Abstract
In order to identify new treatment modalities and targets for the treatment of bladder cancer (BLC), we have searched the literature (PubMed) for circular RNAs (circRNAs) that mediate efficacy in preclinical BLC-related in vivo systems. Pathogenesis-affecting circRNAs can be up-regulated or down-regulated depending on their function as oncogenes or tumor suppressors. We have grouped the identified circRNAs according to functional aspects or protein categories, such as involvement in drug resistance, transmembrane proteins, secreted proteins, mediators of signaling, enzymes with pathogenic potential, transcription factors, as well as circRNAs involved in microRNA (miR) processing and epigenetic modifications. The identified up-regulated targets can be modulated with small molecules or antibody-based drugs depending on their druggability. Down-regulated circRNAs can potentially be reconstituted by replacement therapy, whereas up-regulated circRNAs can be inhibited by nucleic acid (NA)-based inhibitors. The validity of the approach of exploring circRNAs and their corresponding targets for therapeutic intervention was underlined by the identification of circRNAs that up-regulate fibroblast growth factor receptors, which can be inhibited by erdafitinib, an approved agent for the treatment of bladder cancer.
- Antibody-based drugs
- reconstitution therapy
- regulatory RNA
- small molecule inhibitors
- target validation
- xenografts
- review
Introduction
Bladder cancer (BLC) is the tenth most prevalent malignancy, with approximately 570,000 annual new cases and 210,000 deaths worldwide (1). Ninety-five percent of BLCs are derived from urothelial epithelial cells, and non-muscle invasive (NMIBC) and muscle-invasive bladder cancers (MIBC) have been identified (2). Transcriptional profiling has revealed three subtypes of NMIBC and six subtypes of MIBC (3). Activating mutations have been found in telomerase reverse transcriptase (TERT), fibroblast growth factor receptor 3 (FGFR3), tumor protein 53 (TP53), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and cohesin complex component STAG2, and frequently, de-regulation of enzymes involved in chromatin modification has been observed (4). In addition to chemotherapy and immunotherapy with Bacillus Calmette-Guérin (BCG) vaccine for NMIBC, several new therapeutic agents have been approved for the treatment of MIBC. These include erdafitinib, a pan fibroblast growth factor receptor (FGFR) inhibitor, immune checkpoint inhibitors, and antibody-drug conjugates (5). The immune checkpoint inhibitory agents target programmed cell death protein 1 (PD1) and programmed cell death ligand 1 (PD-L1) and include monoclonal antibodies (mAbs) such as Keytruda (Pembrolizumab), which is directed against PD1, and Nivolumab (Opdivo), which targets PD-L1 (6). Additionally, two antibody-drug conjugates (ADCs) have been approved: Enfortumab vedotin (EV), which targets the transmembrane protein nectin 4 (7), and Sacituzumab govitecan (SG), which is directed against trophoblast antigen 2 (TROP2) (8). However, the therapeutic benefit is limited; the expression of the corresponding antigens does not necessarily correlate with response, and the identification of biomarkers correlating with therapy response is still pending (9, 10). Taken together, the identification of new targets and treatment modalities for BLC is an important medical issue. For these reasons, we have searched PubMed for circRNAs that are deregulated in BLC and mediate efficacy in preclinical in vivo models of BLC. The role of circRNAs in BLC as prognostic markers and potential therapeutic targets has also been discussed in (11, 12), but in this review, we focus on the role of circRNAs as therapeutic targets in BLC.
Circular RNAs
CircRNAs are expressed in normal and pathologic tissues in a cell-type and tissue-specific manner and are involved in a plethora of physiological functions such as early development, immune responses, neurogenesis, and tumorigenesis (13, 14). They can be up- or down-regulated in tumors in comparison to matching normal tissues (15) and are generated by backsplicing of pre-mRNA, creating specific new junctions that can be exploited for therapeutic intervention (16). CircRNAs are stable single-stranded molecules that can contain a single exon, exon-intron sequences, or intronic RNA and lack polyA and cap structures. They are able to sponge miRs, can bind to proteins, act as scaffolds, and some of them encode proteins mediated by internal ribosome entry sites (17, 18). In cancer, they can affect functions such as proliferation, migration, metastasis, and angiogenesis, as well as processes such as transcriptional initiation, splicing, and translation, resulting in oncogenic or tumor-suppressive functions (19, 20). In vivo functions were first demonstrated for circRNA Cdr1s, which was shown to contain 70 binding sites for miR-7 and to modulate synaptic responses in vivo in mice (21). The role of circRNAs in cancer was validated with a synthetic circRNA sponging tumor-suppressive miR-21, resulting in inhibition of gastric carcinoma cell proliferation (22). Furthermore, it was shown that circNRIP1 promoted tumor growth in patient-derived xenograft (PDX) models, substantiating the role of circRNAs in cancer (23). Recently, it was demonstrated that circRNAs can be engineered for protein production for many biotechnological applications (24).
CircRNAs Modulating Drug Resistance
Circ0058063 mediates cisplatin (cis-Pt) resistance by up-regulation of β2-microglobulin (β2M). Circ0058063 (Figure 1) was up-regulated in cis-Pt resistant BLC tissues and cell lines and correlated with poor prognosis (25). Knockdown of circ0058063 in cis-Pt resistant BLC cell lines T24/DCCP and 5637/DCCP inhibited cis-Pt resistance in vitro and in vivo in nude mice. Circ0058063 sponged miR-335-3p and up-regulated β2M (25). The latter interacts with major histocompatibility complex (MHC) class I proteins and is also involved in survival, proliferation, and metastasis of tumor cells (26-28).
Circular RNAs mediating drug resistance with efficacy in preclinical bladder cancer-related xenograft models. Upward arrows indicate up-regulation, downward arrows indicate down-regulation. circLIFR: Circ leukemia inhibitory factor receptor; circ PTK2: circ protein tyrosine kinase 2; circVANGL1: circ VANGL planar cell polarity protein 1; circZNF606: circ zinc finger 606; β2M: β2 microglobulin; miR: micro RNA; CDC25B: cell division cycle 25B; MSH2: MutS homolog 2; p73: protein 73; PABPC1: poly(A) binding protein cytoplasmic 1; SETDB1: SET domain bifurcated histone methyltransferase 1; SOX4: SRY-box transcription factor 4.
Circ zinc finger 606 (circZNF606) mediates cis-Pt resistance by up-regulating cell division cycle 25B (CDC 25B). Increased expression of circZNF606 (Figure 1) was associated with worse prognosis in BLC patients (29). In vitro and in nude mice, circZNF606 enhanced proliferation, migration, and cis-Pt resistance in BLC cell lines by sponging miR-1200 and up-regulation of CDC 25B (29). The latter is a phosphatase that promotes cell-cycle progression by activating cyclin B/cyclin-dependent kinase 1 (CDK1) and represents a potential target for anti-cancer therapy (30).
Circ leukemia inhibitory factor receptor (circLIFR) inhibits cis-Pt resistance by binding of MutS homolog 2 (MSH2). CircLIFR (Figure 1) was down-regulated in BLC patients, and down-regulation correlated with poor prognosis (31). CircLIFR interacted with MutS homolog 2 (MSH2) and mediated cis-Pt sensitivity in vitro and in nude mice by recruiting DNA mismatch recognition protein MutSα and serine kinase ataxia telangiectasia (ATM), resulting in activation of protein 73 (p73) (31). MutSα acts as a mismatch repair protein (32). ATM signaling is involved in the DNA damage response (33), and p73 exerts pro-apoptotic functions (34). In PDX models, circLIFR high and MSH2 high xenografts responded better to cis-Pt than circLIFR low and MSH2 low xenografts (31). It was shown that MSH2 is involved in cis-Pt mediated cell death in MIBC (35). Furthermore, it was found that p73 induction was lost in a cis-Pt resistant BLC cell line (36).
Circ protein tyrosine kinase 2 (circPTK2) mediates gemcitabine (GEM) resistance by up-regulation of SET domain bifurcated histone methyltransferase 1 (SETDB1). CircPTK2 (Figure 1) was up-regulated in BLC tissues (37). CircPTK2 increased cell viability, migration, and GEM resistance in UM-UC-3 and T24 BLC cells in vitro. In nude mice, circPTK2 induced lymph node metastasis after injection of T24 BLC cells into the footpads (37). It was shown that circPTK2 binds to poly(A) binding protein cytoplasmic 1 (PABPC1) (38), which interacted with SETDB1 mRNA, leading to its stabilization and increased expression of SETDB1 (37, 39). PABPC1 is involved in mRNA processing and stabilization (38). SETDB1 is deregulated in many types of cancer and can activate ser/thr kinase AKT1 (39, 40).
Circ VANGL planar cell polarity protein 1 (circVANGL1) mediates doxorubicin (DOX) resistance by up-regulation of SRY-box transcription factor 4 (SOX4). CircVANGL1 (Figure 1) was increased in BLC tissues and cell lines (41). In BLC cell lines J82 and T24, circVANGL1 mediated cell viability, decreased apoptosis, and induced DOX resistance. In nude mice, circVANGL1 knockdown in J82 and T24 BLC xenografts resulted in inhibition of tumor growth and increased sensitivity to DOX after subcutaneous implantation. CircVANGL1 sponged miR-145-5p, leading to up-regulation of SOX4 (41). The latter represents a transcription factor related to tumor growth and development (42). In BLC, SOX4 promotes proliferation, metastasis, stem cell properties, and its expression predicts poor patient outcomes (43, 44).
CircRNAs Up-regulating Transmembrane Proteins
Circ0007813 up-regulates insulin-growth factor receptor 2 (IGFR2). High expression of circ0007813 (Figure 2A) correlated with poor prognosis of BLC patients (45). Circ0007813 mediated proliferation, migration, invasion, and autophagy of T24 and UM-UC-3 BLC cells in vitro and in nude mice. This was due to sponging of miR-361-3p and up-regulation of IGFR2 (45). The latter is a multi-faceted receptor that is also known as the cation-dependent mannose-6-phosphate receptor (46). However, opposite findings correlating the loss of IGFR2 with poor prognosis of BLC patients are also available (47).
Circular RNAs targeting transmembrane receptors and secreted proteins with efficacy in preclinical bladder cancer-related xenograft models. (A): transmembrane receptors, (B): secreted proteins. Upward arrows indicate up-regulation, downward arrows indicate down-regulation. circEHBP1: Circ EH domain binding protein 1; circHGS: circ hepatocyte growth factor-regulated tyrosine kinase substrate; circHIPK3: circ homeodomain interacting protein kinase 3; circKIF4A: circ kinesin family member 4A; circNIPBL: circ nipped-B-like protein; circPICALM: circ phosphatidylinositol binding clathrin assembly protein; circTAF4B: circ TATA-box binding protein associated factor 4b; circUVRAG: circ UV radiation resistance-associated gene protein; circZFR: circ zinc finger RNA binding protein; FGFR2,3: fibroblast growth factor receptor 2, 3; HPSE: heparanase; IGFR2: insulin-growth factor receptor 2; miR: microRNA; MMP9: matrix metalloproteinase 9; NOTCH2: neurogenic locus notch homolog protein 2; KCNJ12: ATP-sensitive inward rectifier potassium channel 12; ROBO1: roundabout homology 1; STEAP4: six-transmembrane epithelial antigen of prostate 4; TGFα: transforming growth factor α; TGFβR1: transforming growth factor β receptor 1; VEGF: vascular endothelial growth factor; VEGFC: vascular endothelial growth factor C; WNT5A: WNT-ligand 5A.
Circ kinesin family member 4A (circKIF4A) up-regulates neurogenic locus notch homolog protein 2 (NOTCH2). CircKIF4A (Figure 2A) was up-regulated in BLC tissues and cell lines (48). In vitro, knockdown of circKIF4A inhibited proliferation, migration, and colony formation of 5637, RT-112, and BIU-87 BLC cells by sponging miRs-375 and -1231 and subsequent up-regulation of transmembrane receptor NOTCH2 (48). Intra-tumoral injection of si-circ KIF4A inhibited tumor growth in nude mice with RT-112 and BIU-87 BLC xenografts. RT-112 and BIU-87 cells transfected with si-circ KIF4A showed reduced lung metastasis after tail vein injection (48). NOTCH2 is a key driver of angiogenesis and maintenance of cancer stem cells (48). However, clinical trials with NOTCH inhibitors in cancer patients have been disappointing (49). Independently, it has been shown that NOTCH2 can act as an oncogene in BLC (50, 51).
Circ0014130 up-regulates ATP-sensitive inward rectifier potassium channel 12 (KCNJ12). Circ0014130 (Figure 2A) was up-regulated in BLC patients, and its ectopic expression enhanced proliferation, invasion, and migration of BLC cells in vitro and their growth in xenograft models in mice. Circ0014130 sponged miR-132-3p, up-regulated KCNJ12, and activated the glycogen synthase kinase (GSK)/AKT pathway (52). KCNJ12 represents an inwardly rectifying K-channel that promotes the flow of K-ions out of cells and is expressed in muscle and neuronal cells (53). It has been shown that a mutation in KCNJ12 causes familial dilated cardiac myopathy (54). Therefore, inhibition of KCNJ12 might be critical concerning toxicity issues.
Circ0001495 up-regulates roundabout homology 1 (ROBO1). Circ0001495 (Figure 2A) was up-regulated in BLC tissues and cell lines and correlated with poor prognosis (55). It promoted proliferation, migration, and invasion of BLC cell lines in vitro and tumor growth and metastasis of BLC xenografts in nude mice. Circ0001495 sponged miR-517, resulting in up-regulation of ROBO1 (55). The latter is a member of the immunoglobulin superfamily and is one of the four ROBO receptors that interact with Slits, three secreted extracellular matrix proteins involved in axon guidance and cell adhesion (56). Furthermore, it has been shown that blockage of ROBO1 inhibits the growth of T24 BLC xenografts (57).
Circ EH domain binding protein 1 (circEHBP1) up-regulates transforming growth factor β receptor 1 (TGFβR1). CircEHBP1 (Figure 2A) expression was correlated with lymph node metastasis of BLC patients (58). CircEHBP1 induced tube formation and migration of human endothelial lymphatic cells in vitro, mediated by its expression in T24 and UM-UC-3 BLC cells. It sponged miR-130-3p, resulting in up-regulation of TGFβR1, activation of transforming growth factor β (TGFβ)/SMAD signaling, and secretion of lymph-angiogenesis promoting VEGFD (58, 59). In nude mice, circEHBP1 induced the formation of lymphatic vessels in BLC xenografts and mediated metastasis to the popliteal lymph nodes after footpad injection (58). Furthermore, it has been shown that TGFβR1 can induce metastatic growth in hepatocellular carcinoma (HCC) (60). However, it should be kept in mind that TGFβ signaling, and its physiological outcomes are highly context-dependent (61, 62). Galunisertib, an inhibitor of the ser/thr kinase activity of TGFβR1, did not reach the projected endpoints in clinical trials in cancer patients (63).
Circ UV radiation resistance-associated gene protein (circUVRAG) up-regulates fibroblast growth factor receptor 2 (FGFR2). Circ UVRAG (Figure 2A) was up-regulated in BLC cell lines, and its down-regulation suppressed proliferation of UM-UC-3 cells in vitro as well as tumor growth and experimental metastasis in nude mice. CircUVRAG sponged miR-223 and up-regulated FGFR2 (64). Four transmembrane FGFRs have been identified, which bind at least 18 fibroblast growth factors (FGFs). They can activate signaling via phospholipase Cγ (PLCγ), RAS, mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) pathways that are deregulated in several types of cancers (65). The role of FGFR2 in BLC remains to be investigated in further detail.
Circ0068871 up-regulates fibroblast growth factor receptor 3 (FGFR3). Circ0068871 (Figure 2A) was overexpressed in BLC tissues and cell lines (66). It mediated proliferation, migration, and protection against apoptosis in EJ and UM-UC-3 BLC cells in vitro and in corresponding xenografts in nude mice by sponging miR-181a-5p and up-regulating FGFR3 and signal transducer and activator of transcription (STAT) signaling (66). FGFR3 deregulation by overexpression or mutations has been observed in 54% of BLCs (67, 68). Erdafitinib, a pan FGFR inhibitor, has been approved for the treatment of locally advanced and metastatic BLC (69, 70).
Circ phosphatidylinositol binding clathrin assembly protein (circPICALM) up-regulates six-transmembrane epithelial antigen of prostate 4 (STEAP4). CircPICALM (Figure 2A) was down-regulated in BLC tissues and was related to tumor stage, high grade, lymph node metastasis, and poor survival (71). Over-expression of circPICALM inhibited migration, invasion, and wound healing in vitro in T24 and UM-UC-3 BLC cells by sponging miR-1265 and up-regulating STEAP4. The latter bound to focal adhesion kinase (FAK) to prevent auto-phosphorylation at Y397, resulting in inhibition of epithelial-mesenchymal transition (EMT). Overexpression of circPICALM decreased metastasis to the popliteal lymph nodes after the injection of BLC cells into the footpads of nude mice (71). STEAP4 is a member of the STEAP family of transmembrane receptors comprised of four members that control cell proliferation, apoptosis, molecular trafficking, as well as endo- and exocytic pathways (72). STEAP4 expression and functional relevance seem to be cancer-type specific. STEAP4 was found to be overexpressed in prostate cancer, mediated proliferation of corresponding tumor cells, and was associated with poor survival (73). However, in HCC, the down-regulation of STEAP4 correlated with poor survival (74).
CircRNAs Modulating Expression οf Secreted Proteins
Circ nipped-B-like protein (circNIPBL) up-regulates WNT-ligand 5A (WNT5A). Expression of circNIPBL (Figure 2B) was positively associated with BLC metastasis (75). CircNIPBL enhanced migration and invasion of T24 and UM-UC-3 BLC cell lines in vitro by sponging miR-16-2-3p and up-regulation of WNT5A. In nude mice, circNIPBL promoted lung metastasis of UM-UC-3 cells after tail vein injection (75). WNT5A can activate the WNT/β-catenin, WNT/calcium, and planar polarity pathways (76). It has been shown that WNT5A binds to different members of the frizzled transmembrane receptors (FZD) and receptor tyrosine kinase-like orphan receptors (RORs) (77). WNT5A is overexpressed in BLC, can bind to ROR2, and stimulate migration of BLC cell lines (78, 79).
Circ zinc finger RNA binding protein (circZFR) up-regulates WNT5A. CircZFR (Figure 2A) was increased in BLC patients compared to normal bladder tissues and correlated with worse prognosis (80). It promoted proliferation, migration, and invasion of J82 and T24 BLC cell lines in vitro by sponging miRs-545 and -1270 and up-regulation of WNT5A. In nude mice, circZFR increased tumor growth of T24 cells after subcutaneous implantation (80).
Circ TATA-box binding protein associated factor 4B (circTAF4B) up-regulates transforming growth factor α (TGFα). Up-regulation of circTAF4B (Figure 2B) correlated with poor prognosis in BLC patients (81). Down-regulation of circTAF4B abolished proliferation, wound healing, and migration of SW780 and T24 BLC cell lines by sponging miR-1298 and down-regulating TGFα. In nude mice, down-regulation of circTAF4B inhibited growth of SW780 BLC xenografts after subcutaneous implantation (81). Epidermal growth factor receptor (EGFR) and its ligand TGFα have been shown to be overexpressed in BLC (82, 83).
Circ0001429 up-regulates vascular endothelial growth factor A (VEGFA). Circ0001429 (Figure 2B) was up-regulated in BLC tissues (84). It promoted proliferation, invasion, and inhibited apoptosis of T24 and 5637 BLC cells in vitro as well as tumor growth and metastasis in corresponding xenograft models. Circ0001429 sponged miR-205-5p and up-regulated VEGFA (84). VEGFA and vascular endothelial growth factor receptor 2 (VEGFR2) were found to be significantly up-regulated in BLC and are useful diagnostic biomarkers for this disease (85).
Circ hepatocyte growth factor-regulated tyrosine kinase substrate (circHGS) up-regulates vascular endothelial growth factor C (VEGFC). Expression of circHGS (Figure 2B) positively correlated with grade and pathological stage in BLC patients (86). Silencing of circHGS suppressed cell cycle, proliferation, invasion, and migration of T24 and UM-UC-3 BLC cells in vitro. CircHGS sponged miR-513a-5p, up-regulated VEGFC, and activated mammalian target of rapamycin (mTOR)/AKT signaling. In nude mice, circHGS promoted growth of UM-UC-3 xenografts after subcutaneous implantation (86). VEGFC is a multifaceted dimeric glycoprotein that promotes tumor angiogenesis and lymphangiogenesis by interacting with vascular endothelial growth factor receptor 3 (VEGFR3) (87). High expression of VEGFC has been shown to cause chemo-resistance in BLC cells by up-regulation of serine protease inhibitor maspin (88).
Circ0001361 up-regulates matrix metalloproteinase 9 (MMP9). High-level expression of circ0001361 (Figure 2B) correlated with poor survival in BLC patients (89). Circ0001361 sponged miR-491-5p, up-regulated MMP9, and promoted invasion and metastasis of BLC cell lines in vitro and in vivo in nude mice. Circ0001361 did not affect the cell cycle and proliferation of BLC cell lines (89). MMP9 is expressed in high-grade BLC and mediates degradation of the ECM, migration, metastasis, and angiogenesis in BLC-related preclinical models (90). Clinical studies with MMP inhibitors in cancer patients turned out to be disappointing (91, 92). One of the possible reasons might be the pleiotropic functions including tumor suppression by MMPs (93, 94).
Circ homeodomain interacting protein kinase 3 (circHIPK3) up-regulates heparanase (HPSE). CircHIPK3 (Figure 2B) was underexpressed in BLC tissues compared to normal bladder tissues (95). Overexpression of circHIPK3 in T24T and UM-UC-3 BLC cells inhibited migration, invasion, and tube formation of endothelial cells in vitro due to sponging of miR-558 and subsequent up-regulation of HPSE. In nude mice, circHIPK3 inhibited tumor growth of T24T (96) BLC cells after subcutaneous implantation and metastasis to the lungs after tail vein injection (95). Tumors frequently over-express HPSE with concomitant enhanced tumor growth, metastasis, and poor patient survival (97). In BLC, opposing findings have been reported for the role of HPSE in oncogenesis. Inhibition of HPSE was shown to suppress invasion and adhesion capabilities of BLC cells (98), whereas another report describes the function of HPSE as a tumor suppressor in BLC (99).
CircRNAs that Modulate Signaling Pathways
Circ carbonic anhydrase 12 (circCA12) up-regulates the RAS family of proteins. CircCA12 (Figure 3) was up-regulated in BLC tissues and cell lines, and its silencing repressed proliferation and colony-forming capability in BIU-87 and RT-112 BLC cell lines in vitro (100). In nude mice, circCA12 increased growth and metastasis of BIU-87 xenografts after subcutaneous implantation. CircCA12 sponged miR-1184 and up-regulated RAS proteins Kirsten rat sarcoma (KRAS), Harvey rat sarcoma (HRAS), and neuroblastoma RAS viral homolog (NRAS) (100). The RAS family of small GTPases promotes oncogenesis and cell survival (101, 102). RAS proteins are members of the RAS superfamily and have been shown to be implicated in BLC progression (103).
Circular RNAs targeting components of the signaling system with efficacy in preclinical bladder cancer-related xenograft models. Upward arrows indicate up-regulation, downward arrows indicate down-regulation. circBPTF: Circ bromodomain PHD finger transcription factor; circCA12: circ carbonic anhydrase 12; circCEP128: circ centrosomal protein 128; circFNDC3B: circ fibronectin type III domain containing 3B; circITCH: circ ubiquitin protein ligase; circPSMA7: circ proteasome subunit alpha type-7; circSETD3: circ SET domain containing 3; circSLC38A1: circ solute carrier family 38 member 1; circSOBP: circ sine oculis-binding protein homolog; circVANGL1: circ VANGL planar cell polarity protein 1; G3BP2: Ras GTPase-activating protein-binding protein 2; H-RAS: Harvey rat sarcoma virus; ILF3: interleukin enhancer binding factor 3; ILK: integrin-linked kinase; KRAS: Kirsten rat sarcoma virus; MAPK1: mitogen-activated protein kinase 1; miR: microRNA; MYD88: myeloid differentiation primary response 88; PTEN: phosphatase and tensin homolog; NRAS: neuroblastoma RAS viral oncogene homolog; RAB27A: RAS-associated binding protein 27A; SMAD2: mothers against decapentaplegic homolog 2.
Circ bromodomain PHD finger transcription factor (circBPTF) up-regulates RAS-associated binding protein 27A (RAB27A). High expression of circBPTF (Figure 3) correlated with higher tumor grades, recurrence, and poorer prognosis in BLC patients (104). Its knockdown inhibited proliferation, migration, and wound healing in UM-UC-3 and T24 BLC cells in vitro. CircBPTF sponged miR-31-5p and up-regulated RAB27A. In nude mice, circBPTF knockdown in UM-UC-3 xenografts attenuated tumor growth after subcutaneous implantation (104). RABs are small GTPases, and over seventy members have been identified in humans (105). They are overexpressed in several types of cancer, function as oncogenes, and control cell proliferation, invasion, signal transduction, and protein transport (106, 107). RAB27 isoforms A or B have been identified to be overexpressed in several types of cancer, and RAB27A has been shown to promote proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and chemo-resistance by stimulating nuclear factor κB (NFκB) signaling in BLC cells (108, 109).
Circ0000515 up-regulates integrin-linked kinase (ILK). Circ0000515 (Figure 3) was up-regulated in BLC tissues and cell lines, and its knockdown repressed growth and migration of RT-4 and RT-112 BLC cell lines in vitro, whereas its overexpression had opposite effects (110). In nude mice, knockdown of circ0000515 inhibited pulmonary metastases of RT-4 cells after tail vein injection. Circ0000515 sponged miR-542-3p and up-regulated ILK (110). The latter represents an ankyrin repeat containing ser/thr kinase that interacts with the cytoplasmic domains of integrins β1 and β3. ILK is frequently up-regulated in cancer tissues and has multiple functions in apoptosis, proliferation, motility, activation of downstream pathways, cancer development, and progression (111). In BLC cells, it has been shown that ILK is involved in proliferation, EMT, and inhibition of apoptosis via the ILK/PI3K/AKT pathway (112, 113).
Circ0002623 up-regulates mothers against decapentaplegic homolog 2 (SMAD2). Circ0002623 (Figure 3) was up-regulated in BLC tissues and cell lines and correlated with lymph node metastasis and overall shorter survival (114). In 5637, J82, and T24 BLC cell lines, circ0002623 promoted proliferation, cell cycle progression, and migration in vitro. In nude mice, circ0002623 stimulated growth of T24 xenografts after subcutaneous implantation and lung metastasis after tail vein injection. Circ0002623 sponged miR-1276, resulting in up-regulation of SMAD2 leading to secretion of TGFβ and WNT1 (114). SMAD2 functions as an intracellular signal transducer of receptor tyrosine kinases activated by TGFβ and activin type 1 receptors and induces proliferation, migration, and cell cycle progression (115). SMAD2 is a key component of canonical TGFβ signaling in BLC, and expression of TGFβ1 predicts poor outcomes in BLC patients (116, 117). The other up-regulated component by circ0002623, WNT1, is a component of the WNT/β-catenin pathway, which is frequently up-regulated in BLC (118).
Circ solute carrier family 38 member 1 (circSLC38A1) induces signaling by binding to interleukin enhancer-binding factor 3 (ILF3). CircSLC38A1 (Figure 3) was up-regulated in BLC tissues (119). It promoted migration, invasion, and EMT in J82 and UM-UC-3 BLC cells but did not affect proliferation. CircSLC38A1 stimulated metastasis to the lungs of T24 BLC cells after tail vein injection into nude mice. From a mechanistic point of view, circSLC38A1 bound to ILF3 and stabilized it by inhibiting the ubiquitinylation process. ILF3 interacted with the promoter of the TGFβ gene and activated its transcription (119). ILF3 is a protein that can bind to DNA and RNA, regulate splicing, bind to chromatin, and trigger transcription (120, 121).
Circ centrosomal protein 128 (circCEP128) up-regulates myeloid differentiation primary response 88 (MYD88). CircCEP128 (Figure 3) was up-regulated in BLC tissues, and its silencing in T24 BLC cells restrained viability and motility, induced cell cycle arrest, and accelerated apoptosis in vitro (122). Knockdown of circCEP128 in T24-derived xenografts restrained tumor growth in nude mice after subcutaneous implantation. CircCEP128 sponged miR-145-5p and up-regulated MYD88, resulting in activation of MAPK signaling (122). MYD88 is an adaptor protein that interacts with interleukin-1 receptor-associated kinases 1 and 4 (IRAK1 and IRAK4) to activate both the NFκB and interferon pathways through TNF receptor-associated factor 6 (TRAF6) as an adaptor protein (123). It bridges between anti-inflammatory signaling by toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) and RAS oncogenic signaling (124). The aforementioned MAPK signaling has been found to be activated in BLC (125).
Circ proteasome subunit alpha type-7 (circPSMA7) up-regulates mitogen-activated protein kinase 1 (MAPK1). Expression of circPSMA7 (Figure 3) in BLC was associated with higher tumor grade and stage (126). CircPSMA7 promoted proliferation and invasion in UM-UC-3 cells by regulation of cell cycle and EMT in vitro and in nude mice. Insulin growth factor mRNA binding protein 3 (IGF2BP3) bound to N6-methyladenosine (M6A) modified circPSMA7, resulting in its stabilization. CircPSMA7 sponged miR-128-3p, leading to up-regulation of MAPK1 (126, 127). It has been shown that MAPK1 promotes BLC cell growth, migration, and tumorigenesis (128, 129).
Circ VANGL planar cell polarity protein 1 (circVANGL1) up-regulates VANGL1. CircVANGL1 (Figure 3) was highly expressed in BLC tissues compared to corresponding normal tissues (130). Knockdown of circVANGL1 inhibited proliferation, cell cycle progression, and migration of BLC in vitro and attenuated tumor growth of BLC xenografts in nude mice. CircVANGL1 sponged miR-605-5p and up-regulated VANGL1 (130). The latter functions as a scaffold for WNT/planar cell polarity (PCP) signaling (131). VANGL1 localizes to actin-rich cellular protrusions, and its expression contributes to malignancy by promoting proliferation and migration (132, 133).
Circ fibronectin type III domain containing 3B (circFNDC3B) up-regulates Ras GTPase-activating protein-binding protein 2 (G3BP2). Down-regulation of circFNDC3B (Figure 3) correlated with pathological T-stage, grade, lymphatic invasion, and survival of BLC patients (134). Overexpression of circFNDC3B in UM-UC-3 and T24 cells decreased proliferation in vitro. In nude mice, intra-footpad injection of corresponding UM-UC-3 xenografts inhibited lymphatic metastasis to popliteal lymph nodes. CircFNDC3B sponged miR-1178 and up-regulated G3BP2, which decreased SRC/FAK signaling by promoting phosphorylation of these proteins (134). G3BP2 can function as an RNA binding protein, is involved in stress granule assembly, and has an impact on cell growth, migration, and protein metabolism. It binds to the SRC homology 3 domain (SH3) structural domain of RAS-GTPase activating protein (RAS-GAP) and inhibits SRC/FAK signaling (135-137).
Circ sine oculis-binding protein homolog (circSOBP), circ itchy E3 ubiquitin protein ligase (circ ITCH), circ solute carrier family 8 member A1 (circSLC8A1), and circ SET domain containing 3 (circSETD3) up-regulate phosphatase and tensin homolog (PTEN). These circRNAs (Figure 3) were down-regulated in BLC patients, and decreased expression correlated with worse prognosis (138-141). They inhibited proliferation, migration, EMT, and stemness in T24, 253, EJ, 5637, and UM-UC-3 cells in vitro by sponging miRs 200a-3p, -224, -494, and -641, respectively, and up-regulated PTEN. In vivo, they attenuated the growth of T24 xenografts after subcutaneous implantation into nude mice. CircSOBP also decreased lung metastasis of T24 xenografts after tail vein injection into nude mice (138). PTEN acts as a tumor suppressor with triphosphate phosphatidyl-3,4,5 phosphatase activity which inhibits PI3K signaling. It mediates phosphatase-dependent and - independent functions and affects cancer-related functions such as proliferation, migration, cell survival, metastasis, and genomic stability (142) and was found to be down-regulated in BLC (143).
CircRNAs Targeting Enzymes with Patho-physiological Functions
Circ ubiquitin-associated protein 2 (circUBAP2) up-regulates DNA topoisomerase IIα (TOP2A). CircUBAP2 (Figure 4) was highly expressed in BLC patients, and its expression levels correlated with shorter survival (144). In J82 and SW780 BLC cells, inhibition of circUBAP2 suppressed growth, migration, invasion, and aerobic glycolysis in vitro. In nude mice, inhibition of circUBAP2 decreased the growth of SW780 xenografts after subcutaneous implantation. CircUBAP2 sponged miR-496 and up-regulated TOP2A (144). Topoisomerases are a family of six enzymes with roles in transcription, DNA replication, and chromatin remodeling (145, 146). The TOP2 inhibitor doxorubicin (DOX) is an approved agent for the treatment of BLC (147).
Circular RNAs targeting pathogenic enzymes with efficacy in preclinical bladder cancer-related xenograft models. Upward arrows indicate up-regulation, downward arrows indicate down-regulation. circHP1BP3: Circ heterochromatin protein 1 binding protein 3; circNCOR1: circ nuclear receptor co-repressor 1; circUBAP2: circ ubiquitin-associated protein 2; circUBXN7: circ UBX domain protein 7; circRPS6: circ ribosomal protein S6; circXRN2: circ 5′-3′ exoribonuclease 2; DHCR24: 24-dehydrocholesterol reductase; B4GALT3: beta-1,4-galactosyltransferase 3; C1GALT1: core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1; hnRNPL: heterogeneous nuclear ribonucleoprotein L; SMAD7: mothers against decapentaplegic homolog 7; LATS: serine/threonine-protein kinase LATS1; miR: microRNA; TOP2A: topoisomerase IIα.
Circ000321 up-regulates 24-dehydrocholesterol reductase (DHCR24). Circ000321 (Figure 4) was up-regulated in BLC tissues, and down-regulation in 5637 and T24 BLC cells impeded proliferation, motility, and glycolysis in vitro (148). 5637-based xenografts with down-regulated circ000321 exhibited reduced growth after subcutaneous implantation into nude mice. Circ000321 sponged miR-892-2b and up-regulated DHCR24 (148). The latter is involved in cholesterol biosynthesis. Cholesterol metabolism produces essential membrane compounds, promotes cancer progression, and suppresses immune responses (149). It has been shown that DHCR24 stimulates lymphangiogenesis and lymph-node metastasis of BLC (150). Expression of DHCR24 predicts poor clinico-pathological features in patients with BLC (151).
Circ ribosomal protein S6 (ciRs-6) up-regulates E3 ubiquitin ligase MARCH1. Down-regulation of ciRs-6 (Figure 4) in BLC patients correlated with poor prognosis (152). It suppressed growth of T24 and UM-UC-3 BLC cells in vitro and growth of corresponding xenografts in nude mice after subcutaneous implantation. ciRs-6 sponged miR-653 and up-regulated MARCH1 (152). The latter acts as a regulator of the immune system by mediating lysosomal degradation of MHC in antigen-presenting cells (APC) and prevents their recycling (153). However, its impact on tumor pathogenesis depends on the type of tumor. In contrast to its function in BLC, MARCH1 has been shown to promote proliferation, migration, and invasion by activating NFκB and WNT/β-catenin signaling in ovarian cancer (154).
Circ 5′-3′ exoribonuclease 2 (circXRN2) up-regulates tumor-suppressor serine/threonine-protein kinase LATS1. CircXRN2 (Figure 4) was down-regulated in BLC tissues and cell lines (155). It inhibited proliferation and migration of T24 BLC cells in vitro. In nude mice, circXRN2 decreased the growth of corresponding xenografts after subcutaneous implantation and lung metastasis after tail vein injection (155). CircXRN2 bound to ser/thr protein kinase LATS1 (156) and prevented its degradation by E3 ubiquitin ligase SPOP (157), resulting in activation of HIPPO signaling (155). HIPPO signaling inhibited H3K18 lactylation (158) and expression of lipocalin 2 (LCN2) (155). The latter is a secreted glycoprotein that is highly expressed in several types of cancer and mediates cell proliferation, invasion, metastasis, angiogenesis, and membrane transport (159, 160).
Circ nuclear receptor co-repressor 1 (circNCOR1) up-regulates mothers against decapentaplegic homolog 7 (SMAD7). CircNCOR1 (Figure 4) was negatively associated with BLC lymph node metastasis (161). CircNCOR1 suppressed lymphangiogenesis in vitro by co-culturing T24 and UM-UC-3 BLC cells with human lymphatic endothelial cells. CircNCOR1 inhibited popliteal lymph node metastasis of UM-UC-3 cells after their implantation into the footpads of nude mice (161). In PDX models, circNCOR1 decreased tumor growth and lymph node metastasis. Nuclear circNCOR1 bound to heterogeneous nuclear ribonucleoprotein L (hnRNPL) and epigenetically induced SMAD7 transcription by promoting hnRNPL-induced H3K9 acetylation of the SMAD7 promoter, leading to inhibition of the TGFβ-SMAD signaling pathway (161). SMAD7 acts as a negative regulator of TGFβ signaling (161). Nuclear retention of circNCOR1 was regulated by small ubiquitin-like modifier (SUMOylation) of ATP-dependent RNA helicase DDX39, a regulator of the nuclear export of circNCOR1 (161-163).
Circ heterochromatin protein 1 binding protein 3 (circHP1BP3) up-regulates core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 (C1GALT1). CircHP1BP3 (Figure 4) was up-regulated in BLC tissues, cell lines, and plasma samples (164). Silencing of circHP1BP3 suppressed proliferation and migration of YTS-1 BLC cells in vitro. CircHP1BP3 promoted the growth of YTS-1 xenografts after subcutaneous implantation, and in a splenic metastasis model, it increased liver metastases in nude mice. Furthermore, circHP1BP3 promoted popliteal lymph node metastasis after intra-footpad injection of YTS-1 cells and growth of BLC PDX models in nude mice. CircHP1BP3 sponged miR-1-3p and up-regulated C1GALT1 (164). The latter is involved in the addition of N-acetylgalactosamine (GalNAc) to serine or threonine residues by a glycosidic bond and modifies substrates such as mucin 16, integrins, and O-glycans Thomsen-Nouveau (Tn), sialyl Tn (sTn), and T antigen (165-167). C1GALT1 can exert oncogenic as well as tumor-suppressive functions depending on the type of tumor and its state of progression (168).
Circ UBX domain protein 7 (circUBXN7) up-regulates beta-1,4-galactosyltransferase 3 (B4GALT3). Decreased circUBXN7 (Figure 4) was associated with pathological state, grade, and poor prognosis in BLC patients (169). CircUBXN7 inhibited proliferation, migration, and invasion of T24 and UM-UC-3 BLC cells in vitro and suppressed the growth of UM-UC-3 xenografts in nude mice. It sponged miR-1247-3p and up-regulated B4GALT3 (169). The latter is a member of a family of seven genes and is located in the Golgi apparatus (170). In contrast to its tumor-suppressive function in BLC, in glioblastoma (171) and cervical cancer (172), an oncogenic role for B4GALT3 has been reported.
CircRNAs Regulating Expression of Transcription Factors and Epigenetic Modifier Proteins
Circ ribonuclease P RNA component H1 (circRPPH1) up-regulates signal transducer and activator of transcription 3 (STAT3). Circ RPPH1 (Figure 5) was up-regulated in BLC cell lines compared to normal urothelial cells (173). Down-regulation of circRPPH1 inhibited proliferation, migration, and invasion in T24 and 5637 BLC cells. CircRPPH1 mediated tumor growth of 5637 cells after subcutaneous implantation and increased lung metastasis after tail vein injection into nude mice. It sponged miR-296-5p and up-regulated transcription factor STAT3 (173). CircRPPH1 also interacted with RNA-binding protein fused in sarcoma (FUS) (174) and facilitated the translocation of phosphorylated STAT3 into the nucleus (173). STAT3 can activate several oncogenes such as c-MYC, c-jun, polo-like kinase 1 (PLK1), ser/thr kinases PIM-1 and -2, B-cell lymphoma 2 (BCL2), VEGF, and basic fibroblast growth factor (bFGF) (175). Activation of STAT3 has been shown to be crucial for BLC growth and survival (176). STAT3 can be directly inhibited via the src homology domain 2 (SH2), DNA binding domain, and coiled-coil domain, and several small molecule inhibitors are in clinical trials in cancer patients (177).
Circular RNAs targeting transcription factors with efficacy in preclinical bladder cancer-related xenograft models. Upward arrows indicate up-regulation, downward arrows indicate down-regulation. circACVR2A: Circ activin receptor type-2A; circFAM114A2: circ family with sequence similarity 114 member A2; circFUT8: circ fucosyltransferase 8; circLAMA3: circ laminin subunit alpha-3; circNR3C1: circ nuclear receptor subfamily 3 group C member 1; circRPPH1: circ ribonuclease P RNA component H1; circPTPRA: circ receptor-type tyrosine-protein phosphatase alpha; circSTK39: circ serine/threonine kinase 39; circSTX6: circ syntaxin 6; BRD4: bromodomain-containing protein 4; c-MYC: transcription factor c-MYC; FBXQ1: forkhead box Q1; HMGA2: high mobility group AT-hook 2; EYA4: eyes absent homolog 4; miR: microRNA; MTGR1: myeloid transforming gene-related protein-1; KLF9,10: Krüppel-like factor 9,10; MYBL2: myeloblastosis oncogene-like 2; MYCN: N-myc proto-oncogene; NR3C2: nuclear receptor subfamily 3 group C member 2; NR4A3: nuclear receptor subfamily 4 group A member 3; SND1: staphylococcal nuclease and tudor domain containing 1; RUNX2: runt-related transcription factor 2; STAT3: signal transducer and activator of transcription 3; SUZ12: polycomb protein SUZ12; YB-1: Y box binding protein 1; ΔNP63: p63 transcription factor isoform ΔNP63.
Circ006332 up-regulates myeloblastosis oncogene-like 2 (MYBL2). High circ006332 (Figure 5) correlated with tumor-node metastasis and muscular invasion in BLC patients (178). Its knock-down decreased proliferation, colony formation, invasiveness, and EMT in T24 and UM-UC-3 cells in vitro. Circ006332 knock-down gave rise to smaller tumors in T24 xenografts. Circ006332 sponged miR-143 and up-regulated transcription factor MYBL2 (178). The MYB gene transcription factor family is composed of three members (MYB, MYBL1, MYBL2), and MYBL2 is involved in cell-cycle progression, cell survival, and differentiation (179). In BLC, MYBL2 mediates proliferation and metastasis by activation of cell division cycle associated protein A3 (CDCA3) (180). Focal amplifications of CDCA3 in BLC patients correlate with recurrences (181).
Circ100984 up-regulates Y box binding protein 1 (YB-1). Circ100984 (Figure 5) was up-regulated in BLC (182). Silencing of circ100984 repressed proliferation, migration, and EMT in BIU-87 and HTB9 BLC cells in vitro. In nude mice, inhibition of circ100984 reduced tumor growth and pulmonary metastases of BIU-87 xenografts. Circ100984 sponged miR-432-3p and up-regulated transcription factor YB-1 (182). The latter is a member of the cold heat shock family and is involved in proliferation, survival, drug resistance, and chromatin regulation in cancer cells (183, 184). YB-1 promotes tumor growth and glycolysis in BLC (185) and is a potential factor for worse prognosis in BLC (186).
Circ0088036 up-regulates forkhead box Q1 (FOXQ1). Circ0088036 (Figure 5) was up-regulated in BLC tissues and correlated with clinico-pathological characteristics and poor overall survival (187). It promoted growth, migration, and invasion in T24 and UM-UC-3 cells in vitro. In T24 xenografts, circ0088036 mediated tumor growth and lung metastasis after subcutaneous implantation into nude mice. Circ0088036 sponged miR-140-3p and up-regulated transcription factor FOXQ1 (187). The FOX gene family consists of 43 members (188). FOXQ1 is composed of 403 aa and is involved in tumor initiation, invasion, metastasis, and WNT signaling (189). It has been shown that FOXQ1 promotes proliferation and invasion in BLC (190).
Circ laminin subunit alpha3 (circLAMA3) down-regulates N-MYC proto-oncogene (MYCN). CircLAMA3 (Figure 5) was down-regulated in BLC tissues (191). It inhibited proliferation and invasion of J82 and T24 BLC cells in vitro and growth of T24 xenografts subcutaneously implanted into nude mice. CircLAMA3 bound to MYCN mRNA and inhibited its stability. MYCN mediated transcription of CDK6, promoting cell-cycle progression. MYCN together with MYC and MYCL forms the MYC transcription factor family and promotes tumor cell proliferation by regulating the cell cycle and cell division (191, 192). MYCN is mainly deregulated in childhood neurological tumors and rhabdomyosarcoma (193).
Circ family with sequence similarity 114 member A2 (circFAM114A2) up-regulates p63 transcription factor isoform ΔNP63. CircFAM114A2 (Figure 5) was down-regulated in BLC, and its expression correlated with pathological Tumor, Nodes, Metastasis (TNM) stage (194). It inhibited proliferation, invasion, and migration of 5637 and T24 cells in vitro and growth of T24 xenografts in nude mice after subcutaneous implantation. CircFAM114A2 sponged miR-762 and up-regulated ΔNP63 (194). The latter is an isoform of TP63, a member of the transcription factor family composed of TP53, TP63, TP73 (195). Several isoforms of TP63 are expressed with opposing functions (196). In BLC, reduced expression of ΔNP63 was associated with higher relapse (197).
Circ0000144 up-regulates runt-related transcription factor 2 (RUNX2). Circ0000144 (Figure 5) was highly expressed in BLC tissues, and its knockdown suppressed proliferation and invasion of T24 and UM-UC-3 cells in vitro and growth of corresponding xenografts in nude mice after subcutaneous implantation. Circ0000144 sponged miR-217 and up-regulated RUNX2 (198). RUNX2 is a member of a family of three transcription factors (RUNX1, RUNX2, RUNX3) and affects several oncogenic pathways such as TGFβ, NOTCH, WNT/β-catenin, HIPPO, and MAPK signaling (199). RUNX proteins can function as oncogenes and tumor suppressors depending on the type of tumor (200).
Circ receptor-type tyrosine-protein phosphatase alpha (circPTPRA) up-regulates krueppel-like factor 9 (KLF9). Low expression of circPTPRA (Figure 5) correlated with poor prognosis, advanced tumor stage, and larger tumors in BLC patients (201). CircPTPRA inhibited proliferation of T24 and UM-UC-3 BLC cells in vitro and growth of UM-UC-3 xenografts after subcutaneous implantation into nude mice. CircPTPRA sponged miR-636 and up-regulated KLF9 (201). The KLF family of transcription factors in humans includes 17 members with a conserved DNA binding domain, three zinc fingers, and a variable N-terminal domain responsible for recruiting co-factors (202, 203). KLF9 has been shown to suppress proliferation and migration of BLC cells (204).
Circ fucosyltransferase 8 (circFUT8) up-regulates krueppel-like factor 10 (KLF10). Down-regulation of circFUT8 (Figure 5) in BLC patients correlated with worse prognosis, histological grade, and lymph node metastasis (205). CircFUT8 inhibited migration, invasion, and EMT in T24 and UM-UC-3 BLC cells and metastasis of corresponding xenografts to the popliteal lymph nodes after footpad injection in nude mice. CircFUT8 sponged miR-570-3p and up-regulated KLF10 (205). The latter has been shown to suppress PI3K/AKT signaling in BLC (206). Also, circITGAF7 has been shown to up-regulate KLF10 in BLC (207).
Circ0000658 up-regulates high mobility group AT-hook 2 (HMGA2). Circ0000658 (Figure 5) was highly expressed in BLC tissues and cell lines (207). It promoted proliferation, invasion, and migration of 5637 and T24 BLC cells in vitro and growth of T24 xenografts after subcutaneous implantation into nude mice. Circ0000658 sponged miR-498 and up-regulated HMGA2 (208). Three subfamilies of HMGs have been identified (HMGA, HMGB, and HMGN) as non-histone components of chromatin acting as regulators of transcription (209). It has been shown that HMGA2 promotes cancer metastasis by regulation of EMT (210). Also in BLC, HMGA2 promotes metastasis (211).
Circ003058 up-regulates nuclear receptor subfamily 4 group A member 3 (NR4A3). Circ003058 (Figure 5) was down-regulated in BLC tissues and suppressed proliferation and stemness of 5637 and UM-UC-3 BLC cells in vitro and growth of corresponding xenografts in nude mice after subcutaneous implantation. It sponged miR-665, up-regulated NR4A3, and suppressed ERK signaling (212). NR4A3 is part of a family of highly conserved orphan nuclear receptors that act as tumor suppressors in hematological malignancies and BLC (213, 214).
Circ serine/threonine kinase 39 (circSTK39) up-regulates nuclear receptor subfamily 3 group C member 2 (NR3C2). Expression of circSTK39 (Figure 5) was reduced in BLC tissues, and lower expression correlated with worse prognosis (215). Ectopic expression of circSTK39 inhibited proliferation, invasion, colony formation, and EMT in BLC cell lines and in vivo in nude mice. CircSTK39 sponged miR-135a-5p and up-regulated NR3C2 (215). The latter binds mineralocorticoids and glucocorticoids with equal affinity and is down-regulated in several types of tumors (216). NR3C2 has been shown to suppress migration, invasion, and angiogenesis by regulating glucose metabolism due to phosphorylation of AMP-activated protein kinase (AMPK) and inhibition of AKT/ERK signaling (217, 218).
Circ activin receptor type 2A (circACVR2A) up-regulates eyes absent homolog 4 (EYA4). CircACVR2A (Figure 5) was lower expressed in BLC tissues compared to matching normal tissues and correlated with aggressive clinicopathological characteristics (219). CircACVR2A decreased proliferation, migration, and invasion of UM-UC-3 and T24 BLC cells in vitro. It inhibited popliteal metastasis after injection of UM-UC-3 cells into the footpads of nude mice. CircACVR2A sponged miR-626 and up-regulated EYA4 (219). The latter is a member of a family of four members of the eyes absent family, which are composed of ser/thr kinase and phosphatase domains (220). They exhibit tumor-suppressive as well as oncogenic properties depending on the type of tumor. In BLC, hypermethylation of the EYA4 gene has been reported (221).
Circ0008532 up-regulates myeloid transforming gene-related protein 1 (MTGR1). Circ0008532 (Figure 5) was up-regulated in BLC tissues and cell lines (222). It promoted migration and invasion in EJ and T24 BLC cells and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro. Circ0008532 mediated the formation of lung metastasis in EJ cells after tail vein injection. It sponged miRs-155-3p and -330-5p, resulting in the up-regulation of MTGR1 and inhibition of NOTCH signaling (222). MTGR1 is a member of the myeloid translocation gene family, which acts as transcriptional repressors, does not bind to DNA, and inhibits NOTCH signaling (223). It has been shown that NOTCH signaling is down-regulated in BLC, exerting a tumor-suppressive role in BLC (224).
CircRNAs Regulating other Target Categories
Circ0001583 up-regulates staphylococcal nuclease and tudor domain containing 1 (SND1). Circ0001583 (Figure 5) was highly up-regulated in BLC tissues (225). It increased colony formation, invasion, and migration, but not proliferation, in T24T BLC cells (225). In nude mice, circ0001583 promoted lung metastasis of T24T cells after tail vein injection. Circ0001583 bound to SND1, preventing it from degradation, and due to the exonuclease activity of SND1, decreased expression of miR-126-3p, resulting in the up-regulation of disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) (225). SND1 generally acts as a co-factor of the RNA-induced silencing complex (RISC), affecting the processing of miRs (226). ADAM9 is overexpressed in several types of cancer and correlates with their aggressiveness (227). In BLC, it has been shown that inhibition of SND1 overcomes chemoresistance by promoting ferroptosis (228).
Circ nuclear receptor subfamily 3 group C member 1 (circNR3C1) interacts with bromodomain-containing protein 4 (BRD4). CircNR3C1 (Figure 5) was down-regulated in BLC tissues (229). In T24T cells, circNR3C1 induced cell cycle arrest in vitro and reduced tumor growth of corresponding xenografts in nude mice (229). CircNR3C1 interacted with BRD4 and dissociated the BRD4/c-MYC complex, resulting in inhibition of c-MYC transcription. The C-terminus of BRD4 promotes transcription and is involved in transcriptional elongation (230). It binds to acetylated lysine residues of target proteins, including histones (231). Several BRD4 inhibitors are presently being evaluated in clinical studies in cancer patients (232, 233).
Circ syntaxin6 (circSTX6) up-regulates polycomb protein SUZ12. CircSTX6 (Figure 5) was up-regulated in BLC tissues and promoted migration and invasion of EJ and UM-UC-3 BLC cells in vitro (234). Furthermore, circSTX6 stimulated the growth of EJ xenografts after subcutaneous implantation into nude mice. A two-fold mechanism of action for circSTX6 was revealed: it sponged miR-515-3p, resulting in the up-regulation of SUZ12, and in addition, it interacted with poly(A) binding protein cytoplasmic 1 (PABPC1), leading to increased stability of SUZ12 mRNA (234). PABPC1 acts as an RNA-binding protein enhancing mRNA stability and translation and is involved in tumorigenesis (235). SUZ12 is a component of the polycomb repressive complex 2 (PRC2) and can mediate proliferation, migration, and invasion of tumor cells (236). PRC2 contributes to chromatin compaction and catalyzes the methylation of histone H3 at lysine 27 (237).
We have identified additional circRNAs which target pathways not matching with the outlined categories as described above and therefore are not discussed in detail. Circ lysine-specific histone demethylase 1A (circKDM1A) up-regulates p53 by sponging RNA binding protein CPEB3 (238). Circ protein arginine methyltransferase (circPRMT5) promotes BLC metastasis by inducing EMT (239). Circ ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 6 (circST6GALNAC6) was shown to affect the BLC cytoskeleton by regulation of stathmin (240). In addition, circRNA LOC729852 has been implicated in macrophage polarization (241).
Technical Issues
We have identified up- and down-regulated circRNAs with efficacy in preclinical BLC-related in vivo models. The targets of up-regulated circRNAs can be tackled with antibody-related moieties, chimeric antigen receptors (CARs), protein degraders (242, 243), or small molecules. The corresponding circRNAs can be inhibited with antisense oligonucleotides (ASOs), siRNA, small hairpin RNAs (shRNAs), or CRISPR-CAS-based interference due to the unique junctions generated during the biogenesis of circRNAs (244-246). Because of their tumor-specific junctions, targeting of circRNAs might give rise to limited side effects in comparison to intervention with other types of targets.
The targets of down-regulated circRNAs can be reconstituted by gene therapy with plasmids, virus-based vectors (247, 248), or up-regulated with small molecules. The caveat of this approach is the need to deliver these compounds to all target cells. Up-regulation of targets with small molecules is hampered by specificity issues of the identified compounds and the need for target deconvolution. As previously outlined, it has been shown that transfection of gastric cancer cells with a synthetic circRNA sponging oncogenic miR-21 can inhibit the growth of tumor cells (23).
The critical issues of therapeutic manipulation of circRNA depend on the specific approach and include pharmacodynamic and pharmacokinetic issues, immunogenicity, tumor-specific and high-efficiency delivery, rapid renal clearance, as well as side effects due to unspecific delivery (249, 250). These aspects are not discussed in detail in this review. Considerable progress has been achieved by the development of new delivery vehicles such as liposome-based nanoparticles (251-254), cell- and organ-specific delivery through targeting of organ-specific receptors (253, 254), and improvement of cellular uptake and endosomal release of nucleic acid (NA)-based therapeutics (255).
Conclusions and Further Remarks
The identified circRNAs can be grouped with respect to affecting the following function-related or protein-class specific categories: drug resistance, transmembrane and secreted proteins, mediators of signaling, enzymes, transcription factors, and epigenetic modifiers (Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5). As a proof of concept for circRNAs as drivers of BLC pathogenesis, FGFR-inducing circRNAs were identified. FGFRs are targets of the BLC-approved agent erdafitinib. The ranking of the identified circRNAs and their corresponding targets for further drug development will depend on extended preclinical target validation experiments. We have excluded circRNAs that affect regulators of the cell cycle, such as cyclins, cdks, p21, and p27, which are deregulated in many types of tumors. Of note, four down-regulated circRNAs have been identified that up-regulate the tumor suppressor PTEN, emphasizing its role in BLC (Figure 3).
However, there are several limitations inherent to the outlined approaches of circRNA identification. One of the critical issues is the small number of BLC cell lines investigated. In many cases, only T24 and UM-UC-3 BLC cell lines were explored. Cell lines and their characteristics used in BLC research have been summarized in (256). An assignment of the identified circRNAs and their corresponding targets to the molecular subtypes of BLC and their possible impact on personalized treatment of BLC is not available. The use of BLC cell lines for evaluation of in vitro properties of the corresponding circRNAs will not cover targets which are affected by the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and immune cells such as tumor-infiltrating lymphocytes (TILs), cytotoxic T cells, regulatory T cells (Tregs), and dendritic cells (DCs). The use of immunodeficient mice for in vivo evaluation of the corresponding circRNA limits the assessment of immunomodulatory properties of the identified circRNAs. Therefore, the circRNAs identified according to the outlined approaches comprise only limited categories of oncogenic or tumor-suppressive BLC-related targets.
Sequencing-based analysis of the expression of selected circRNAs in circRNA database version 2 common data set (CSCD2) (257) confirmed overexpression of circ0001361, circNIPBL, and circEHBP1 targeting MMP9, WNT5A, and TGFβR1 respectively (Figure 6). However, only overexpression of circEHBP1 was statistically significant. Despite the limitations outlined above, many of the identified circRNAs and their corresponding targets deserve further investigations with respect to target validation experiments. Further information on the role of circRNAs in cancer can be found on our reviews on acute myeloid leukemia (AML), prostate- and breast cancer (258-261).
Differential expression of selected circRNAs in bladder cancer tissues versus normal bladder tissues. 95 bladder cancer tissues and 5 normal bladder tissues (n=100) were analyzed by RNA sequencing. The data were retrieved from the Cancer-Specific CircRNA Database v. 2 Common Data Set (CSCD2) (253) and originally detected with a chiastic clipping signal-based algorithm (CIRI). Expression levels are presented as log10 transcripts per 1000 circRNAs (log10TPK) within the sample. The data are visualized using box plots, where the black line 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. Data points for tumor and normal tissues are shown in red or blue, respectively. We calculated the significance of differential circRNA expression between cancer and normal samples with the Mann-Whitney U test. CircEHBP1 expression was significantly different between the two groups (p=0.02), whereas circ0001361 and circNIPBL expression differences were not significant (p=0.44 and p=0.14, respectively). *: Statistically significant; ns: non-significant.
Footnotes
Conflicts of Interest
SN is and UHW was an employee of Roche.
Authors’ Contributions
UHW performed the literature analysis, designed and composed the manuscript. SN explored the circRNA database landscape, designed the differential expression analysis, conducted statistical analysis for Figure 6, created all figures, and commented on the manuscript.
- Received April 17, 2025.
- Revision received May 13, 2025.
- Accepted June 6, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
References
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- Circular RNAs
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- CircRNAs Targeting Enzymes with Patho-physiological Functions
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