Abstract
We have reviewed the literature for circular RNAs (circRNAs) with efficacy in preclinical pancreatic-cancer related in vivo models. The identified circRNAs target chemoresistance mechanisms (n=5), secreted proteins and transmembrane receptors (n=15), transcription factors (n=9), components of the signaling- (n=11), ubiquitination- (n=2), autophagy-system (n=2), and others (n=9). In addition to identifying targets for therapeutic intervention, circRNAs are potential new entities for treatment of pancreatic cancer. Up-regulated circRNAs can be inhibited by antisense oligonucleotides (ASO), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) or clustered regularly interspaced short-palindromic repeats-CRISPR associated protein (CRISPR-CAS)-based intervention. The function of down-regulated circRNAs can be reconstituted by replacement therapy using plasmids or virus-based vector systems. Target validation experiments and the development of improved delivery systems for corresponding agents were examined.
- Micro RNA (miR) sponging
- replacement therapy
- target validation
- small-interfering RNA (siRNA)
- short-hairpin RNA (shRNA)
- xenograft models
- review
Pancreatic cancer represents the third overall leading cause of cancer death with a worldwide incidence of 600 000 per year in 2022 (1). The median five years survival after surgery is approximately 20% and only 15-20% of patients are eligible for surgery at diagnosis (2). Presently, Abraxane, a paclitaxel-albumin stabilized nanoparticle formulation, capecitabine, 5-fluoro-uracil (5-FU), gemcitabine (GEM), erlotinib (ERL) and sunitinib are approved agents for treatment of pancreatic cancer, however, their therapeutic efficacy is very limited (3). Approximately 90% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC) (4). Four clusters of transcriptional subtypes have been defined: squamous, immunogenic, pancreatic progenitor and aberrantly differentiated exocrine (ADEX) (5). Approximately 90% of PDACs carry Kirsten rat sarcoma (KRAS) mutations and p53, mothers of decapentapledgic 4 (SMAD4), and cyclin-dependent kinase inhibitor 2A (CDKN2A) are frequently inactivated (6). Treatment of PDAC is hindered by its high content of desmoplastic tissue, metabolic reprogramming of the glucose, glutamine and lipid metabolism and hyperactivation of nutrient scavenging mechanisms, such as autophagy and macropinocytosis (7, 8). In addition, PDACs generate an immuno-suppressive tumor microenvironment (TME), especially mediated by cancer-associated fibroblasts (CAFs). Mutated KRAS plays a crucial role in all of these phenomena. Promising therapeutic approaches have been reported recently. Adagrasib, an inhibitor of mutated KRAS, has been approved for non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) (9). Furthermore, a personalized neoantigen vaccine based on mRNA has been shown to stimulate T-cells in patients with pancreatic cancer (10). However, identification of new targets and treatment modalities is of paramount importance. In this review we focus on circRNAs and corresponding targets for treatment of PDAC.
Circular RNA
CircRNAs can be generated by intron-pairing, lariat-induced, and RNA-binding protein-mediated circularization (11). They are stable, single-stranded circular entities with unique junctions due to the circularization process. CircRNAs can vary in size from less than 100 nucleotides (nts) to over 4 kb (12). Advances in high-throughput sequencing have allowed the identification of more than 400,000 cytosolic and nuclear circ RNAs in humans (13). They have a broad impact on physiological functions, such as organogenesis, differentiation and on the pathophysiology of neurodegeneration, cardiac fibrosis, diabetes and cancer.
In cancer, circRNAs can exert oncogenic as well as tumor-suppressive functions (14, 15). An impact on processes, such as proliferation, cell-cycle progression, migration, invasion, metastasis, transcription, translation, angiogenesis, apoptosis, autophagy, regulation of the tumor microenvironment (TME) and drug-resistance have been noted (14, 15). A seminal finding elucidating their function in cancer was the demonstration that circRNAs can sponge miRs, neutralizing their mRNA-degrading function (16, 17). Additional functionalities of circRNAs have been revealed in cancer: stabilization of mRNA, involvement as scaffolds in protein complexes, modulation of post-translational and epigenetic modifications and as entities which can encode proteins (18, 19). CircRNAs also can be transferred from cell-to-cell by exosomes (20). The role of circRNAs as biomarkers, prognostic markers, and therapeutic targets in PDAC has been summarized by other reviews (21, 22), but here we focus on their role as therapeutic targets with in vivo activity in preclinical PDAC-related models.
Due to the unique sequences generated by their formation, up-regulated circRNAs can be inhibited by ASOs, siRNA, shRNA or CRISPR-CAS (23, 24). Down-regulated circRNAs can be reconstituted by expression vectors such as plasmid- or virus-based vectors (25).
Circular RNAs Deregulated in Pancreatic Cancer
circRNAs Mediating Gemcitabine (GEM) and Erlotinib (ERL) Resistance
circFERM, ARH/Rho GEF and pleckstrin homology domain protein 1 (circFARP1) up-regulates leukemia inhibitory factor (LIF). circFARP1 (Figure 1) was synthesized in cancer-associated fibroblasts (CAFs) and induced GEM resistance in PDAC cells (26). In nude mice, PANC-1 cells co-injected with CAFs gave rise to larger tumors and reduced response to GEM in comparison to controls without CAFs. circFARP1 sponged miR-660-3p, resulting in up-regulation of LIF. In addition, circFARP1 blocked interaction between caveolin 1 (CAV1) and E3 ubiquitin ligase ZNFR1 preventing its degradation. Secretion of LIF by CAFs stimulated signal transducer and activator of transcription 3 (STAT3) signaling after interaction with LIF receptor (LIFR) on tumor cells. LIF has been identified as a regulator of renewal of cancer stem cells and survival in PDAC (27, 28). CAV1, an integral membrane protein involved in endocytosis, is a constituent of caveolae (29). In patient-derived xenografts (PDX) from GEM-resistant patients with PDACs, si-circFARP1 and anti-LIF neutralizing antibody improved therapy response (26).
Circular RNAs involved in drug-resistance with activity in preclinical pancreatic cancer in vivo models. Upward arrows: up-regulated, downward arrows: down-regulated. CAV1: Caveolin 1; circACTR2: circ actin-related gene 2; circFARP1: circ FERM, ARH/RHO GEF and pleckstrin homolog domain protein 1; circHIPK3: circ homeodomain interacting protein kinase 3; E-cad: E-cadherin; ERL-R: erlotinib resistance; ESPR1: epithelial splicing regulatory protein1; GEM-R: gemcitabine resistance; LIF: leukemia inhibitory factor; miR: micro RNA; RASSF1: RAS associated domain containing 1.
Circ homeodomain interacting protein kinase 3 (circHIPK3) up-regulates RAS association domain containing 1 (RASSF1). circHIPK3 (Figure 1) was increased in PDAC tissues and shRNA directed against circHIPK3 inhibited proliferation, migration, and invasion of GEM resistant PANC-1 and SW1990 PDAC cells (30). Down-regulation of circHIPK3 enhanced sensitivity of GEM-resistant SW1990 PDAC cells to GEM after injection into the abdominal cavity of nude mice (30). It was shown that circHIPK3 sponges miR-330-5p, resulting in up-regulation of RASSF1. Contrasting to these findings, RASSF1 also has been identified as a tumor suppressor gene in PDAC (31). With respect to drug resistance, it has been shown that RASSF1 interacts with xeroderma pigmentosum A (XPA) and coordinates the balance between apoptotic DNA response and DNA repair (32).
Circ actin-related gene 2 (circACTR2) up-regulates phosphatase and tensin homolog (PTEN). Down-regulation of circACTR2 (Figure 1) correlated with poor prognosis and GEM resistance in patients with PDAC (33). circACTR2 sponged miR-221-3p, resulting in up-regulation of PTEN and inhibition of phosphoinosite-3-kinase (PI3K)/AKT signaling. PTEN dephosphorylates inositol-phosphates and proteins and decreases PI3K signaling (34). In nude mice, circACTR2 retarded GEM resistance of SW1990 cells after subcutaneous implantation.
circ0092367 up-regulates epithelial splicing regulatory protein 1 (ESRP1). circ0092367 (Figure 1) was down-regulated in PDAC tissues and mediated GEM- resistance in PDAC cells (35). Over-expression of circ0092367 inhibited invasion, EMT, and GEM resistance in MIA PaCa-2 PDAC cells and attenuated tumor growth and GEM-resistance after s.c. implantation into nude mice. circ0092367 sponged miR-1206 and up-regulated ESRP1. The latter functions as an RNA binding protein and is a member of the heterogenous nuclear ribonucleoprotein family (36). ESPR1 is involved in regulation of alternative splicing, inhibition of proliferation, epithelial-mesenchymal transition (EMT), metastasis as well as modulation of apoptosis and autophagy (37, 38).
circ0013587 up-regulates E-cadherin (E-Cad). circ0013587 (Figure 1) mediated Erlotinib (ERL) resistance in PDAC cells (39). Its down-regulation induced proliferation, invasion and EMT of AsPC-1 PDAC cells. In nude mice, over-expression of circ0013587 enhanced ERL sensitivity in ERL-resistant AsPC-1 xenografts. circ0013587 sponged miR-1277, resulting in up-regulation of E-Cad. ERL, a small molecule inhibitor of epidermal growth factor receptor (EGFR), is an approved agent for treatment of PDAC (40). E-Cad is down-regulated in PDAC, and its down-regulation induces EMT (41).
circRNAs Involved in the Expression of Secreted and Transmembrane Proteins
Circ low density lipoprotein-receptor class A, domain 3 (circLDLRAD3) up-regulates pleiotrophin (PT). circLDLRAD3 (Figure 2) was over-expressed in PDAC patients and correlated with poor prognosis (42). Down-regulation of circLDLRAD3 suppressed proliferation, migration, and invasion of PANC-1 and SW1990 PDAC cells and inhibited their growth in nude mice. circLDLRAD3 sponged miR-137-3p and up-regulated PT, which acts as a mitogenic cytokine and neurotrophic factor. PT exhibits a high affinity to the polysaccharide glycosaminoglycan and interacts with transmembrane receptor tyrosine phosphatase β/ζ (42). PT is over-expressed in several types of cancer and stimulates angiogenesis (43, 44). In PDAC, PT has been shown to induce perineural invasion and to be correlated with poor prognosis (45, 46).
Circular RNAs regulating expression of secreted and transmembrane proteins with activity in preclinical pancreatic cancer in vivo models. Upward arrows: up-regulated, downward arrows: down-regulated. circADAM9: circ disintegrin and metalloprotease domain containing protein 9; circASHL2: circ histone lysine N-methyltransferase; circBFAR: circ bifunctional apoptosis regulator; circCCT3: circ chaperone containing TCP1 subunit 3; circEIF6: circ eukaryotic translation initiation factor 6; circLDLRAD3: circ low density lipoprotein complex class A, domain 3; circSEC24A: circ sec 24 homolog A; circSTK39: circ ser-thre kinase 39; circRNF13: circ ring finger 13; CD80: cluster of differentiation 80; c-MET: receptor tyrosine kinase c-MET; IL6R: interleukin 6 receptor; IGFR1: insulin-like growth factor receptor 1; MET: metastasis; miR: micro RNA; NOTCH1: neurogenic locus notch homolog protein 1; NPRS1: neuropeptide S receptor 1; PDL1: programmed cell death ligand 1; PRSS3: serine protease 3; PT: pleiotrophin; SLC4A4: solute carrier family 4, member 4; SLC7A11: solute carrier family 7, member 11; TG: tumor growth; TGFBR2: transforming growth factor ß receptor 2; TRAM2: translation-associated membrane protein 2; VEGFA: vascular endothelial growth factor A; VEGFR2: vascular endothelial growth factor receptor 2.
Circ disintegrin and metalloproteinase domain-containing protein 9 (circADAM9) up-regulates serine protease 3 (PRSS3). circADAM9 (Figure 2) was up-regulated in PDAC patients and predicted a poor prognosis (47). It inhibited migration and invasion of MIA PaCa-2 and Capan-1 PDAC cells by sponging miR-217. circADAM9 over-expression stimulated growth of Capan-1-based xenografts after s.c. implantation into nude mice. The target of circADAM9 was revealed as PRSS3 which up-regulated vascular endothelial growth factor (VEGF) and the extracellular regulated kinase (ERK) pathway (47). PRSS3 functions as a serine protease and is a member of the trypsin family of proteases. It is expressed in brain and pancreas and is over-expressed in 54% (n=74) of PDACs (48). PRSS3 is a trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice (49).
Circ chaperon containing TCP1 subunit 3 (circCCT3) up-regulates VEGFA and VEGFR2. circCCT3 (Figure 2) was increased in PDAC tissues and correlated with vascular invasion and peritoneal metastasis (50). circCCT3 promoted cell proliferation, migration, and invasion of PDAC cell lines in vitro and growth of Panc02 xenografts after s.c. implantation into nude mice. circCCT3 sponged miR-613, resulting in up-regulation of its targets VEGFA and vascular endothelial growth factor receptor 2 (VEGFR2) (50). VEGFA/VEGFR2 signaling has been shown to mediate motility of PDAC cells (51). However, anti-angiogenic approaches in patients with PDAC did not fulfill the expectations in clinical studies (52).
Circ bifunctional apoptosis regulator (circBFAR) up-regulates receptor tyrosine kinase c-MET. circBFAR (Figure 2) promoted proliferation, migration, and invasion of PANC-1 and CFPAC-1 PDAC cells in vitro (53). In nude mice, circBFAR induced growth and metastasis in PANC-1 cells after s.c. implantation. Circ BFAR sponged miR-34b-5p and up-regulated c-MET. The cross-talk between hepatocyte growth factor (HGF) expressed by stromal cells and c-MET on tumor cells has been shown to support progression of PDAC (54, 55). HGF can be produced by pancreatic stellate cells (PSC) and stimulate formation of collagenous desmoplasia by stromal cells after interaction with c-MET on tumor and endothelial cells. In addition, HGF/c-MET interaction facilitates perineural invasion in PDAC by activation of the mammalian target of rapamycin (mTOR)/nerve growth factor (NGF) axis (56).
Circ ring finger 13 (circRNF13) up-regulates insulin-like growth factor 1 receptor (IGFR1). circRNF13 (Figure 2) accelerated proliferation, migration and invasion of PANC-1 and MIA-PaCa-2 PDAC cells in vitro (57). Intra-tumoral injection of siRNA-circRNF13 inhibited growth of corresponding xenografts in nude mice. circRNF13 sponged miR-139-5p, resulting in up-regulation of IGFR1. Activation of the insulin growth factor (IGF)/IGFR1 system in PDAC correlates with poor survival (58). Also, IGF-driven cancer stromal interactions were shown to support growth of PDAC (59). Preclinical experiments have shown that targeting IGFR1 inhibits growth and metastasis of PDAC cells (60). However, targeting of IGF/IGFR1 in clinical studies in cancer patients did not fulfill the expectations (61).
Circ eukaryotic translation initiation factor 6 (circEIF6) and circ0072008 up-regulate solute carrier family 7, member 11 (SLC7A11). circEIF6 (Figure 2) knockdown hampered proliferation, migration, and invasion, and induced apoptosis in Hs766T and SW1990 PDAC cells in vitro and suppressed growth of SW1990 xenografts in nude mice (62). Mechanistically, this was due to sponging of miR-557, activation of PI3K/AKT signaling and up-regulation of SLC7A11. Increased expression of circ0072008 was associated with high tumor-node-metastasis (TNM) stage in patients with PDAC (63). Knockdown of circ0072008 suppressed cell viability, migration, invasion, ECM acidification, lactate production, glucose uptake, and ATP generation, and promoted apoptosis in SW1990 and PANC-1 PDAC cells in vitro. Inhibition of circ0072008 in PANC-1 cells reduced their growth as a xenograft in nude mice. circ0072008 sponged miR-545-3p and up-regulated SLC7A11. The latter is member of a gene family of 458 target proteins grouped into 65 families, which represent transmembrane proteins in organelles and the plasma membrane (64). SLC7A11 is a 12 transmembrane domain protein that is expressed in the brain and regulates synaptic activity. It is part of a heterodimeric amino acid transport system, which is highly specific for cysteine and glutamate and can be inhibited by small molecules (65, 66). SLC7A11 has been shown to promote proliferation, invasion, and metastasis of PDAC by activating PI3K/AKT signaling (67).
circ001587 up-regulates solute carrier family 4, member 4 (SLC4A4). Down-regulation of circ001587 (Figure 2) correlated with a poor prognosis in patients with PDAC and was weakly expressed in corresponding tissues (68). Over-expression of circ001587 inhibited migration-, invasion-, angiogenesis-inducing capability of PANC-1 cells and attenuated their growth in nude mice (68). circ001587 sponged miR-223, resulting in over-expression of SLC4A4. The latter functions as a bicarbonate transporter, which is expressed in astrocytes, and is the most expressed bicarbonate transporter in PDAC. SLC4A4 inhibition mitigates the acidosis of the TME, decreases T-cell mediated immune responses and breaks macrophage-mediated immune suppression. SLC4A4 targeting and immune checkpoint blockade overcomes immuno-therapy resistance and prolongs survival in mice (69, 70).
Circ histone lysine N-methyltransferase 2 (circASHL2) up-regulates neurogenic locus notch homolog protein 1 (NOTCH1). Increased expression of circASHL2 (Figure 2) correlated with poor survival in patients with PDAC (71). circASHL2 promoted proliferation, invasion, and angiogenesis in Capan-1 PDAC cells in vitro. In vivo, circASHL2 increased growth and liver metastasis of Capan1 cells orthotopically injected into nude mice. circASHL2 up-regulated NOTCH1 by sponging miR-34a. NOTCH 1 stimulated cell proliferation, invasion, EMT induced by c-MET and transcription factor SNAIL expression as well as angiogenesis by VEGFA (71). The NOTCH family of transmembrane receptors consists of four paralogs, which signal by translocating the cytoplasmic domains into the nucleus, resulting in transcriptional activation. The outcome of NOTCH signaling varies and is highly dependent on the context (71, 72). In PDAC, NOTCH paralogs can mediate oncogenic as well as tumor-suppressive properties (73, 74). NOTCH 1 is over-expressed in 50% of PDACs, is activated during the late stages of PDAC and promotes proliferation (75). The role of NOTCH inhibitors including γ-secretase inhibitors in PDAC remains to be investigated in further details.
circ100782 up-regulates interleukin 6 receptor (IL6R). circ100782 (Figure 2) was up-regulated in PDAC tissues (76). circ100782 promoted cell proliferation in BxPC-3 PDAC cells in vitro and its knockdown repressed growth of corresponding xenografts in nude mice. circ100782 sponged miR-124 and up-regulated IL6R stimulating the interleukin 6 (IL6)-Janus kinase 2 (JAK2)-STAT3 pathway. This pathway plays an important role in many types of cancers (77, 78). IL6 is a pleiotropic cytokine which contributes to the pathogenesis of PDAC by creating an inflammatory TME by activation of STAT3 through cis- and trans-signaling (79). Elevation of IL6 is a negative prognostic marker in patients with PDAC (80). In preclinical PDAC-related models, antibodies directed against IL6 inhibit tumor growth and IL6-antibodies combined with antibodies directed against death ligand 1 (PDL1) enhanced growth inhibition of PDAC xenografts (79, 81).
circ0058058 up-regulates programmed death ligand 1 (PDL1). circ0058058 (Figure 2) was highly expressed in PDAC tissues and cell lines (82). Its knockdown inhibited proliferation, invasion, angiogenesis and immune escape, and promoted apoptosis of PDAC cells. circ0058058 sponged miR-577 and up-regulated PDL1 on tumor cells. circ005058 promoted the growth of PDAC-related xenografts in nude mice. Targeting of PD1/PDL1 interaction in clinical studies of patients with PDAC did not result in praxis-changing results (83).
circ102049 up-regulates cluster of differentiation 80 (CD80). circ102049 (Figure 2) was up-regulated in PDAC cell lines (84). siRNA directed against circ102049 inhibited growth, migration, invasion, and secretion of inflammatory factors, and induced apoptosis in SW1990 and PANC-1 PDAC cell lines. Among the inflammatory factors, tumor necrosis factor α (TNFα), interleukin 1β (IL1β), interleukin 8 (IL8) and interleukin 17 (IL17) were identified. circ102049 sponged miR-455-3p and up-regulated CD80 (B7.1). In nude mice, knockdown of circ102049 inhibited growth of PANC-1 xenografts. It has been shown that interleukin 12 (IL12) and CD80 co-expression mediate tumor growth in a PDAC-related in vivo model after i.t. injection (85). CD80 represents a complex target because it is expressed on activated B-cells, macrophages, T-cells and can interact with co-stimulatory receptors cluster of differentiation CD28 (CD28), cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and neurotrophic receptor p75 (86, 87).
circ0050102 up-regulates neuropeptide S receptor 1 (NPSR1). circ0050102 (Figure 2) was higher expressed in PDAC tissues compared to adjacent tissues (88). In PANC-1 and CFPAC-1 PDAC cell lines, circ0050102 mediated proliferation, cell-cycle progression, migration, and invasion, and inhibited apoptosis in vitro. In PANC-1 and CFPAC-1-derived xenografts, inhibition of circ005102 decreased tumorigenesis in nude mice. circ0050102 sponged miR-1182 and up-regulated NPSR1. The latter is a G-protein coupled receptor, interacts with neuropeptide S (NPS), a 20 amino acid peptide. It is widely expressed in neurons and endocrine tumors (89, 90). NPS promotes arousal and anxiolithic effects (89, 90). It increases cyclic AMP levels and activates the mitogen activated protein kinase (MAPK) pathway (91). Taken together, the NPS/NPSR1 interaction might play a tumor-driving role not only in islet cell tumors of the pancreas, but also in PDACs.
Circ sec24 homolog A (circSEC24A) up-regulates transforming growth factor ß receptor 2 (TGFBR2). circSEC24A (Figure 2) was highly expressed in PDAC tissues (92). In PANC-1 and MIA PaCa-2 PDAC cells, circSEC24A mediated cell proliferation, migration, and invasion in vitro. In nude mice, sh-circSEC24A inhibited growth of PANC-1 xenografts after s.c. implantation. circSEC24A bound to miR-606, up-regulated TGFBR2 and activated AKT signaling. A complex between TGFBR1, transforming growth factor β (TGFβ) and TGFBR2 is formed and activates SMAD2, SMAD3 and SMAD4 to regulate gene expression. However, the signaling output is highly context dependent (92, 93). In PDAC, TGFBR1/TGFBR2, SMAD2, SMAD3 signaling has been shown to mediate invasiveness (94). Galunisertib, an oral inhibitor of the kinase activity of TGFRB2 gave rise to promising clinical activity in patients with unresectable PDAC, however, clinical development of this compound was cancelled, probably due to safety issues (95). Inhibition of TGFβ signaling together with anti-programmed death1 (PD1) antibody durvalumab showed promising clinical efficacy in patients with metastatic PDAC (96, 97).
Circ serine-threonine kinase 39 (circSTK39) up-regulates translocation associated membrane protein 2 (TRAM2). circSTK39 (Figure 2) was higher expressed in PDAC tissues compared to adjacent tissues (98). In PANC-1 and BxPC-3 PDAC cell lines, si-circ-STK39 inhibited cell proliferation and invasion in vitro and growth of PANC-1 xenografts in nude mice. circSTK39 sponged miR-140-3p and up-regulated TRAM2. The latter is a translocation component that controls post-translational processing of nascent secretory and membrane proteins of the endoplasmic reticulum and is expressed in many human tissues (98). TRAM2 has been identified as a key mediator of proliferation, invasion and matrix metalloproteinase activation, yes-associated protein (YAP)-induced oncogenesis and as an activator of the PI3K/AKT/mTOR pathway (99-101).
Circular RNAs Involved in the Expression of Transcription Factors
Circ EYA transcriptional co-activator and phosphatase 3 (circEYA3) and circ SLIT homolog 2 (circSLIT2) up-regulate transcription factor c-MYC. circEYA3 (Figure 3) over-expression was associated with poor prognosis in patients with PDAC and correlated with advanced lymph node invasion and tumor-node-metastasis (TNM) status (102). Knockdown of circEYA3 suppressed proliferation, migration, invasion, ATP production, and lactate dehydrogenase A (LDHA), induced apoptosis in PANC-1 and MIA PaCa-2 PDAC cells in vitro, and inhibited growth in PANC-1 xenografts in nude mice (102). circEYA3 sponged miR-1294 and up-regulated c-MYC. Expression of circSLIT2 was up-regulated and correlated with poor prognosis in patients with PDAC (103). circSLIT2 promoted proliferation and anaerobic glycolysis in PDAC cells in vitro and stimulated growth of PDAC-derived xenografts in nude mice. circSLIT2 sponged miR-510 and up-regulated c-MYC. The function of c-MYC as a driver of proliferation, growth and metastasis has been documented (104-106). OMO-103, a cell penetrating peptide, which inhibits interaction of c-MYC with several proteins, is presently clinically evaluated in patients with cancer and stable disease was noted in half of pancreatic cancer patients (107, 108). Inhibition of MYC-based spheres-formation is presently evaluated as a new approach for cancer therapy (109).
Circular RNAs regulating expression of transcription factors with activity in preclinical pancreatic cancer in vivo models. Upward arrows: up-regulated, downward arrows: down-regulated. BTG2: B-cell translocation gene 2; BPTF: bromodomain PHD finger; circ CDR as: circ antisense to the cerebellar degeneration-related protein 1; circEYA3: circ EYA transcriptional co-activator and phosphatase 3; circPDK1: circ pyruvate dehydrogenase lipoamide kinase isoenzyme 1; circSLIT2: circ slit homolog 2; c-MYC: transcription factor c-MYC; E2F2: transcription factor E2F2; E2F3: transcription factor E2F3; HIF-1α: hypoxia-inducible factor 1α; MET: metastasis; miR: micro RNA; SOX4: SRY-box transcription factor 4; NR3C1: nuclear receptor subfamily 3, group C, member 1; TG: tumor growth.
Circ pyruvate dehydrogenase lipoamide kinase isoenzyme 1 (circ PDK1) up-regulates c-MYC. circPDK1 (Figure 3) was highly expressed in PDAC and correlated with poor prognosis (110). circPDK1 was found in exosomes from PDAC cell lines and promoted proliferation, migration, and glycolysis of MIA PaCa-2 and PANC-1 cells in vitro, and growth of MIA PaCa-2 cells was enhanced in nude mice i.v. injected with exosomes containing circ PDK1. Sh-circ-PDK1 inhibited lung metastasis of MIA PaCa-2 cells in nude mice. A twofold mode of action of circPDK1 was identified. mir-628-3p was sponged by circPDK1, resulting in up-regulation of bromodomain PHD finger (BPTF), a transcription factor which up-regulates c-MYC (111). In addition, circPDK1 functioned as a scaffold for the interaction between E2 ubiquitin-conjugating enzyme UBE2D (112) and bridging integrator 1 (BIN1), mediating its degradation. BIN1 acts as pro-apoptotic tumor suppressor that binds to and inhibits MYC transcription factors (113).
circ0066147 up-regulates transcription factor E2F2. circ0066147 (Figure 3) was over-expressed in PDAC tissues and cell lines (114). In PANC-1 PDAC cells, knockdown of circ0066147 suppressed proliferation, migration, and invasion, and induced apoptosis in vitro. In nude mice, sh-circ0066147 inhibited the growth of SW1990 and PANC-1 PDAC xenografts. These observations are due to sponging of miR-326 by circ0066147 and up-regulation of E2F2. E2Fs are a family of transcription factors, which consist of a DNA binding domain, a dimerization domain, a transactivation domain and a tumor suppressor protein association domain and can both repress and activate E2F responsive genes (115, 116). E2F2 promotes proliferation and invasion in pancreatic cancer, but also in normal cells (117). It regulates PI3K/AKT and mTOR signaling (118) as well as angiogenesis in PDAC (119).
Circ antisense to the cerebellar degeneration-related protein 1 (circCDR1as) up-regulates transcription factor E2F3. circCDR1as (Figure 3) was highly expressed in PDAC tissues and cell lines (120). Knockdown of circCDR1as inhibited proliferation, invasion, and migration, and promoted apoptosis in AsPC-1 and PANC-1 PDAC cell lines in vitro and decreased of their growth after administration into the peritoneal cavity of nude mice (120). circCDR1as sponged miR-432-5p and up-regulated E2F3. The latter has a cyclin-binding domain, which binds to the retinoblastoma protein and promotes proliferation and invasion of PDAC cells (121, 122).
circ0001666 up-regulates SRY-Box transcription factor 4 (SOX4). circ0001666 (Figure 3) was highly expressed in PDAC tissues and cell lines and correlated with short survival times (123). In MIA PaCa-2 and AsPC1 PDAC cells, circ0001666 mediated proliferation, invasiveness and EMT in vitro. Knockdown of circ0001666 had opposite effects. In nude mice, siRNA directed against circ0001666 inhibited growth of MIA PaCa-2 cells. circ0001666 sponged miR-1251 and up-regulated SOX4. The latter functions as a developmental transcription factor, which mediates stemness, differentiation, progenitor development, PI3K, wingless/integrated (WNT) and TGFβ signaling (124, 125). SOX4 has been shown to mediate proliferation, invasion, and PI3K/AKT signaling, and to correlate with poor prognosis in PDAC patients (126, 127). In addition, SOX4 is involved in expression of multiple semaphorin 3A (SEM3A)/plexin family members and promotes tumor growth in PDAC (128).
circ03955 up-regulates hypoxia-inducible factor 1α (HIF-1α). circ03955 (Figure 3) was up-regulated in PDAC clinical samples and cell lines and increased expression was associated with poor clinical outcomes (129). circ03955 stimulated proliferation of PDAC cell lines, inhibited apoptosis in vitro and promoted their growth in nude mice. It sponged miR-3662 leading to up-regulation of transcription factor HIF-1α which is over-expressed in PDAC. HIF-1α is a key factor in the regulation of cell adaption to hypoxia and plays an important role in progression of PDAC (130). HIF-1α is expressed in hypoxic niches, heterodimerizes with HIF-1ß and induces genes involved in proliferation, invasion, EMT, metastasis, autophagy, desmoplasia, chemo- and radiotherapy resistance, tumor stem cell maintenance and immune evasion of PDAC (131, 132). Inhibitors of HIF-1α have been clinically evaluated in patients with cancer, but none has been approved yet (131, 132).
circ000854 up-regulates B-cell translocation gene 2 (BTG2). circ000854 (Figure 3) was poorly expressed in PDAC tissues and corresponding cell lines (133). Over-expression of circ000854 in AsPC-1 PDAC cells inhibited proliferation, invasion, cell-cycle, apoptosis and growth of corresponding xenografts in nude mice after s.c. implantation. circ000854 sponges miR-361-3p and up-regulates BTG2. This protein belongs to the BTG family of anti-proliferative transcriptional co-regulators, which through protein-protein interactions bind to DNA transcription factors and regulate their activity (134). BTG2 has been shown to act as a tumor suppressor in several types of tumors (134).
circ0128846 up-regulates nuclear receptor subfamily3, group C, member 1 (NR3C1). circ0128846 (Figure 3) was increased in PDAC tissues and corresponding cell lines (135). In PANC-1 and SW1980 PDAC cell lines, silencing of circ0128846 repressed growth and migration, but accelerated apoptosis in vitro. circ0128846 promoted development of corresponding tumors in nude mice. It sponged miR-1270 and up-regulated NR3C1, the glucocorticoid receptor, which contains an N-terminal domain, a DNA binding domain and a ligand binding domain. Activation of NR3C1 by dexamethasone is used for treatment of hematological tumors; however, its role in solid tumors seems dependent on the specific type of tumor (136). It has been shown that activation NR3C1 by dexamethasone inhibits PDAC-patient derived xenografts by suppression of nuclear factor 
B (NF
B), EMT, IL6 and VEGF (137), indicating that the role of NR3C1 in PDAC is controversial.
Circ RNAs Involved in the Expression of Components of Signaling Pathways
Circ nuclear factor NFIB1 (circNFIB1) up-regulates phosphoinosite-3-kinase-reglatory subunit 1 (PI3KR1). circNFIB1 (Figure 4) was down-regulated in PDAC tissues and negatively correlated with lymph node metastasis in patients with PDAC (138). Conditioned media from PANC-1 and Capan-2 PDAC cells with silenced circNFIB1 promoted tube formation and migration of human lymphatic endothelial cells. circNFIB1 suppressed lymph node metastasis of PANC-1 cells in nude mice. circNFIB1 sponged miR-486-5p and up-regulated PIK3R1, which inhibits PI3K/AKT signaling, vascular endothelial growth factor C (VEGFC) expression and lymphangiogenesis. VEGFC was found to be over-expressed in PDAC (139, 140).
Circular RNAs regulating signaling with activity in preclinical pancreatic cancer in vivo models. Upward arrows: up-regulated, downward arrows: down-regulated. ARL4C: ADP ribosylation factor 4C; BNIP3: BCL2/adenovirus E1B 19 kD interacting protein 3; CAFs: cancer-associated fibroblasts; circ APAPC7: circ anaphase promoting complex subunit 7; circCUL2: circ cullin 2; circDLSP22: circ dual specificity phosphatase 22; circFGFR1: circ fibroblast growth factor receptor 1; circNFIB1: circ nuclear factor IB1; circPVRL3: circ poliovirus receptor related 3; circSFMBT1: circ scam-like with four malignant brain tumor domains; circUHRF1: circ ubiquitin-like with PH finger domain 1; LAMTOR3: lysosomal adaptor MAPK and mTOR activator 3; MET: metastasis; miR: micro RNA; MYD88: myeloid differentiation primary responders 88; PAK1: p21 activated protein kinase 1; PIK3B: phosphatidyl-4,5-biphosphate-3-kinase, catalytic subunit ß; PI3KR1: phosphoinosite-3-kinase catalytic subunit ß; PHLPP2: P domain and leucine rich protein phosphatase 2; SOCS2,5: suppressor of cytokine signaling 2 or 5; TG: tumor growth; YAP1: yes-associated protein 1.
Circ fibroblast growth factor receptor 1 (circFGFR1) up-regulates phosphatidylinositol-4,5-biphosphate-3-kinase catalytic subunit β (PIK3CB). circFGFR1 (Figure 4) up-regulated cell growth, migration and invasion of PDAC cells in vitro and inhibited tumor growth and lung metastasis in nude mice (141). It sponged miR-532-3p and up-regulated PIK3CB. Several inhibitors of this pathway have been identified: Pan-PI3K inhibitors, dual PI3K/mTOR inhibitors and isoform-specific inhibitors (142). A PI3Kα isoform-specific inhibitor has been approved for treatment of breast cancer as well as a PI3Kθ specific inhibitor for treatment of B-cell malignancies (143).
Circ anaphase promoting complex subunit 7 (circAPAPC7) up-regulates P domain and leucine rich repeat protein phosphatase 2 (PHLPP2). circAPAPC7 (Figure 4) over-expression in MIA PaCa-2 and AsPC-1 PDAC cells suppressed proliferation and colony formation (144). In orthotopic in vivo models in nude mice, circAPAPC7 inhibited growth of MIA PaCa-2 and AsPC-1 xenografts. It sponged miR-373 and up-regulated PHLPP2. The latter dephosphorylated AKT, inhibited cell proliferation via down-regulation of cyclin D1 and prevented muscle wasting by decreasing secretion of TGFβ. PHLPP2 is part of the PHLPP phosphatase family comprised of two members; it decreases proliferation and promotes apoptosis in cancer cells (145). Its paralog, PHLPP1, has been shown to regulate the Ser-Thr kinase AKT2, to inhibit proliferation and to induce apoptosis in PDAC cells (146).
Circ scm-like with four malignant brain tumor domains (circSFMBT1) up-regulates p21 activated kinase 1 (PAK1). circSFMBT1 (Figure 4) was up-regulated in PDAC tissues and corresponding cell lines (147). It promoted proliferation, migration, invasion and EMT of PDAC cell lines in vitro and its knockdown inhibited growth and metastasis of PDAC xenografts in nude mice. circSFMBT1 sponged miR-330-5p and up-regulated PAK1, a member of a family of six proteins (148). PAKs can activate pathways, such as RAS/mitogen-activated protein kinase kinase (MEK)/ERK, AKT/mTOR, WNT/β-catenin and JNK/NF
B. PAK1 is involved in proliferation pathways, apoptosis resistance, restoration of DNA damage, creation of an inflammatory environment, and immune suppression (149, 150). PAK1 inhibitor CP734 showed promising in vitro and in vivo results in a preclinical model of PDAC (151).
Circ cullin-2 (circCUL2) up-regulates myeloid differentiation primary responders 88 (MYD88). High expression level of MYD88 in PDAC tissues correlated with poor prognosis (152). circCUL2 (Figure 4) was expressed in CAFs, sponged miR-203a-3p, and up-regulated adaptor protein MYD88. circCUL2-transduced fibroblasts promoted lung metastasis of PANC-1 and MIA PaCa-2 PDAC cells in nude mice. In an orthotopic model with these cell lines, increase of tumorigenesis and higher abdominal metastasis was observed. These effects were inhibited with an antibody directed against interleukin 6 (IL6). In a PDX model, si-circ CUL2 and an IL6 antibody reduced tumor growth (152). The role of CAFs in promoting progression of PDAC by creating an inflammatory, desmoplastic and immune-suppressive environment has been described (153, 154). The IL6/JAK2/STAT3 pathway plays an important tumor-promoting role in several types of cancer (155).
circ007367 up-regulates yes-associated protein-1 (YAP-1). circ007367 (Figure 4) was up-regulated in PDAC tissues and cell lines and correlated with aggressive clinico-pathologic characteristics (156). circ007367 mediated proliferation, migration, and invasion in PANC-1 and AsPC-1 PDAC cells in vitro. It sponged miR-6820-3p and up-regulated YAP-1, which is a downstream target of the Hippo pathway (157, 158). YAP-1 acts as a transcriptional co-regulator, which promotes transcription of genes involved in proliferation and inhibits apoptotic genes (157, 158). In PDAC, YAP-1 modulates the function of stellate cells, recruitment of tumor-associated macrophages and myeloid-derived suppressor cells and is associated with poor prognosis (159).
circ ubiquitin-like with PH and ring finger domain 1 (circUHRF1) up-regulates ADP-ribosylation factor-like protein 4C (ARL4C). circUHRF1 (Figure 4) was highly expressed in PDAC tissues (160). In PANC-1 and AsPC-1 cells, circUHRF1 mediated proliferation, invasion, migration, and EMT in vitro. si-circ-UHRF1 inhibited growth of PANC-1 xenografts after s.c. implantation into nude mice. circUHRF1 sponged miR-1306-5p and up-regulated GTPase ARL4C. The latter is a member of the ADP-ribosylation factor family of GTP-binding proteins, which acts as a key regulator of tubulogenesis and tumorigenesis and as target of WNT/ß-catenin and growth factor-RAS signaling (161). ARL4C also promotes growth and drug resistance of PDAC by regulating tumor-stromal interactions (162).
Circ dual-specificity phosphatase 22 (circDUSP22) up-regulates BCL-2/adenovirus E1B 19kD protein interacting protein 3 (BNIP3). circDUSP22 (Figure 4) was decreased in PDAC tissues and cell lines (163). Ectopic expression of circDUSP22 blocked proliferation, arrested cell-cycle, and induced apoptosis in PDAC cell lines in vitro and inhibited growth of PDAC-related xenografts in nude mice (163). circDUSP22 sponged miR-1178-3p and up-regulated BNIP3, a member of the BCL2 protein family (163). BNIP3 modulates the permeability of the outer mitochondrial membrane by forming homo- and hetero-oligomers in the membrane (164). Loss of BNIP3 is a late event in PDAC progression, contributes to resistance against 5-FU and GEM, and correlates with worse prognosis (165, 166).
circ0075829 up-regulates lysosomal adaptor MAPK and mTOR activator 3 (LAMTOR3). circ0075829 (Figure 4) was increased in PDAC tissues and cell lines and its expression correlated with clinical features of PDAC (167). circ0075829 suppressed proliferation, migration, and invasion of PDAC cells both in vitro and in vivo in nude mice after s.c. implantation. It sponged miR-1287-5p and up-regulated LAMTOR3/pERK signaling. LAMTOR3 acts as an adaptor protein, which hyperactivates MAPK signaling (168). In PDAC, LAMTOR3 has been shown to activate MAPK and mTOR signaling to promote PDAC tumorigenesis (169).
Circ poliovirus receptor related-3 (circPVRL3) up-regulates suppressor of cytokine signaling 2 (SOCS2). circPVRL3 (Figure 4) sponged miR-194-5p and up-regulated SOCS2 (170). circPVRL3 inhibited proliferation and migration of CFPAC-1 and PANC-1 PDAC in vitro and in vivo after s.c. implantation into nude mice. mir-194-5p was up-regulated in PDAC tissue compared to tumor-adjacent tissue. SOCS2 is member of a family of eight proteins, which inhibit cytokine signaling and modulate immune responses (171, 172). They can interfere with JAK/STAT signaling by binding to phosphorylated residues on target proteins via their src homology 2 (SH2) domains and inhibit jun-activated kinase (JAK) activity through their N-terminal motif resulting in degradation by the proteasome (171, 172). Up-regulation of SOCS2 in cancer causes mitochondrial dysfunction and promotes apoptosis (173). The SOCS2 gene was found to be methylated by DNA methyltransferase 3A (DNMT3A) in PDAC (174).
circ0047744 up-regulates suppressor of cytokine signaling 5 (SOCS5). circ0047744 (Figure 4) was down-regulated in PDAC tissues and down-regulation correlated with lymph node metastasis and overall survival of patients (175). Functionally, circ0047744 inhibited invasion of MIA PaCa-2 and PANC-1 PDAC cells in vitro and in vivo in nude mice by sponging miR-21 and up-regulation of SOCS5. miR-301 has been shown to promote pancreatic cancer invasion and metastasis through JAK/STAT3 signaling by targeting SOCS5 (176).
Circ RNAs Involved in the Expression of Components of the Ubiquitination System
Circ syntaxin 6 (circSTX6) up-regulates non-muscle myosin 9 (MYH9) and cullin 2 (CUL2). circSTX6 (Figure 5) was found over-expressed in PDAC tissues (177). In vitro, circSTX6 promoted proliferation, migration, and invasion of CFPAC-1 and PANC-1 PDAC cells. circ STX6 increased growth and metastasis to the lungs and liver of CFPAC-1 and PANC-1 cells after s.c. implantation into nude mice. circSTX6 sponged miR-449b-5p, resulting in up-regulation of MYH9. In addition, it interacted with CUL2, which is part of the CUL2-E3 ubiquitin ligase complex and prevented degradation of HIF-1α (178), which drives expression of MYH9. The latter is expressed in PDAC and acts as a mechano-responsive protein, which promotes metastatic behavior (179).
Circular RNAs regulating ubiquitination, autophagy and components of other categories with activity in preclinical pancreatic cancer in vivo models. Upward arrows: up-regulated, downward arrows: down-regulated. circATG7: circ autophagy-related 7; circMBOAT2: circ O-acetyltransferase domain containing 2; circNEIL3: circ Nei-like DNA glycosylase 3; circPTPRA: circ protein tyrosine phosphatase receptor type A; circRHOBTB3: circ RHO-related BTB-domain-containing protein 3; circSTX6: circ syntaxin 6; circRTN4: circ reticulon 4; Col11A: collagen 11A; CUL2: cullin 2; FSCN1: fascin 1; GOT1: glutamic oxaloacetate transminase 1; HIPK2: homeobox-interacting protein kinase 2; HOTTIP: lnc RNA HOXA transcript at the distal tip; HuR: human antigen R; MYH9: non-muscle myosin 9; LMNB1: laminin B1; MET: metastasis; miR: microRNA; NACC1: nucleus accumbens associated 1; RAB11FIP1: RAB 11 family interacting protein 1; S100P: S100 protein P; STAMP: STAM binding protein 2; STIL: SCL/TAL1 interrupting locus; TG: tumor growth.
circ 0007334 up-regulates STAM binding protein (STAMBP). circ0007334 (Figure 5) was over-expressed in PDAC tissues in comparison to adjacent normal tissues and correlated with poor prognosis (180). In BxPC-3 and SW1990 PDAC cells, circ0007334 promoted invasion in vitro and tumor growth in nude mice. circ0007334 was found to up-regulate STAMBP via binding of miR-1272 and to activate NF
B signaling (180). STAMBP functions as an endosome-associated deubiquitinase, binds to STAM, a component of the endosomal sorting complex and regulates cytokine secretion in PDAC through the NOD-, LRR- and pyrin-domain containing protein (NLRP) inflammasome (181). STAMBP has been shown to promote lung adenocarcinoma metastasis through activation of the EGFR/MAPK signaling pathway (182).
Circ RNAs Involved in the Expression of Autophagy-related Components
Circ autophagy-related 7 (circATG7) up-regulates ATG7 and recruits human antigen R (HuR). circATG7 (Figure 5) was increased in PDAC tissues in comparison to adjacent normal tissues and was found in the cytoplasm as well as in the nucleus (183). It promoted proliferation, motility, and autophagy in MIA PaCa-2 and PANC-1 PDAC cells. In corresponding xenografts in nude mice, circATG7 increased growth after s.c. implantation and metastasis to the lungs after tail vein injection. circATG7 sponged miR-766-5p and up-regulated ATG7. In addition, it increased the stability of ATG7 mRNA by recruiting HuR. ATG7 is an E1-like ligase that plays an essential role in autophagosome biogenesis by conjugating ATG5 to ATG12 (184). HuR functions as an mRNA binding protein, regulates its stability and nucleus-to-cytoplasm shuttling (185).
Circ RHO-related BTB-domain-containing protein 3 (circRHOBTB3) up-regulates nucleus accumbens associated 1 (NACC1). circRHOBTB3 (Figure 5) expression levels were correlated with worse prognosis in PDAC patients (186). It promoted proliferation and autophagy of MIA PaC-2 and PANC-1 cells in vitro and in vivo in nude mice. circRHOBTB3 sponged miR-600 and up-regulated NACC1. This protein acted as a promoter of autophagy and AKT/mTOR signaling and bound to actin to regulate cytokinesis (186, 187). Autophagy is a mechanism by which cellular macromolecules are delivered to lysosomes for degradation to generate energy. In ovarian cancer, NACC1 has been shown to mediate invasion and migration (188). For PDAC, also opposite findings claiming that low expression of NACC1 predicts a poor prognosis are available (189). Overall, one should keep in mind that stimulation or inhibition of autophagy can have tumor promoting or inhibitory effects in a context-dependent manner (190). It has been reported that dysfunctional autophagy plays a role in PDAC (191).
Circ RNAs Mediating Expression of Proteins of Other Categories of Targets
Circ protein tyrosine phosphatase receptor type A (circPTPRA) up-regulates laminin B1 (LMNB1). circPTPRA (Figure 5) was up-regulated in PDAC tissues and over-expression was closely associated with lymph node invasion and poor prognosis in patients (192). It promoted proliferation, invasion and EMT in vitro in AsPC-1 and PANC-1 cells and growth of corresponding xenografts after s.c. implantation into nude mice. circPTPRA sponged miR-140-5p and up-regulated LMNB1. Laminins are a family of extracellular matrix glycoproteins located in the basement membrane, which are composed of three non-identical chains α, β and γ (193). LMNB1 was shown to be involved in processes, such as proliferation, chromosome distribution and DNA repair (194). In PDAC, LMNB1 correlates with low-grade differentiation and poor prognosis and its knockdown in PDAC cells inhibits proliferation, invasion, tumorigenicity, and metastasis (195).
circ-0005105 up-regulates collagen 11A1 (COL11A1). circ0005105 (Figure 5) was up-regulated in PDAC tissues and correlated with poor outcome in patients (196). In PANC-1 and SW1990 cells, circ-0005105 promoted proliferation, invasion, and migration in vitro. Silencing of circ-005105 in PANC-1 cells inhibited tumor growth after s.c. implantation and metastasis after tail vein injection in nude mice. circ-005105 sponged miR-20-3p and up-regulated COL11A, a collagen subtype critical for fiber assembly. Collagens are the major component of the TME, participate in cancer fibrosis, contain at least one triple helix and interact with fibronectin, hyaluronic acid, laminin and metallo-proteinases (197). COL11A has been identified as a marker for poor prognosis in PDAC (198).
circ 0099999 up-regulates fascin bundling protein 1 (FSCN1). circ0099999 (Figure 5) was up-regulated in PDAC tissues and cell lines (199). Knockdown of circ0099999 impeded proliferation, migration, glycolysis, promoted apoptosis of PDAC cells in vitro and inhibited their growth as xenografts in nude mice. circ0099999 sponged miR-330-5p and up-regulated FSCN1. This protein cross-links F-actin filaments into tight parallel bundles and induces membrane protrusions, migration. motility, adhesion, formation, stabilization of filipodia, EMT, and autophagy (200). FSCN1 is over-expressed in many types of cancer and correlates with poor prognosis (201). In PDAC, FSCN1 has been shown to promote invasion and metastasis (202).
Circ-membrane bound O-acyltransferase domain containing 2 (circMBOAT2) up-regulates glutamic-oxaloacetic transaminase 1 (GOT1). circMBOAT2 (Figure 5) was up-regulated in tissues and cell lines of PDAC (203). In PANC-1 and SW1990 cells, silencing of circ-MBOAT2 repressed proliferation, migration, invasion, and glutamine metabolism, and induced apoptosis. Knockdown of circ-MBOAT2 in SW1990 xenografts inhibited tumor growth in nude mice after s.c. implantation. circ-MBOAT2 sponged miR-433-3p and up-regulated GOT1. This enzyme mediates proliferation, is involved in amino acid and especially in glutamine metabolism and is a potential target for cancer treatment (204-206). GOT1 inhibition promotes cell death and ferroptosis in pancreatic tumor cells (207).
circ0012634 up-regulates homeobox-interacting protein kinase 2 (HIPK2). circ0012634 (Figure 5) was down-regulated in PDAC tissues (208). In Capan-2 and PANC-1 cells, circ0012634 and exosomal RNA containing circ0012634 reduced proliferation, colony formation and glucose uptake in vitro. In nude mice, circ0012634 inhibited growth of PANC-1 xenografts and PANC-1 cells co-injected with exosomes containing circ0012634 showed reduced tumor growth. circ0012634 sponged miR-147b and up-regulated HIPK2. This enzyme is member of the HIPK family of ser-thr kinases which regulate mediators of transcription and chromatin modifiers (209). HIPK2 can be up- and down-regulated or mutated depending on the type of tumor and can function as an oncogene or as a tumor suppressor in a context-dependent manner (209). In PDAC, HIPK2 hinders proliferation and anaerobic glycolysis (210) and its down-regulation correlates with poor prognosis (211).
Circ Nei-like DNA glycosylase 3 (circNEIL3) up-regulates adenosine deaminase acting on RNA-1 (ADAR-1). circNEIL3 (Figure 5) levels were associated with poor prognosis and TNM stage in patients with PDAC and were an independent risk factor for survival (212). circNEIL3 promoted proliferation, migration, and invasion of CFPAC-1 and MIA PaCa-2 cells in vitro. In nude mice, circNEIL3 facilitated growth and metastasis of CFPAC-1 and MIA PaCa-2 cells after s.c. implantation. circNEIL3 sponged miR-432-5p and up-regulated ADAR-1. It enhanced transcription factor GLI-family of zinc finger 1 (GLI-1) A-to-I double-stranded RNA editing, stimulating the cell-cycle and the EMT pathway (212). ADAR-1 mediated RNA editing has been shown to exert a pro-tumorigenic role (213, 214). It has been reported that ADAR-1 can enhance the transcriptional activity of GLI-1, a downstream effector of hedgehog signaling by A-to-I editing (215, 216). GLI-1 has been shown to promote progression and metastasis of PDAC and to exert a negative impact on patient survival (217).
Circ reticulon 4 (circRTN4) up-regulates lnc RNA HOXA transcript at the distal tip (HOTTIP) and induces epithelial mesenchymal transition (EMT). circRTN4 (Figure 5) was up-regulated in PDAC tissues (218). In vitro, circRTN4 promoted growth and clonogenic ability of CFPAC-1 PDAC cells. circRTN4 stimulated growth of CFPAC-1 cells after s.c. implantation and metastasis to the liver after orthotopic implantation into the pancreas of nude mice. Mechanistically, a two-fold mode of action was revealed. circRTN4 sponged miR-497-5p, resulting in up-regulation of lncRNA HOXA transcript at the distal top (HOTTIP), which up-regulated transcription factor HOXA13. In addition, circRTN4 interacted with RAB11 family interacting protein 1 (RAB11FIP1) protecting it from degradation, resulting in up-regulation of EMT-mediating transcription factors, such as SLUG, SNAIL1, TWIST and ZEB1. lncRNA HOTTIP also modulates stem cell properties of PDAC by regulating transcription factor HOX9 (219). RAB11FIP1 has been shown to mediate EMT in esophageal cancer (220).
circ0000069 up-regulates SCL/TAL1 interrupting locus (STIL). circ0000069 (Figure 5) was markedly up-regulated in PDAC tissues and cell lines (221). Its down-regulation in MIA PaCa-2, PANC-1 and SW1990 cells inhibited proliferation, migration, invasion, and induced apoptosis. In nude mice, down-regulation of circ0000069 decreased tumor growth after s.c. implantation of PDAC cancer cells. circ0000069 sponged miR-144 and up-regulated STIL. This protein functions as a regulator of the mitotic centrosome to promote cell cycling (222). STIL can bind to cytoplasmic suppressor of fused (SUFU), which heterodimerizes with GLI-1, allowing GLI-1 to translocate into the nucleus (223). GLI-1 functions as a promoter of PDAC (224).
circ0092314 up-regulates S100 protein P (S100P). circ0092314 (Figure 5) was highly expressed in PDAC tissues and cells (225). It enhanced proliferation and EMT in AsPC-1 and PaCa-2 cells in vitro. Over-expression of circ0092314 in AsPC-1 cells accelerated tumor growth after s.c. implantation into nude mice. circ0092314 sponged miR-671 and up-regulated S100 protein S100P. S100 proteins, a family of 21 proteins, function as intracellular Calcium-binding proteins and as extracellular factors (226). S100P can advance progression and metastasis of PDAC (227). In PDAC, expression of S100P is associated with drug resistance and correlates with worse clinical outcome (228).
Technical Issues
Up-regulated circRNAs can be inhibited by ASO, siRNA, shRNA or CRISPR-CAS technology (229-232). Down-regulated circRNAs can be reconstituted with plasmid- or viral-vector based gene transfer (233). There are still several hurdles, which have to be solved depending on the specific approach under consideration. These include pharmaco-kinetic and pharmaco-dynamic-, specificity- and delivery-issues (234, 235). These topics are not discussed in further detail in this review. The delivery issues have been approached by development and optimization of polymer- and lipid-based nanoparticles (LNP) (236). The latter are composed of phospholipids, such as phosphatidyl-choline and phosphatidyl-ethanol-amine, cholesterol or polyethylene glycol (PEG)-functionalized lipids. By variation of the composition of the LNPs, selective organ-targeting (SORT) vehicles have been developed targeting liver, lungs and spleen (237-239). Muscle-specific delivery has been achieved by conjugating LNPs with antibody fragments (240). Liver-specific targeting also has been demonstrated by conjugating N-acetylgalactosamine (GalAc) to LNPs, which are internalized by the asialoglycoprotein receptor (241).
Regarding the identified targets, previously untargetable proteins can be degraded by proteolysis-targeting chimeras (PROTACS), which are bifunctional moieties composed of a target binding moiety and a component mediating degradation by the proteasome (242, 243). Also, mRNA-based vaccination of PDAC patients might result in improvement of therapeutic intervention in PDAC patients (10).
Target-related Conclusions
We have identified circRNAs and their corresponding targets with activity in PDAC-related preclinical models. They affect chemo-resistance (n=5), secreted proteins and transmembrane receptors (n=15), transcription factors (n=9), signaling components (n=11), targets of ubiquitination (n=2) and autophagy (n=2), as well as components belonging to other categories (n=9). Chemo-resistance (GEM, ERL) is mediated by a complexity of mechanisms (Figure 1). Three circRNAs affect SLC receptors, such as SLC4A4 and SLC7A11 (Figure 2). The target relevance and tractability of the transcription factors c-MYC, E2F2, E2F3 and HIF-1α should be explored in further detail (Figure 3). As outlined in Figure 4, identified targets support interference with PI3K, hedgehog, mTOR and PAK1 signal transduction. Since the outcome of interference with ubiquitination and autophagy is context-dependent, further target validation experiments are necessary to support therapeutic intervention in PDAC (Figure 5). Interference with glutamine metabolism with GOT1 as a target is justified by preclinical evidence. However, it is unclear whether a therapeutic window can be defined due to the general physiological relevance of this pathway (Figure 5). Inhibition of RNA editing by interference with ADAR-1 is a preclinically underexplored mechanism for attenuating of PDAC progression (Figure 5). Modulation of lncRNA HOTTIP remains another strategy for treatment of PDAC (Figure 5) to be investigated in further detail.
Footnotes
Conflicts of Interest
AN is and UHW was an employee of Roche.
Authors’ Contributions
AN and UHW contributed equally to all aspects of the manuscript.
- Received May 7, 2024.
- Revision received June 3, 2024.
- Accepted June 4, 2024.
- Copyright © 2024, 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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