Abstract
Hepatocellular carcinoma (HCC) ranges as number two with respect to the incidence of tumors and is associated with a dismal prognosis. The therapeutic efficacy of approved multi-tyrosine kinase inhibitors and checkpoint inhibitors is modest. Therefore, the identification of new therapeutic targets and entities is of paramount importance. We searched the literature for up-regulated circular RNAs (circRNAs) which mediate efficacy in preclinical in vivo models of HCC. Our search resulted in 14 circRNAs which up-regulate plasma membrane transmembrane receptors, while 5 circRNAs induced secreted proteins. Two circRNAs facilitated replication of Hepatitis B or C viruses. Three circRNAs up-regulated high mobility group proteins. Six circRNAs regulated components of the ubiquitin system. Seven circRNAs induced GTPases of the family of ras-associated binding proteins (RABs). Three circRNAs induced redox-related proteins, eight of them up-regulated metabolic enzymes and nine circRNAs induced signaling-related proteins. The identified circRNAs up-regulate the corresponding targets by sponging microRNAs. Identified circRNAs and their targets have to be validated by standard criteria of preclinical drug development. Identified targets can potentially be inhibited by small molecules or antibody-based moieties and circRNAs can be inhibited by small-interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) for therapeutic purposes.
HCC is an aggressive malignancy with a poor prognosis and is the second leading cause of cancer-related mortality (1, 2) with 830,000 casualties annually worldwide (3). Risk factors are chronic hepatitis B and C infection, aflatoxin exposure, obesity, nonalcoholic fatty liver disease and chronic alcohol consumption (1). HCC is a very heterogeneous type of tumor and responds poorly to chemotherapy and radiation (1, 2). Many pathways, such as rapidly growing fibrosarcoma (RAF)/mitogen activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK), phosphpoinosite-3 kinase (PI3K)/serine-threonine kinase AKT/mammalian target of rapamycin (mTOR), WNT/β catenin, janus kinase (JAK)/signal transducer and activator of transcription (STAT), Hippo, Ubiquitin-proteasome and Hedgehog signaling have been found to be deregulated in HCC (1, 2). Nevertheless, only sorafenib, regorafenib, levatinib, cabozantinib, small-molecule anti-angiogenic multi-tyrosine kinase inhibitors, anti-angiogenic antibodies, such as bevacizumab [vascular endothelial growth factor A (VEGFA)] and ramicurumab [vascular endothelial growth factor receptor 2 (VEGFR2)], as well as immune-checkpoint inhibitory antibodies, such as Keytruda [anti-programmed cell death protein 1 (PD1)], Nivolumab (anti-PD1) and Atezolizumb [anti-programmed cell death ligand (PD-L1)] have been approved for treatment of HCC. Overall, the therapeutic benefit with respect to survival is limited (4, 5). Therefore, the identification of new modalities and targets for treatment of HCC is an issue of high priority (6).
In this regard, we searched the literature for circRNAs, which are up-regulated in HCC and have pro-tumoral properties in preclinical in vivo models of HCC. This approach allows the compilation of potential therapeutic targets for inhibition with small molecules, antibody-based or other entities and of circular RNAs which can be inhibited by siRNAs or shRNAs (7, 8). In this review we focus on circRNAs which sponge microRNAs (miRs) leading to up-regulation of the corresponding targets.
The Role of Circular RNA in Cancer
A CircRNA is generated by lariat driven circularization and back-splicing (9). It is transcribed by RNA polymerase II and does not contain a cap structure and polyA extensions. CircRNAs are present in all tissues and are deregulated in cancer. Up-regulated circRNAs can function as oncogenes, while down-regulated circRNAs can act as tumor suppressors (10). They are mainly located in the cytoplasm; only a minority is found in the nucleus. One of their functions is sponging of miRs leading to up-regulation of the corresponding substrates of miRs (11). They also can interact with RNA binding proteins, function as protein scaffolds and in rare cases can contain open reading frames encoding small proteins (12). They have been shown to be involved in regulation of transcription by epigenetic mechanisms, splicing, translation and processes, such as angiogenesis, proliferation, apoptosis, epithelial mesenchymal transition (EMT) and drug resistance. They can be expressed in tumor cells and cells of the tumor micro-environment and can often be found in exosomes (13-15). They are potential biomarkers for cancer and can be detected in body fluids, such as saliva and blood (16). The role of circRNAs in HCC has previously been summarized (17, 18). In this review, we focus on circRNAs in HCC which function by sponging miRs and have a pro-tumoral function in preclinical, HCC-related models, because they can be inhibited with nucleic acid-based agents.
CircRNAs Up-regulating Transmembrane Receptors
Circ103809 up-regulates fibroblast growth factor receptor 1 (FGFR1). Circ103809 (Figure 1) was overexpressed in HCC patients and its knockdown inhibited HCC cell proliferation, cell cycle progression and invasion in vitro, while tumor growth of HCC cells with knockdown of circ103809 was inhibited in immunocompromised mice (19). Circ103809 sponged miR-377-5p and up-regulated FGFR1 (19).
Circular RNAs (circRNAs) up-regulating transmembrane receptors. All shown circRNAs are up-regulated in liver cancer and mediate efficacy in preclinical xenograft models. CD44v6: Cluster of differentiation 44, variant 6; c-MET: transmembrane tyrosine kinase c-MET; circFBLIM1: circular filament binding LIM protein 1; circGSE1: circular genetic suppressor element 1; circLARPB1: circular LA ribonucleoprotein; circPTGR1: circular prostaglandin reductase 1; circSTIL: circular SCL-locus interrupting protein; FGFR1: fibroblast growth factor receptor 1; HEG1: HEG homolog1; IGF-1R: insulin growth factor receptor 1; MET; metastasis; miR: microRNA; PD-L1: programmed death ligand 1; PTPRE: receptor tyrosine phosphatase ε; TG: tumor growth; TGFBR1: TGF-β receptor 1.
Circ0015756 up-regulates FGFR1. Circ0015756 (Figure 1) was up-regulated in HCC tissues and serum as well as in HCC cells (20). It mediates cell proliferation and invasion in SNU-387 and Huh-7 HCC cells and promotes their growth in immunocompromised mice (20). Circ001756 sponges miR-610 and up-regulates FGFR1 (20). The fibroblast growth factor (FGF)/FGFR signaling system is composed of 22 human FGFs encoded by different genes and four types of FGFRs (FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c and FGFR4) (21, 22). Heparin mediates dimerization and activation of FGFRs. Pathways, such as mitogen-activated protein kinase (MAPK), PI3K, phospholipase gamma (PLCγ), STAT and angiogenesis are induced by FGFRs (21, 22). Levantinib inhibits HCC by suppressing the FGF/FGFR system in addition to other targets, such as VEGFRs, platelet-derived growth factor receptor alpha (PDGFRα), Ret proto-oncogene (RET) and receptor tyrosine kinase KIT (23). Ongoing therapeutic strategies for inhibition of FGF/FGFR signaling with small molecules and antibody-derived entities in HCC are summarized in (24).
Circular La Ribonucleoprotein 1B (circLARP1B up-regulates insulin-like growth factor 1 receptor (IGF-1R). CircLARP1B (Figure 1) was highly expressed in HCC tissues and cells and was associated with poor outcome in HCC patients (25). Knock-down in Hep3B and Huh7 HCC cells decreased proliferation and invasion and enhanced apoptosis and radiation sensitivity. Silencing of circLARP1B impeded tumor growth and radiation sensitivity of Hu-7 xenografts in nude mice. CircLARP1 sponged miR-578, resulting in up-regulation of IGF-1R.
Circ0001459 up-regulates IGF-1R. Circ000149 (Figure 1) was increased in patients with HCC and promoted growth, migration, and invasion of HepG2 and Huh7 HCC cells (26). Circ0001459 promoted tumor growth and lung metastasis of these cell lines after injection into the flanks of nude mice (26). Circ0001459 sponged miR-6165 and up-regulated IGF-1R (26). Abnormal regulation of IGF/IGF-1R has been observed in HCC through IGF1 and IGF2, the ligands of IGF-1R and by insulin growth factor binding proteins (IGFBPs) (27). Viral infections promote deregulation of IGF1/IGF-1R signaling in HCC (28). IGF/IGF1R signaling induces stemness-related proteins, such as homeobox factor NANOG, octamer binding transcription factor 4 (OCT4), nuclear factor kappa B (NF
B), STATs, cyclo-oxygenase 2 (COX2) and yes-associated protein (YAP) (27). Stemness of HCC contributes to the limitation of therapies, such as sorafenib and is responsible for acquired drug resistance. Several antagonistic monoclonal antibodies (mAbs) directed against IGF-1R have been evaluated in combination with sorafenib in clinical Phase I and II studies in HCC patients with disappointing outcome (27). Selection of patients according to viral infection and administration via the hepatic artery are the next steps for clinical evaluation of IGF-1R mAbs.
Circular prostaglandin reductase 1 (circPTGR1) up-regulates tyrosine kinase c-MET. Exosomal circPTGR1 (Figure 1) was associated with poor outcome in HCC patients (29). Knock-down of circPTGR1 inhibited migration and invasion, and supported cell-cycle arrest at S-phase and apoptosis in LM3 and HepG2 HCC cells in vitro. After implantation of PTGR1-knockdown LM3 cells into the liver of nude mice, fewer metastatic nodules to the mesentery were detected (29). CircPTGR1 sponged miR-449 and up-regulated c-MET. The latter is overexpressed in many types of cancer, is involved in their initiation and development, mediates proliferation, migration, and invasion, while some cancers are addicted to hepatocyte growth factor (HGF)/c-MET interaction (30-32). Several c-MET inhibitors are investigated in patients with HCC (33, 34).
Circ005397 targets HEG homolog 1 (HEG1). Circ005397 (Figure 1) was highly expressed in HCC tissues and cell lines (35). Its silencing inhibited viability, migration, invasion, and angiogenesis in Huh-7 and SNU-387 HCCs in vitro, as well as tumor growth of corresponding Huh-7 xenografts in nude mice. Circ005397 sponged miR-1283 resulting in up-regulation of HEG1. The latter was identified as a novel mucin-like transmembrane protein as a potential diagnostic and therapeutic target in malignant mesothelioma (36). In mice, HEG1 has been shown to be involved in cardiovascular development (37). HEG1 contains a Ser/Thr rich region, three epidermal growth factor (EGF)-like domains, two extracellular juxtamembrane domains, a transmembrane and a cytoplasmic domain and O-linked glycans in the Ser/Thr rich region (36). HEG1 indicates poor prognosis and promotes HCC invasion, metastasis and EMT by activation of WNT/β-catenin signaling (38, 39).
Circ000328 up-regulates programmed death-ligand 1 (PD-L1). Circ000328 (Figure 1) was increased in HCC tissues and knockdown in HepG2 and Huh7 HCC cells inhibited EMT, migration and invasion via the PI3K/AKT signaling pathway in vitro (40). Circ000328 was shown to sponge miR-145 which caused up-regulation of PD-L1. Circ000328 expression was positively correlated with PD-L1 in HCC tissues (40). Knockdown of circ000328 in HepG2 cells inhibited growth in BALBc nude mice with increases of E-cadherin in the xenografts. It is unclear how PD-L1 acts as a mediator of tumor growth in this system.
Circ0048674 up-regulates PDL1. Circ0048674 (Figure 1) was up-regulated in HCC tissues and cells (41). Knockdown of circ0048674 in Huh7 and HCCLM3 HCC cells suppressed proliferation, migration, invasion, and tube formation. Circ0048674 sponged miR-223 and up-regulated PD-L1. In BALBc nude mice, knockdown of circ0048674 restrained the tumorigenicity of Huh7 cells (41). The mechanistic basis for tumor growth inhibition, however, is not clear. Knockdown of circ0048674 also promoted function of natural killer (NK) cells as shown by co-culture of Huh7 and HCCLM3 cells with NK cells (41). PD-L1 binds to its PD1 receptor and inhibits T-cell activation (42). Several PD-1 and PD-L1 related antibodies have been approved for HCC, however, the group of responders and the clinical benefit are limited (43-45).
Circular genetic suppressor element 1 (circGSE1) up-regulates transforming growth factor β receptor 1 (TGFBR1). CircGSE1 (Figure 1) was up-regulated in T-cells incubated with exosomes from Huh-7 and HepG2 HCC cells (46). Exosomes from these cell lines promoted differentiation of CD4+ T-cells into Tregs and enhanced proliferation, migration, and invasion of HCC cell lines. Circ GSE1 sponged miR-324-5p and activated the TGFBR1/small mothers against decapentapledgic3 (SMAD3) pathway in T-cells. Huh-7 cells together with conditioned T-cells injected into the tail vein of C57 male mice gave rise to significant metastases in the lungs (46). CircGSE1 functioned as an inducer of immune escape through Tregs (46). The role of Tregs in inhibition of an anti-tumoral immune response is well documented (47-49).
Circular SCL-interrupting locus protein (circSTIL) up-regulates aquaporin 3 (AQP3). CircSTIL (Figure 1) was reported to be up-regulated in HCC tissues and cells (50). Knockdown reduced HCC proliferation, migration, invasion and promoted HCC apoptosis in vitro, while it inhibited growth of HCC-related xenografts in nude mice after subcutaneous implantation. CircSTIL sponged miR-345-5p resulting in the up-regulation of AQP3 (50). Aquaporins (AQPs) are a family of plasma membrane receptors with six transmembrane domains which assemble as tetramers. They are involved in processes, such as angiogenesis, cellular dissociation, EMT, cell polarization and protrusion (51, 52). AQP3 has been shown to be involved in metastasis (53). Several low molecular weight AQP3 inhibitors have been identified and are evaluated in preclinical cancer-related systems (51-53).
Circ0073181 up-regulates receptor tyrosine phosphatase epsilon (PTPRE). Circ0073181 (Figure 1) was up-regulated in tissues of HCC patients and corresponding cell lines (54). Knockdown of circ0073181 suppressed proliferation, migration, invasion and promoted apoptosis of HCC cell lines in vitro and cell growth and metastasis in nude mice. Circ0073181 sponged miR-548p and up-regulated PTPRE (54). The latter is a transmembrane protein which is highly expressed in brain, testes, lymph nodes and lungs (55). PTPRE can activate non-receptor tyrosine kinases rac, syk, src, yes, fyn and pyk2 (32). PTPRE can dysregulate src (56), and act as an oncogene in mammary tumors (57).
Circ0003998 up-regulates cluster of differentiation 44, variant 6 (CD44v6). Circ0003998 (Figure 1) was overexpressed in HCC patients and promoted proliferation, mobility, EMT in vitro and in MHCC97H and HepG2 xenografts in nude mice (58). It promoted metastasis into the lungs in the tail vein injection model and into the livers after injection into the spleen of MHCC97H cells (58). A two-pronged mode of action has been identified. It sponged miR-143-3p resulting in up-regulation of FOS-like 2 (FOSL2) (59) and bound to poly (rC)-binding protein 1 (PCBP1) (60) mediating expression of CD44v6. The latter is correlated with poor prognosis in patients with HCC (61, 62).
Circ0001955 up-regulates frizzled 4 (FZD4). Circ0001955 (Figure 1) was reported to be highly expressed in HCC tissues and cells and its knockdown inhibited, invasion, migration, angiogenesis, and enhanced apoptosis of HCC cells (63). It sponged miR-646 causing up-regulation of FZD4. In immunocompromised mice, silencing of circ000195 restrained the growth of HCC-related xenografts (63). FZDs are GPCRs with seven transmembrane domains comprising ten members in humans (64). WNT ligands bind to FZD receptors and low-density lipoprotein receptor-related proteins 5 and 6 (LRP5, 6) to induce WNT/β-catenin signaling (65). In HCC, FZDs activate β-catenin inducing proliferation, EMT, stemness and chemo-resistance (66). OMP-18R5, a mAb interacting with 5 out of 10 FZD receptors is presently evaluated in patients with solid tumors (67).
Circular RNA filament binding LIM potein1 (FBLIM1) up-regulates LDL- receptor related protein 6 (LRP6). Circular FBLIM1 (Figure 1) was reported to be highly expressed in HCC exosomes and cells (68). Its inhibition confined HCC glycolysis and progression and blocked tumorigenesis of HCC cells in immunocompromised mice. CircFBLIM1 sponged miR-338 with subsequent up-regulation of LRP6, which is involved in Wnt signaling (69). LRP6 is critical in the anabolic response of bone to parathyroid response (70) and has been associated with progression of TNBC, primary chronic lymphatic leukemia (CLL), non-small cell lung cancer (NSCLC), lung squamous cell carcinoma (LSCC) and HCC (71).
CircRNAs Up-regulating Secreted Proteins
Circ0001178 up-regulates VEGFA. Circ0001178 (Figure 2A) was found to be greatly elevated in HCC tissues (72). In HepG2 and Huh7 HCC cells, knockdown of circ0001178 led to inhibition of proliferation, invasion, migration, cell cycle arrest and increased apoptosis in vitro and tumor growth of HCC xenografts in nude mice. Circ0001178 sponged miR-382 and concomitantly up-regulated VEGF-A. The effect could be rescued by overexpression of miR-382. FDA-approved agents for treatment of HCC, such as small molecule multikinase inhibitors (sorafenib, cabozantinib, lenvatinib and regorafenib) as well as mAbs, such as avastin (VEGF-A) and ramicurumab (VEGFR2) are targeting components of the VEGF-signaling system (73).
Circular RNAs (circRNAs) up-regulating secreted factors, virus-related components, and high mobility group proteins. All shown circRNAs are up-regulated in liver cancer and mediate efficacy in preclinical xenograft models. (A) circRNAs up-regulating secreted factors. (B) circRNAs up-regulating virus-related components. (C) circRNAs up-regulating high mobility group proteins. AFP: α fetoprotein; APRIL: proliferation-inducing ligand; circBACH1: circular BTB domain and CNC homolog1; circCDR1 as: circular antisense RNA cerebellum-related antigen 1; circCSSP1: circular spindle-pole associated protein 1; circNFIX: circular nuclear factor 1, X-type; circRNF13: circular ring finger protein 13; circTMEM45A: circular transmembrane protein 45; circ ZFR: circular zinc finger RNA binding protein; HMGA2: high mobility group A2; HMGB1: high mobility group B1; IGF2: insulin-like growth factor 2; MAP3K2: mitogen-activated protein kinase 2; MET: metastasis; miR: microRNA; STC2: stanniocalcin 2; TGIF2: TGFβ-induced factor homeobox2; TG: tumor growth; VEGFA: vascular endothelial growth factor A.
Circular transmembrane protein 45A (TMEM45A) up-regulates insulin growth factor 2 (IGF-2). Circular TMEM45A (Figure 2A) was up-regulated in HCC and positively correlated with clinicopathological features and poor prognosis in patients with HCC and is found in exosomes in the serum of patients (74). Silencing of circTMEM45A in MHCC97H cells arrested the cell-cycle in the G1 phase, whereas overexpression in Hep3B HCC cells induced G1/S cell cycle progression, colony formation and motility in vitro (74). In nude mice, circTMEM45A promoted growth of Hep3B xenografts. Circ TMEM45A sponged miR-665 and subsequently up-regulated IGF2. The IGF system plays an important role in drug discovery of HCC (75, 76).
Circular antisense RNA cerebellum-related antigen 1 (circCDR1as) up-regulates α-fetoprotein (AFP). CircCDR1as (Figure 2A) was up-regulated in HCC tissues and induced proliferation and migration in HepG2 and SMMC-7211 HCC cells in vitro (77). In nude mice, SMMC-7211 cells transfected with circCDR1as showed increased tumor growth after subcutaneous implantation and after tail vein injection and the number and size of lung metastases were increased (77). CircCDR1as sponged miR-1270 which resulted in up-regulation of AFP. Exosomes extracted from cells overexpressing circCDR1as accelerated proliferation and migration of surrounding normal cells (77). Abnormally high AFP concentrations have been found in the serum of HCC patients (78). AFP is a glycoprotein of 70 kD which transports steroids, retinoids, heavy metals, bilirubin, fatty acids, dioxin, and flavonoids (79). It remains controversial whether AFP can be a therapeutic target for the treatment of HCC (79, 80). It is well documented that overexpression of AFP has an impact on angiogenesis, tumor progression, stem cell properties, invasion, and metastasis of HCC; however, the observed effects depend on the concentration of AFP and its cytoplasmic or circulating status (79, 80).
Circ0011385 up-regulates stanniocalcin 2 (STC2). Circ0011385 (Figure 2A) was up-regulated in HCC tissues and cell lines (81). Inhibition of circ0011385 decreased proliferation of Huh7 and HepG2 HCC cells in vitro and tumor growth in nude mice after subcutaneous implantation. It sponged miR-361-3p with subsequent up-regulation of STC2. The latter is a glycosylated, disulphide-linked homodimeric hormone, which is expressed in a variety of tissues, and mediates calcium regulation, phosphorous metabolism, osteoblast differentiation, glucose homeostasis, protects against oxidative stress and promotes angiogenesis, metastasis, and immune avoidance (82, 83). STC2 can up-regulate cyclin D1 and activate ERK (84). STC2 is up-regulated in HCC and is correlated with a poor prognosis (85). The receptor of STC2 has not yet been identified.
Circ0005785 up-regulates a proliferation-inducing ligand (APRIL). Circ0005785 (Figure 2A) is overexpressed in HCC tissues (86). Its knockdown in Huh7 and SKHep1 HCC cells inhibited proliferation, migration, and invasion in vitro. In nude mice, growth of SK-Hep1 treated with sh circ0005785 was suppressed. Circ0005785 sponged miR-578 resulting in up-regulation of APRIL. The latter is a member of the tumor necrosis factor (TNF) superfamily and is known for its role in hematological cancers (87). APRIL is produced by myeloid cells and their precursors in the bone marrow. It binds to transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (BCMA), two transmembrane receptors. TACI mediates T-cell dependent and independent antibody secretion and B-cell differentiation, whereas interaction with BCMA has the opposite effects (87). In solid tumors, APRIL also can bind to heparan proteoglycans. It has been shown that APRIL is expressed in HCC cells and is involved in angiogenesis (88, 89). It can induce proliferation, inhibit apoptosis, and promote metastasis (87-89). It is unclear which kind of APRIL receptors mediate the observed effects. Underlining the possible pleiotropic effects of APRIL, it has been shown that APRIL can bind to BCMA on HCC cells and induce G2/M cell growth arrest (90).
CircRNAs Promoting Viral Hepatocellular Carcinogenesis
Circular BTB domain and CNC homolog 1 (circBACH1) induces mitogen-activated protein kinase 2 (MAP3K2). Expression of circBACH1 (Figure 2B) was increased in HCC tissues and HBV-transduced hepatoma cells (91). Knockdown of BACH1 suppressed proliferation, migration, invasion, and virus replication in Huh 7-1.3 and HepG 2.2.15 cells. Tumor growth of HepG 2.2.15 cells was inhibited in immunocompromised mice after knockdown of circBACH1. The latter sponges miR-200a-3p which led to induction of MAP3K2, an enzyme which activates other kinases of the MAPK signaling pathway (91, 92).
Circular ring finger protein 13 (circRNF13) up-regulates TGF-β induced factor homeobox 2 (TGIF2). CircRNF13 (Figure 2B) was up-regulated in HBV-associated HCC tissues and cells (93). Knockdown of circRNF13 inhibits proliferation, virus replication, migration and invasion in Huh7-HBV and Hep3B-HBV cells in vitro and retards their growth in immunocompromised mice after subcutaneous implantation. CircRNF13 sponges miR-434-5p and induces TGIF2 (93). The latter promotes cell proliferation and inhibits apoptosis in HBV-virus related HCC (94). TGIF2 has been shown to induce tumor invasion and metastasis in gastric cancer (95). TGIF2 acts as a homeobox transcriptional repressor and is amplified in ovarian carcinoma cell lines (96).
Circular RNAs Up-regulating High Mobility Group (HMG) Proteins
Circular nuclear factor 1 X-type (NFIX) targets HMGA2. CircNFIX (Figure 2C) was up-regulated in patients with HCC and indicated a bad prognosis (97). Its knockdown suppressed cell growth and glutaminolysis in Hep3B and HuH7 HCC cell lines and xenograft growth of HuH7 cells in immunocompromised mice. Circ NFIX sponged miR-3064-5p resulting in up-regulation of HMGA2 (97).
Circular ZFR (circ zinc finger RNA binding protein) targets HMGA2. CircZFR (Figure 2C) was up-regulated in HCC patients and its silencing inhibited cell proliferation, glycolysis and promoted apoptosis in HCC cells. It sponged miR-375 with concomitant up-regulation of HMGA2 (98). Down-regulation of miR-375 inhibited growth of HCC-related xenografts after subcutaneous implantation in immunocompromised mice (98).
Circular centrosome and spindle-pole associated protein (circCSPP1) up-regulates HMGB1. CircCSPP1 (Figure 2C) was up-regulated in HCC patients and its down-regulation inhibited proliferation, invasion, and migration of HCC cells in vitro and tumor growth in nude mice (99). CircCSSP1 sponged miR-493-5p resulting in up-regulation of HMGB1 (99). HMGs are small nuclear proteins with high mobility. HMG1A, HMB1B and HMG1C are generated by alternative splicing, HMGA2 is another member of this family (100). They bind to duplex DNA increasing the binding of transcription factors (100). They can also be secreted to the extracellular matrix (ECM) by creating pro-inflammatory auto- and paracrine loops by interacting with receptors such of rapid glycation end products (RAGE), toll-like receptors (TLRs) and C-X-C motif chemokine receptor 4 (CXCR4) (100, 101). They modulate many pathways, such as TNF, NF
B, EGFR, Hippo, RAS/ERK, AKT, WNT/β-catenin and PI3K/AKT. HMGA1 leads to resistance to chemotherapeutic drugs by maintaining the stemness properties of tumor cells, while an autocrine loop between secreted HMGA1 and RAGE has been shown to drive progression of triple-negative breast cancer (TNBC) (100, 101). From a therapeutic point of view, HMG proteins can be targeted with small molecules and with antibody-derived entities (100, 101).
CircRNAs up-regulating components of the ubiquitin system. The ubiquitin-based modification system consists of E1, E2 and E3 ligases and deubiquitinases (DUBs); it is deregulated in cancer and is involved in tissue homeostasis, metabolism, cell-cycle progression, and signal transduction (102-105). The ubiquitination process starts by generating a thioester-linked E1-Ubiquitin (Ub) conjugate; the activated Ub is then transferred to a E2 Ub conjugating enzyme by a transthiolation reaction and finally to an E3 Ub-ligase which ubiquitinates defined substrates at the ε-amino group of the target protein, forming an isopeptide bond. The human genome contains two E1, 50 E2, 700 E3 genes and more than a hundred DUBs, which reverse ubiquitination. Many of these enzymes are potential targets for the treatment of cancer (102-105). The topology of the Ub chains dictates the fate of the substrates, marking them for recognition and degradation by the proteasome or assembly into functional complexes (104). Ub-ligases and DUBs can exert oncogenic as well as tumor-suppressive functions (104, 105).
Circ0001394 up-regulates ubiquitin conjugating enzyme 2A (UBE 2A). Circ0001394 (Figure 3A) was up-regulated in HCC specimens and cell lines and high circ0001394 expression correlated with poor survival (106). Circ0001394 promoted the proliferation, invasion, and migration of Hep3B and Huh7 HCC cells in vitro. It sponged miR-527 and up-regulated E2 ligase UBE2A which promotes deregulation of p53 (106). In vivo, knockdown of UBE2A in Huh7 xenografts inhibited tumor growth after subcutaneous implantation into nude mice (107). High expression of UBE2A correlates with poor prognosis in HCC patients (107).
Circular RNAs (circRNAs) up-regulating components of the ubiquitin system and ras-associated proteins (RABs). All shown circRNAs are up-regulated in liver cancer and mediate efficacy in preclinical xenograft models. (A) circRNAs up-regulating components of the ubiquitin system. (B) circRNAs up-regulating RAS associated proteins (RABs). circDB: Circular deubiquitinylation; circLNTEP: circular leucyl and cystinyl aminopeptidase; circMYLK: circular myosin light chain kinase; CircLRIG3: circular leucine-rich repeats and immunoglobulin-like domains protein 3; circUGT2: circular UDP-glucose glycoprotein; MET: metastasis; miR: micro RNA; RAB1A, RAB9A, RAB21, RAB23: RAS related proteins 1A, −9A, −21 and −23; RNF38: ringer finger protein 38; TG: tumor growth; UBA-2A, −2T: ubiquitin conjugation enzyme 2A, 2T; USP-7, −21: ubiquitin specific peptidase −7, −21.
Circ00090049 up-regulates ubiquitin-conjugating enzyme 2T (UBE-2T). Circ00090049 (Figure 3A) was up-regulated in HCC and its silencing reduced proliferation, migration, invasion, tumor spheroid formation in Huh7 and HCCLM3 HCC cells in vitro (108). Circ00090049 sponged miR-605 and miR-548c-3p and up-regulated UBE-2T. Its silencing inhibited tumor growth of corresponding xenografts in nude mice. It was shown that UBE-2T promotes proliferation via the G2/M checkpoint in HCC (109). Also, it has been shown that UBE-2T promotes ubiquitinylation of p53 (110). Up-regulation of UBE-2T predicts poor prognosis and promotes HCC progression (111).
Circ0000291 up-regulates ubiquitin-conjugating enzyme 2T (UBE-2T). Circ0000291 (Figure 3A) was up-regulated in HCC and its overexpression correlated with worse prognosis (112). Its knock-down suppressed proliferation, migration, invasion, stemness and induced apoptosis of Huh7 and LM6 HCC cells. It sponged miR-1322 and up-regulated UBE-2T. Knock-down of circ0000291 reduced the tumorigenic potential of Huh7 HCC cells after subcutaneous injection into nude mice (112). In addition to ubiquitinylation of p53, as outlined previously (110), UBE-2T can promote HCC carcinogenesis via cell-cycle promotion and inhibition of apoptosis (113).
Circular leucine-rich repeats and immunoglobulin like domains protein 3 (circLRIG3) up-regulates E3 ligase ring finger protein 38 (RNF38). CircLRIG3 (Figure 3A) was up-regulated in HCC (114). Knockdown of circLRIG3 suppressed proliferation, migration, invasion and facilitated apoptosis of HCC cells in vitro and blocked growth of HCC xenografts in nude mice. CircLRIG3 sponged miR-449a resulting in up-regulation of RNF38. The latter enhances transforming growth factor β (TGF-β) signaling by ubiquitinylation and degradation of neuroblast differentiation associated protein AHNAK (115). RNF38 encodes a nuclear E3 ligase that modifies p53 and alters its location to discrete promyelocytic leukemia (PML) nuclear bodies (116). RNF38 and TNFRβ1 expression relate to short overall survival and high cumulative recurrence of HCC patients (115).
Exosomal circular deubiquitination (circDB) up-regulates ubiquitin-specific peptidase 7 (USP-7) in HCC cells. Adipose derived exosomes were found to promote growth and inhibit DNA damage of HepG2 cells (117). CircDB (Figure 3A) sponged miR-34a and up-regulated cyclin A2. CircDB was up-regulated in HCC with higher body fat ratios (117). Obesity is an independent risk factor for HCC (118). It was shown that adipose-derived exosomes can deliver circDB into mouse-derived HCC Hep1-6 tumors in mice and to double liver metastasis, whereas knockdown of circDB suppressed liver metastasis by the mechanism described above. USP7 acts as an oncogene by decreasing ubiquitinylation of a number of proteins including cyclin A2. USP7 has been shown to regulate the Hippo pathway through ubiquitination of the transcriptional activator Yorkie (119), to promote pathological lipogenesis through promotion of transcription of ZNF 638 (120), while its expression has been found to correlate with poor overall survival in HCC patients (121).
Circ0039053 up-regulates ubiquitin-specific peptidase 21 (USP-21). Circ0039053 (Figure 3A) was up-regulated in HCC tissues and cell lines and promoted HCC progression (122). Down-regulation of circ0039053 decreased proliferation and invasion of Hep3B and HepG2 HCC cells in vitro and tumor growth in nude mice after intraperitoneal injection. Circ003905 sponged miR-637 resulting in up-regulation of USP-21 (122). The latter has been shown to stabilize dual specificity mitogen-activated protein kinase 2 (MEK2) (123).
Circular RNAs activating ras-related small GTPases (RABS). RABS are members of a superfamily composed of 66 proteins in humans (124). These proteins are constituents of vesicles which are involved in the transport of early and late endosomes, mediating transport from the endoplasmic reticulum to the Golgi apparatus and many other transport processes by binding to different membrane components. They are also regulators of signal transduction, cell growth and differentiation and can also interact with actin and motor proteins, such as myosins and kinesins (124, 125). RABs interact with vesicles and bind to effector proteins after activation by loading with GTP. Their activity is regulated by guanine exchange factors (GEF) and GTPase activating factors (126). In HCC, RABs promote replication of HBV and HCV, and their involvement of membrane trafficking can stimulate cell proliferation, invasion, metastasis, and drug resistance (127).
Circular UDP-glucose glycoprotein glucosyl-transferase 2 (circUGGT2) up-regulates ras-related protein 1A (RAB1A). CircUGGT2 (Figure 3B) was up-regulated in HCC tissues and cells (128). Knockdown of circUGGT2 inhibited proliferation, colony formation, cell cycle progression, migration, and invasion of Huh-7 and SK-HEP1 HCC cells in vitro and tumor growth in nude mice after subcutaneous injection. CircUGGT2 sponged miR-5266-5p and induced expression of RAB1A (128). The latter regulates motility of early endocytic vesicles (129) and is involved in transport of vesicles from the endoplasmic reticulum to the Golgi apparatus (130). In HCC, it has been shown that RAB1A positively regulates growth and metastasis by activating the mammalian target of rapamycin complex 1 (mTORC1) (131).
Circular leucyl and cystinyl aminopeptidase (circLNPEP) up-regulates ras-related protein 9A (RAB9A). Loss of androgen receptor (AR) in HCC cells led to up-regulation of circLNPEP (Figure 3A) (132). The latter sponged miR-532-3p and up-regulated RAB9A resulting in migration and invasion of HCC cells. In vitro and in vivo, overexpression of circLNPEP reverses the suppressive effect of AR. It has been shown that RAB9A promotes proliferation and inhibits apoptosis in Hep3b and HepG2 HCC cells by activating the AKT/mTOR pathway (133).
Circ008043 up-regulates ras-related protein 21 (RAB21). Circ008043 (Figure 3B) was highly expressed in HCC (134). Its knockdown inhibited proliferation, migration, invasion and EMT of Focus and HA22T HCC cell lines in vitro and impeded their tumor growth in vivo after subcutaneous implantation into nude mice. Circ008043 sponged miR-326 and up-regulated RAB21. The latter rescued the miR-326 effect on HCC cells (134). RAB21 mediates structure and function in the Golgi apparatus, affects endosome morphology and function, regulates cell adhesion and controls endosomal traffic of β1 integrins (135, 136). It has been shown that integrin trafficking regulated RAB21 is necessary for cytokinesis (137).
Circular myosin light chain kinase (circMYLK) up-regulates ras-related protein 23 (RAB23). CircMYLK (Figure 3B) was highly expressed in HCC tissues and cell lines and was associated with poor prognosis in HCC patients (138). Its knockdown inhibited proliferation, invasion, and migration of Huh7 and HepG3 cells in vitro. CircMYLK sponged miR-362-3p with subsequent up-regulation of RAB23. In nude mice, knockdown of circ MYLK inhibited the growth of Hep3B cells after subcutaneous implantation into nude mice. RAB23 has been identified as a tumor promoter in several types of malignancies (139). In HCC cells, RAB23 has been shown to mediate migration via ras-related C3 botulinum toxin substrate 1 (RAC1)/TGF-β signaling (140) and to induce sonic hedgehog signaling (141). Therefore, RAB23 is a potential target for treatment of HCC (142).
CircRNAs Targeting Redox-related Proteins
Circ0000517 up-regulates thioredoxin domain containing protein disulfide isomerase 5 (TXNDC5). Circ0000517 (Figure 4A) is up-regulated in HCC tissues and cell lines (143). In SNU-387 and Huh7 HCC cells, its knockdown suppressed cell viability, cell-cycle process, cell colony formation and promoted apoptosis in vitro, while knockdown in Huh7 cells blocked tumor growth in nude mice. Circ0000517 sponged miR-1296-5p and up-regulated TXNPC5.
Circular RNAs (circRNAs) up-regulating redox-related proteins and enzymes. All shown circRNAs are up-regulated in liver cancer and mediate efficacy in preclinical xenograft models. (A) Redox-related proteins. (B) Enzymes. ACER: Alkaline ceramidase 3; circIL4R: circular interleukin receptor 4; circHIPK3: circular homeodomain-interacting protein kinase; circMAT2B: circular metallothionein 2; circPRMT5: circular protein arginine methyltransferase 5; circZNF566: circular zinc finger 566; GPX4: glutathione peroxidase 4; HK2: hexokinase 2; MET: metastasis; miR; microRNA; PDK2: pyruvate dehydrogenase kinase 2; PKM2: pyruvate kinase isoform 2; PLB1: phospholipase B1; miR: microRNA; TDO2: tryptophan 2,3 dioxygenase; TG: tumor growth; TXNDC5: protein disulfide isomerase 5.
Circ104718 up-regulates TXNDC5. Circ104718 (Figure 4A) was up-regulated in HCC and a higher expression correlates with poor prognosis (144). It accelerated HCC cell proliferation, migration and invasion and inhibited apoptosis in vitro. Overexpression in HCC cells increased tumor growth and metastasis in nude mice. Circ104718 sponged miR-218-5p and up-regulated TXNDC5. The latter can protect cancer cells from oxidative stress and can stimulate cancer cell growth and proliferation, facilitate survival and angiogenesis, and modulate the ECM (145, 146). It has been identified as a promoter of HCC (147).
Circular interleukin receptor 4 (circIL4R) up-regulates glutathione peroxidase 4 (GPX4). CircIL4R (Figure 4A) was overexpressed in HCC tissues and cell lines (148). It sponges miR-541-3p and up-regulated GPX4 protecting HCC cells from ferroptosis, an iron-dependent form of cell death in vitro and in xenografts. GPX4 protects against lipid peroxidation which produces reactive hydrogen peroxides which induce DNA damage and ferroptosis (148). Inhibition of ferroptosis plays a critical role in liver diseases and HCC (149, 150). It has been shown that sorafenib induces ferroptosis in liver cancer cells (150).
Circ RNAs Targeting Enzymes
Circ RNAs targeting the lipid metabolism.
Circ0091579 up-regulates phosholipase B1 (PLB1). Alterations in the lipid metabolism, such as fatty acid synthesis and cellular lipid compositions can lead to initiation and progression of HCC (151). Circ0091579 (Figure 4B) was up-regulated in HCC tissues and cell lines (152). Depletion of circ0091579 led to inhibitory effects on proliferation, migration, and invasion of HCC cells in vitro. Circ0091579 sponged miR-1225 which led to up-regulation of PLB1 (152). Circ0091579 could suppress growth of HCC xenografts in nude mice after subcutaneous injection.
Circ0001955 up-regulates alkaline ceramidase 3 (ACER3). Circ0001955 (Figure 4B) was up-regulated in HCC (153). Knockdown of circ0001955 in HCCLM3 and Huh7 cells decreased proliferation, migration and invasion, impaired colony forming abilities, induced cell-cycle arrest, increased apoptosis and decreased the number of branches in human umbilical vein endothelial cells (HUVECs). Circ0001955 targeted miR-655-3p and up-regulated ACER3. Knockdown of circ0001955 suppressed growth of HCC xenograft in nude mice. ACER3 promotes HCC cell survival via sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor 2 (S1PR2)/PI3K/AKT signaling (154).
Circular zinc finger 566 (circZNF566) up-regulates tryptophan 2,3 dioxygenase (TDO2). CircZNF566 (Figure 4B) was up-regulated in HCC tissues and cell lines (155). It promoted proliferation, migration, and invasion in Huh7 and LM3 HCC cells in vitro by sponging miR-4738 and up-regulation of TDO2 which is highly expressed in HCC (155). The latter is involved in Trp catabolism together with indoleamine 2,3 dioxygenases 1 and 2 (IDO1, 2), and its inhibition restored anti-tumor immune responses in preclinical models (156). However, clinical studies with IDO1 inhibitors in cancer patients did not reach the projected clinical endpoints (157).
CircRNAs Involved in Metabolism
Circular protein arginine methyltransferase 5 (circPRMT5) up-regulates hexokinase 2 (HK2). Dysregulation of enzymes involved in glucose, amino acid and glutamine metabolism has been frequently observed in HCC (158). CircPRMT5 (Figure 4B) was up-regulated in HCC tissues and cell lines (159). In SNU-387 and HCCLM3 HCC cells, its knockdown impaired proliferation, migration, and glycolysis. It sponged miR-188-5p and up-regulated HK2. In immuno-deficient mice, tumor growth was impeded by knockout in HCC cell xenografts (159). HK2 plays a critical role in glycolysis (160). It has been shown that inhibition of HK2 suppresses growth of lung cancer (161).
Circular metallothionein 2B (circMAT2B) up-regulates pyruvate kinase isoform 2 (PKM2). High circMAT2B (Figure 4B) shortened survival in HCC patients and up-regulated PKM2 under hypoxic conditions in HCC cells by sponging miR-338-3p (162). The tumor-promoting properties of MAT2B were shown in HCC cells in vitro, in HCC organoids and in vivo by injecting tumor xenotransplants into nude mice. It was shown that PKM2 activates glycolysis (163), promotes metastasis, and inhibits autophagy via the JAK/STAT3 pathway in HCC (164). High PKM2 correlated with decreased survival in HCC (165).
Circular 0091597 up-regulates pyruvate dehydrogenase kinase 2 (PDK2). Circ0091579 (Figure 4B) was highly expressed in HCC and its knockdown suppressed glycolysis and induced apoptosis in MHCC97 and Huh7 HCC cells by sponging miR-1297 and up-regulating PDK2 (166). PDK1 and PDK2 have been shown to activate AKT (167). Circ0091579 accelerated the growth of MHC97 cells in nude mice (167).
Circ005397 up-regulates PDK2. Circ005397 (Figure 4B) was up-regulated in HCC tissues and cells (168). Knockdown inhibited proliferation, migration, invasion and promoted apoptosis by sponging miR-362 and up-regulating PDK2 in vitro. It also suppressed tumor growth after subcutaneous injection into nude mice (168).
Circular homeodomain-interacting protein kinase 3 (circHIPK3) up-regulates PDK2. CircHIPK3 (Figure 4B) was up-regulated in HCC tissues (169). Its knockdown restrained proliferation and invasion of HepG2 and SMMC-7721 HCC in vitro. CircHIPK3 sponged miR-124 and miR-506 with concomitant up-regulation of PDK2. Knockdown of circHIPK3 suppressed growth of HepG2 xenografts in nude mice.
Circular RNAs Involved in Signaling
Circ0036412 up-regulates GLI family zinc finger 2 (GLI2). Circ0036412 (Figure 5) is up-regulated in HCC tissues and its knockdown inhibits proliferation and induces G2/M arrest in Huh7 and Hep3B HCC cells (170). It sponged miR-379-3p and is bound to RNA binding protein ELAV-like binding protein-1 (ELAV-1) (171), resulting in up-regulation of transcription factor GLI2. In nude mice, circ0036412 promoted the growth of Huh7 and Hep3 xenografts after subcutaneous implantation. It has been shown that down-regulation of hedgehog components, such as GLI2 inhibits proliferation of HCC cells and GLI2 expression correlated with poor prognosis in HCC patients (172, 173). The hedgehog pathway induces angiogenesis and sustains cancer stem cell growth in HCC (174).
Circular RNAs (circRNAs) up-regulating components of signaling cascades. All shown circRNAs are up-regulated in liver cancer and mediate efficacy in preclinical xenograft models. CircGpcrc 5a: Circular G-protein coupled receptor class C group 5 member A; circMAST1: circular microtubule-associated ser-threonine kinase 1; circRASGRF2: circular ras-specific guanine-releasing factor 2; circZFR: circular zinc finger binding protein; circZNF 609: circular zinc finger 609; CTNND1: catenin delta 1; FAK: focal adhesion kinase; GLI2: GLI family zinc finger 2; JAK2: Janus kinase 2; MAPK1: mitogen-activated protein kinase 1; MET: metastasis; miR: micro RNA; PIK3K3: phospho-inosite-3 kinase regulatory subunit γ, ROCK1: RHO-associated coiled-coil containing protein kinase 1; TG: tumor growth.
Circular ZNF609 up-regulates GLI2. CircZNF609 (Figure 5) was highly expressed in HCC tissues and its down-regulation impaired proliferation, metastasis and stemness in HCCM3 and MHCC-97H HCC cells (175). It activated the hedgehog pathway by sponging miR-15a-5p/15b-5p and up-regulation of GLI2 (175). Hedgehog inhibitors vismodegib, sonigedib and odomzo, which target Smoothened, have been approved for treatment of basal cell carcinoma (176). Hedgehog inhibitors have not yet been approved for other types of cancer.
Circular microtubule-associated serine-threonine kinase 1 (circMAST1) up-regulates CTNND1. CircMAST1 (Figure 5) was increased in HCC compared to matching adjacent liver tissues (177). It mediates proliferation, cell-cycle progression, and invasion of HepG2 and HCCLM3 HCC cells in vitro by sponging miR-1299 and up-regulation of CTNND1. In nude mice, circMAST1 sustained tumor growth of HCCLM3 xenografts. It has been shown that overexpression of CTNND1 in HCC activates WNT/β-catenin signaling (177, 178).
Circular G-protein coupled receptor class C group5 member A (circ Gprc5a) up-regulates yes associated protein1 (YAP1) and TEAD domain family member 1 (TEAD1). CircGprc5a (Figure 5) was up-regulated in HCC (179). In vitro, it mediated proliferation and inhibited apoptosis in HepG2 and Hep3B HCC cells by sponging miR-1283 and activating the Hippo signaling by up-regulation of YAP1/TEAD1. In nude mice, growth of subcutaneously injected HEP3B xenografts was inhibited by injecting sh circGprc5a. The Hippo pathway, a Ser/Thr kinase cascade is a key regulator of liver size, regeneration, metabolism, and homeostasis and can lead to liver cancer (180, 181). Cluster of differentiation 44 (CD44) can promote HCC progression by up-regulation of YAP1 (182). However, the function of the Hippo pathway in HCC is context-dependent, because a tumor-inhibiting function in HCC has also been described (183).
Circ0061395 up-regulates phosphatidylinositol 3-kinase regulatory subunit γ (PIK3K3). Circ0061395 (Figure 5) was up-regulated in serum exosomes from patients with HCC (184). Knockdown of circ0061395 impeded invasion and migration, induced cell cycle arrest and promoted apoptosis in SNU-387 and Huh7 HCC cells in vitro. Circ0061395 sponged miR-877-3p and up-regulated PIK3R3. Knockdown of circ0061395 in Huh7 HCC cells reduced tumor growth after subcutaneous implantation. It has been shown that PIK3R3 mediates proliferation, migration and invasion and activates and the AKT/mTOR pathway in HCC cells (185, 186). PIK3R3 also stimulates peroxisome proliferator-activated receptor α (PPARα) expression to stimulate fatty acid β-oxidation (187).
Circular zinc finger RNA binding protein (circZFR) up-regulates Ser/Thr kinase AKT1. CircZFR (Figure 5) was up-regulated in HCC patients and its silencing suppressed proliferation, migration and invasion and induced apoptosis in HCC cells (188). It sponged miR-511 and up-regulated AKT1 which activated MYC, cyclin D1 and survivin. Depletion of circ ZFR inhibited tumorigenesis and decreased expression level of AKT in HCC-related xenografts (188). The PI3K/AKT/mTOR pathway plays an important role in HCC (189).
Circ0005075 activates mitogen-activated protein kinase 1 (MAPK1). Circ0005075 (Figure 5) was up-regulated in HCC tissues (190). It sponged miR-375, up-regulated MAPK1 and activated the RAS-MEK-ERK pathway. HCC progression was suppressed by down-regulation of circ0005075 in vitro and in HCC xenografts in immunodeficient mice. The ERK pathway plays a crucial role in HCC through its involvement in survival, proliferation, EMT and drug resistance (191). Activation of the ERK pathway also contributes to the tumor-promoting effects of hepatic stellar cells in HCC (192).
Circ101280 up-regulates Janus kinase 2 (JAK2). Circ101280 (Figure 5) was highly expressed in HCC tissues and cell lines and its silencing restrained proliferation and promoted apoptosis in HCC cells by sponging miR-375 and up-regulation of JAK2 (193). Its silencing inhibited growth of HCC xenografts in nude mice. It has been shown independently that JAK2 mediates proliferation of HCC (194). High expression of JAK2 is associated with poor prognosis in HCC patients (195). JAK2 is a validated target in myeloproliferative diseases and several JAK2 inhibitors are approved (196).
Circular RAS-specific guanine nucleotide-releasing factor 2 (CircRASGRF2) up-regulates focal adhesion kinase (FAK). CircRASGRF2 (Figure 5) was up-regulated in HCC tissues and cell lines. Down-regulation of circRASGRF2 inhibited proliferation, migration, and invasion, EMT and induced apoptosis in Hep3B and MHCC97H HCC cells in vitro. siRASGRF2, injected intraperitoneally, inhibited xenograft growth of these cell lines in nude mice (195). CircRASGRF2 acted by sponging miR-1224 and up-regulating FAK. FAK/src signaling has been shown to be involved in growth, invasion, and metastasis in preclinical models of HCC (197, 198). FAK expression is associated with poorer prognosis in patients with HCC following surgery (199).
Circ0009910 up-regulates Rho-associated coiled-coil containing protein kinase 1 (ROCK1). Circ0009910 (Figure 5) was up-regulated in HCC tissues and cell lines and its knockdown in HepG2 HCC cells suppressed proliferation, invasion, and migration in vitro. It targeted miR-335 and led to the up-regulation of ROCK1. Its knockdown inhibited tumor growth of HepG2 xenografts after subcutaneous inoculation into nude mice (200).
Circ101141 up-regulates ROCK1. Circ101141 (Figure 5) was highly expressed in HCC tissues and cells and its down-regulation suppressed proliferation, migration, and invasion of Huh7 HCC cells in vitro. It sponged miR-1297 and up-regulated ROCK1 (201). Knockdown of circ101141 in Hep3B HCC cells attenuated HCC tumorigenesis in nude mice. ROCK1 is activated when bound to GTP-bound form of RAS homolog family member A (RHOA) and functions as a mediator of cell mobility, metastasis, and angiogenesis in various types of cancer (202). Targeting ROCK1/2 blocks cell division and induces mitotic catastrophe in HCC (203). It functions as a key regulator of actin-myosin contraction and cell polarity (204).
Conclusion
Bioinformatic-based techniques have speeded up the identification of HCC-related circ RNAs (205-207), such as circMRD27 which mediates resistance to levantinib (208). Previously we have reviewed the role of circular RNAs in breast cancer (209, 210), esophageal squamous cell carcinoma (211) and colorectal cancer (212). We have extended this work to HCC and identified circRNAs which promote growth of HCC in preclinical in vivo models by sponging miRs. The potential therapeutic approaches are inhibition of their targets with small molecules, antibody-related approaches, or inhibition of the corresponding circRNAs with siRNA or shRNA. The latter approach is still associated with numerous technical issues which have to be optimized and are not discussed in detail in this review (213-217).
The identified circRNAs can be grouped into the following categories: transmembrane proteins (n=14) (Figure 1), secreted proteins (n=5) (Figure 2A), virus-related proteins (n=2) (Figure 2B), HMG-proteins (n=3) (Figure 2C), ubiquitin-system related proteins (n=6) (Figure 3A), RAS-associated binding proteins (n=7) (Figure 3B), redox-related proteins (n=3) (Figure 4A), metabolic enzymes (n=8) (Figure 4B) and signaling-related proteins (n=9) (Figure 5).
PD-L1 and VEGFA (Figure 1, Figure 2) are targets for mAbs approved for treatment of HCC. Approved small molecule inhibitors for HCC, such as sorafenib, regorafenib, levatinib and cabozantinib target VEGF receptors and additional tyrosine kinase receptor inhibitors. Interestingly, three circRNAs up-regulate components of the IGF-signaling system (Figure 1, Figure 2). As outlined, AFP and APRIL (Figure 2A) are controversial targets for therapeutic intervention in HCC. HEC1 (Figure 1) and STC2 (Figure 2B) deserve further validation, keeping in mind that the receptor for STC2 is not yet identified. Since HBV and HCV viruses are drivers of hepatocarcinogenesis, circBACH1 and RNF13 and their corresponding targets should be evaluated in further details. Inhibition of circRNAs up-regulating HMG proteins might be a promising approach and superior to inhibition of DNA binding or interfering with the secreted versions of HMG proteins (Figure 2C).
The identified ubiquitin ligases and USPs (Figure 3A) are tractable targets for the treatment of HCC and merit further target validation. Four RABs and corresponding circRNAs have been identified (Figure 3B). Recently, inhibitors for mutant RAS proteins have been identified (218), but inhibition of wild-type RABs with small molecules is still elusive. The role of redox-related proteins, such as TXNDC5 and GPX (Figure 4A) deserves further validation. Figure 5 indicates that pathways, such as Hedgehog, WNT, PI3K, AKT; MAPK1 and JAK2 are deregulated in HCC, but inhibition is not yet clinically validated for HCC; the same holds true for inhibition of FAK and ROCK1.
Footnotes
Conflicts of Interest
UHW was and AN is an employee of Roche.
Authors’ Contributions
AN and UHW equally contributed to all aspects of the paper.
- Received July 3, 2023.
- Revision received August 22, 2023.
- Accepted August 30, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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).
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- The Role of Circular RNA in Cancer
- CircRNAs Up-regulating Transmembrane Receptors
- CircRNAs Up-regulating Secreted Proteins
- CircRNAs Promoting Viral Hepatocellular Carcinogenesis
- Circular RNAs Up-regulating High Mobility Group (HMG) Proteins
- CircRNAs Targeting Redox-related Proteins
- Circ RNAs Targeting Enzymes
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- Circular RNAs Involved in Signaling
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