton to the plasma membrane and has been identified as a mediator of invasion and metastasis in glioma (189).

Figure 5.

CircRNAs involved in cytoskeleton dynamics, microtube formation and function, cell-cell interactions and other cellular functions with efficacy in preclinical in vivo systems. The first column outlines the corresponding circRNA, the second column shows involved microRNAs or interacting proteins, and the third column refers to the affected target. circGLIS3: Circ GLIS family zinc finger 3; circPITX1: circ paired-like homeodomain 1; CircPOSTN: circ periostin; circRFX3: circ regulatory factor X3; circSEPT9: circ septin 9; CircU2AF1: circ U2 small nuclear RNA auxiliary factor 1; circVPS18: circ vacuolar protein sorting-associated protein 18; BCAT1: branched-chain amino acid transaminase 1; B7-H6: B7-homolog 6; CAPG: capping actin protein, gelsolin-like; DICER: endoribonuclease DICER; EIF4A3: eukaryotic translation initiation factor 4A3; KIF1B: kinesin family member 1B; KPNB1: karyopherin subunit beta 1; ITGB8: integrin β8; LASP1: LIM and SH3 protein 1; NFATC3: nuclear factor of activated T cells 3; NOVA2: neuro-oncological ventral antigen 2; RBM3: RNA-binding motif 3 protein; ROCK1: rho-associated coiled-coil containing protein kinase 1; TPX2: kinesin-like protein 2; VASP: vasodilator-stimulated phosphoprotein.

Circ0079593 upregulates rho-associated coiled-coil containing protein kinase 1 (ROCK1). Circ0079593 (Figure 5) was elevated in glioma and promoted proliferation and metastasis, while it inhibited apoptosis of glioma cells in vitro and stimulated tumor growth of corresponding xenografts in nude mice (190). It also inhibited the glioma-suppressive effects of the inhalational anesthetic sevoflurane. Circ0079593 sponged miR-633 and upregulated ROCK1 (190). The latter acts as downstream effector of GTPase RHOA and is involved in cytoskeleton organization and actomyosin contractility (191). It has been shown independently that ROCK1 promotes invasiveness of glioma cells (192).

Circ regulatory factor X3 (CircRFX3) upregulates vasodilator-stimulated phosphoprotein (VASP). CircRFX3 (Figure 5) levels were increased in glioma tissues and cells; circRFX3 stimulated proliferation, migration, invasion and inhibited apoptosis in A172 and U251 glioma cells in vitro. It promoted growth of A172 xenografts after subcutaneous implantation into nude mice. CircRFX3 sponged miRs-1179 and -1229 and upregulated VASP (193). The latter is an actin-binding protein which is involved in cytoskeletal regulation (194). VASP has been shown to affect lamellipodia formation which plays a pivotal role in cell migration (195). It has been demonstrated that VASP mediates proliferation, invasion and migration of glioma cells and is associated with poor prognosis in glioma patients (196).

Circ0055412 upregulates capping actin protein, gelsolin-like (CAPG). Circ0055412 (Figure 5) was upregulated in glioma and recruited EIF4A3 to stabilize CAPG mRNA (197). Circ0055412 also served as a sponge for miR-330-3p and upregulated nuclear factor of activated T cells 3 (NFATC3) to induce the transcription of ß-catenin, resulting in activation of WNT/β-catenin signaling. Knockdown of circ0055412 enhanced cis-platin sensitivity of glioma cells in vitro and in nude mice (197). CAPG functions as an actin-binding protein of the gelsolin superfamily, promotes growth and metastasis of glioma cells and its expression is associated with poor prognosis in glioma patients (198, 199).

Circ septin 9 (circSEPT9) upregulates LIM and SH3 protein 1 (LASP1). CircSEPT9 (Figure 5) was highly expressed in glioma tissues and cell lines, promoted proliferation, migration, invasion and glycolysis in vitro as well as growth of corresponding xenografts in nude mice. CircSEPT9 sponged miR-432-5p and upregulated LASP1 (200). This protein has F-actin binding properties and is involved in chemokine receptor and PI3K/AKT signaling (201). In glioma, it has been shown that LASP1 mediates proliferation, invasion, EMT and PI3K/AKT signaling (202, 203).

CircPOSTN upregulates kinesin family member 1B (KIF1B). CircPOSTN (Figure 5) was highly expressed in glioma tissues and cells and its knockdown inhibited glioma cell proliferation, migration and invasion in vitro, as well as growth of corresponding xenografts in nude mice. CircPOSTN sponged miR-185-5p and upregulated KIF1B (204). This protein is part of the family of myosin motors which move on microtubules and form a dynamic network between cellular membrane compartments and the cytoskeleton (205). KIF1B promotes glioma invasion by facilitating surface localization of membrane-type matrix metalloproteinase 1 (MT1-MMP), and its expression is associated with worse prognosis in glioma patients (206).

circPOSTN upregulates Xenopus kinesin-like protein 2 (TPX2). CircPOSTN (Figure 5) was overexpressed in glioma tissues and cells and its knockdown repressed proliferation and aerobic glycolysis, while it induced apoptosis in LN229 and U251 glioma cells and inhibited growth of LN229 xenografts after subcutaneous implantation into mice. CircPOSTN sponged miR-361-5p and upregulated TPX2 (207). The latter has been identified as a microtubule-associated protein which is required for the formation of the mitotic spindle (208). TPX2 promotes glioma cell proliferation and invasion via activation of the AKT signaling pathway (209) and has been shown to correlate with poor prognosis in glioma patients (210).

CircRNAs Regulating Components οf Further Categories

Circ vacuolar protein sorting-associated protein 18 (circVPS18) upregulates branched-chain amino acid transaminase 1 (BCAT1). CircVPS18 (Figure 5) was highly expressed in glioma cell lines and tissues (211). CircVPS18 knockdown inhibited glioma progression by decreasing cell proliferation, migration, invasion, and angiogenesis, and by promoting apoptosis; it attenuated glioma xenograft growth in nude mice. CircVPS18 sponged miR-1229-3p and upregulated BCAT1 (211). Branched-chain amino acids play a crucial role in the reprogramming of tumor metabolism (212). BCAT1 has been shown to promote proliferation and invasion of glioma cells in vitro by promoting secretion of glutamate (213, 214).

Circ paired-like homeodomain 1(circPITX1) upregulates karyopherin subunit beta 1 (KPNB1). CircPITX1 (Figure 5) was overexpressed in glioma tissues and its knockdown suppressed proliferation, angiogenesis, migration, invasion, cell-cycle progression of glioma cells in vitro and growth of corresponding xenografts in nude mice. CircPITX1 sponged miR-584-5p and upregulated KPNB1 (215). This protein is involved in the import of proteins from the cytoplasm to the nucleus by interacting with phenylalanine-glycine repeats of nucleoporins which are functional constituents of the import complex (216). It has been shown that KPNB1 regulates proliferation of human glioma cells via WNT/β-catenin pathway signaling (217).

Circ2082 affects the cellular localization of endoribonuclease DICER. Circ2082 (Figure 5) was overexpressed in glioma cell lines and tissues, promoted growth of glioma-stem cells (GSC) as neurospheres and as xenografts after intracranial implantation into nude mice (218). It was shown that the miR-RNAome was suppressed in GSC due to localization of the miR maturation endonuclease DICER (218, 219) to the nucleus instead of to the cytosol (218). Circ2082 formed a complex with DICER and RNA-binding motif 3 protein (RBM3) in the nucleus (218-220). These findings support that inhibition of miR-maturation can increase oncogenicity of GSCs. The implications of these findings from a general perspective with respect to pathogenesis of glioma remain to be investigated in further detail.

Circ U2 small nuclear RNA auxiliary factor 1 (circU2AF1) upregulates neuro-oncological ventral antigen 2 (NOVA2). CircU2AF1 (Figure 5) was upregulated in glioma cells and tissues, promoted proliferation, invasion, and migration, inhibited their apoptosis in vitro,and promoted the growth of corresponding xenografts after subcutaneous implantation into nude mice. CircU2AF1 sponged miR-7-5p and upregulated NOVA2 (221). The latter is a member of a family of two alternative splicing factors which are expressed in neurons and are essential for angiogenesis (222). It has been shown that NOVA2 controls unique RNA splicing programs in inhibitory and excitatory neurons (223).

Technical Issues

For therapeutic intervention, upregulated circRNAs can be inhibited with antisense oligonucleotides (ASOs), or as demonstrated more frequently, by siRNA (224). siRNAs are 21-23 nt long and exhibit better BBB permeability than ASOs, which are substantially longer. Furthermore, it has been shown that circRNAs can be knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13 (CAS13) technology (225, 226). However, there are many urdles inherent to these therapeutic approaches such as stability of the corresponding agents, organ-specific delivery, renal clearance, immunogenicity, off-target gene silencing, pharmacokinetic and pharmacodynamic parameters, as well as toxicity (227, 228). These issues are not discussed in any further detail here.

In the case of glioma therapy, delivery of circRNA targeting agents to the brain is hampered by the BBB, which is composed of brain capillary endothelial cells, pericytes and astrocytes, and constitutes a highly fenestrated network (229). In addition to intrathecal and intraventricular administration of drugs to the brain, successful targeting of the BBB has been demonstrated. Angiopep2 has been identified as an important mediator of brain delivery by covalent linkage to siRNA or by decoration of nanoparticles with this peptide (230, 231). Angiopep2 is an 18aa peptide which binds to the low-density lipoprotein receptor-related protein 1 (LRP1) and delivers the corresponding therapeutic agents to the brain after transcytosis (232). With this technology, glioma-relevant therapeutic targets such as VEGF and EGFR have been inhibited in orthotopic glioma models in mice (232). Furthermore, Angiopep2-decorated cationic nanoparticles have been shown to deliver a suicide gene into gliomas in an orthotopic glioma model (233). Transferrin receptor (TfR), insulin receptor and leptin receptor are also targets for agents delivering payloads into the brain parenchyma (229). A bispecific antibody directed against TfR and Alzheimer’s β-site amyloid precursor protein-cleaving enzyme (BACE1) was shown to act as a brain shuttle (234). However, all of these technologies have to be continuously optimized with respect to efficacy of delivery, minimization of toxicity alerts and to be extended by new delivery technologies.

Concluding Remarks

In this review, we have focused on upregulated circRNAs which mediate in vivo efficacy in preclinical models. We have covered the following target classes and functional characteristics: TMZ-resistance, transmembrane proteins of the plasma membrane and the endoplasmic reticulum, secreted proteins, WNT-signaling, cytoskeletal and microtube-related, as well as selected circRNAs with functions that do not align with the outlined categories. We did not focus on circRNAs that target transcription factors, components of the RNA processing and ubiquitination system. However, in selected examples, transcriptional components and regulators such as RUNX1, ZIC5, SPI-1, TCF/ßcatenin and DICER were discussed.

The data reveal that TMZ-resistance can be mediated by a plethora of mechanisms (Figure 1). Notably, three of the identified circRNAs affect N-glycosylation through components of the oligosaccharyl-transferase system (Figure 2). Since growth, invasion and therapy-resistance of gliomas are dependent on TMs and TNTs, synaptic signaling, paracrine interactions between glioma cells and synapses as well as glioma cell/ECM adhesion, the corresponding identified circRNAs and targets should be scrutinized further with respect to their role as potential therapeutic targets. Examples are circXRCC5 which affects chloride transporter CIC-3, and circTLK1 which upregulates the expression of the pore-forming protein PNX1. Another example is circRPPH1 which promotes interactions of glioma cells with SDC1 and the ECM (Figure 2). Furthermore, G-protein coupled receptor GPR133 has been identified as target upregulated by circRNA (235). Secreted targets affected by circRNAs such as HDGF, CSF-1, EDN1, SERPINE1 and SPARC (Figure 3) deserve further preclinical validation. In addition, circRNA 005951, affecting heparin-binding epidermal growth factor (HBEGF) has been identified (236). RNA steady-state levels for selected genes encoding transmembrane receptors and secreted proteins based on data derived from The Cancer Genome Atlas (TCGA) database confirmed overexpression in gliomas and glioblastomas in comparison to normal brain tissues (Figure 6).The same holds true for identified targets of the WNT pathway such as WNT7B and FZD6, 7 (Figure 4). Recently it has been shown that FZD6 upregulated by a circRNA can confer resistance to TMZ demonstrating a connection between WNT signaling and TMZ resistance (237). Since cytoskeletal dynamics and connectivity among tumor cells themselves and with cells of the TME, such as nerve cells, are hallmarks of glioma, targets such as ROCK1, EZRIN, VASP, CAPG and LASP1 (Figure 5) and their corresponding circRNAs should be further validated with respect to functional implications and tractability. Also, cytoskeleton associated protein 2 like (CKAP2L) has emerged as a circRNA-regulated target (238). The dependence of glioma on branched-chain amino acids, BCAT1 and its corresponding circVPS18 should be evaluated in further detail for glioma treatment. Identification of glioma subtypes that depend on the mislocalization of DICER to the nucleus resulting in an aberrant oncogenic transcriptional program caused by circ2082 (Figure 5) might lead to new approaches for the treatment of glioma.

Figure 6.

Expression of 28 genes encoding transmembrane receptors and secreted proteins in normal brain and glioma datasets. Uniformly processed RNA-seq data were obtained via recount3 (246) for Genotype-Tissue Expression Project (GTEx) normal brain (n=2391), TCGA low-grade glioma (LGG) (n=532), and TCGA glioblastoma multiforme (GBM) (n=175), datasets. Gene transcription levels were converted to transcripts per million (TPM) and plotted as log2(TPM+1). Each facet represents one gene, and boxplots summarize the distribution across samples. The black line in the boxplot indicates the median value and the black rectangles represent the interquartile range (IQR) encompassing the middle 50% of the data range. Whiskers extend to the most extreme data points within 1.5 times the IQR from the quartiles, excluding outliers, while overlaid points indicate individual specimens. Statistical significance was assessed using pairwise Wilcoxon rank-sum tests between groups for each gene. p-Values were adjusted using Benjamini-Hochberg FDR correction across all tested gene-group contrasts. *q<0.05, **q<0.01, ***q<0.001, and ****q<0.0001. Non-significant pairs are unmarked. Independent y-axis scaling was used to accommodate gene-specific expression ranges. The figure enables comparison of transcript abundance between non-neoplastic brain tissue and glioma subtypes.

However, the studies discussed have important limitations. These include small patient cohorts, reliance on cell lines such as U87, U251, LN299 and A172 that may not capture modern glioma subtypes. In addition, subcutaneous xenografts poorly model brain invasion and immune microenvironment.

Recent strategies for glioma treatment combine standard therapy with compounds such as perampanel, which affects the cancer-neuron synapse by inhibiting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and compounds that interfere with the tumor-tumor and TME network such as gap junction inhibitor meclofenamate. These drugs are currently being evaluated in clinical trials in glioma patients (239-243). Therapy with genetically modified immune cells such as CAR- and NK-cell based therapies or other immunology-based interventions have given rise to promising therapeutic responses in glioma patients and may have a significant impact on its treatment. For translational glioma-related therapeutic approaches one should keep in mind that enhanced neuronal connectivity leads to regional immuno-suppression (244).

The recent identification of circ0059914, which upregulates VEGFA, the target of glioma-approved bevacizumab, validates the approach of target identification by scrutinizing the roles of circRNAs in the pathogenesis and progression of glioma (245).

Conflicts of Interest

SN is and UHW was an employee of Roche.

Authors’ Contributions

SN and UHW equally contributed to all aspects of the paper.

Artificial Intelligence (AI) Disclosure

During the preparation of this manuscript, a large language model (ChatGPT 5.5, OpenAI) was used solely for language editing and stylistic improvements in select sections. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified by generative AI or machine learning–based image enhancement tools.