Roles of circular RNAs in the pathogenesis and treatment of pancreatic cancer

Circular RNAs are single-stranded RNAs with a covalently closed structure formed by the process of back-splicing. Aberrant expression of circular RNAs contributes to the pathogenesis of a wide range of cancers. Pancreatic cancer is one of the most lethal cancers due to diagnostic difficulties and limited therapeutic options. Circular RNAs are emerging as novel diagnostic biomarkers and therapeutic targets for pancreatic cancer. Moreover, recent advances in the therapeutic application of engineered circular RNAs have provided a promising approach to overcoming pancreatic cancer. This review discusses the roles of circular RNAs in the pathogenesis of pancreatic cancer and in potential treatment applications and their usefulness as diagnostic biomarkers.

With clarification of the diversity of circRNAs in the human transcriptome, their biological functions are also being elucidated. This review discusses the roles of circRNAs in the pathogenesis of pancreatic cancer and potential therapeutic applications and their utility as diagnostic markers for pancreatic cancer.
2 Roles of circRNAs in the pathogenesis of pancreatic cancer Although circRNAs have identical exon sequences as those of their linear cognate mRNAs, they have distinct cellular functions. circRNAs can interact with DNA, RNA, and proteins to modulate transcription, act as decoys for miRNAs and proteins, enhance mRNA stability, provide scaffolds for protein complex formation, and serve as templates for translation. In pancreatic cancer, many circRNAs are dysregulated and play important roles in various aspects of cancer progression. Most pathogenic circRNAs function as miRNA decoys, and several circRNAs have been reported to interact with proteins to stabilize mRNA, inhibit posttranslational modifications, and provide scaffolds for formation of protein complexes (Figure 1). This chapter focuses on representative circRNAs involved in the pathogenesis of pancreatic cancer for which the detailed mechanisms have been elucidated, and those that could not be introduced in the text are summarized in Table 1.

Decoys for miRNAs
As first demonstrated with CDR1as, some circRNAs function as decoys that bind to miRNAs and prevent them from binding to their target mRNAs (Hansen et al., 2013;Memczak et al., 2013). Hansen et al. also demonstrated that CDR1as reduced miR-7 activity, whereas the cognate linear RNA of CDR1as had little effect on miR-7 activity, suggesting that circRNAs have unique biological functions that differ from linear RNAs despite having the same sequence. In pancreatic cancer, many pathogenic circRNAs have been reported to function as miRNA decoys, leading to cell proliferation, inhibition of apoptosis, metastasis, chemotherapy resistance, and metabolic reprogramming. For example, circBFAR was identified as an upregulated circRNA in pancreatic cancer, and its overexpression was shown to be correlated with poor prognosis (Guo X. et al., 2020). Mechanistically, circBFAR binds to miR-34b-5p and decreases MET expression, thereby activating the MET/PI3K/Akt signaling pathway and promoting the progression of pancreatic cancer cells. In another example, circSLIT2 was shown to upregulate c-Myc expression by binding to miR-510-5p and to facilitate aerobic glycolysis in pancreatic cancer cells (Guan et al., 2021). In addition, many other circRNAs have been reported to contribute to pancreatic cancer progression in a cell-autonomous manner by disrupting miRNA function.
The circRNAs that are dysregulated in cancer cells not only increase their malignant potential in a cell-autonomous manner but also contribute to the progression of pancreatic cancer in a non-cell-autonomous manner by altering the tumor microenvironment ( Figure 2). circNFIB1 was shown to be downregulated and to be negatively associated with lymph node metastasis in pancreatic cancer patients (Guan et al., 2021). circNFIB1 functions as a decoy for miR-486-5p and antagonizes the miR-486-5p-mediated suppression of PIK3R1 expression. As PIK3R1 acts as an inhibitor of the PI3K/Akt signaling pathway, circNFIB1 inhibits the PI3K/Akt pathway and downregulates its downstream target, VEGF-C, resulting in suppression of lymphangiogenesis and lymph node metastasis.
Cancer-associated fibroblasts (CAFs) are the predominant cell type in the stroma of pancreatic cancer and play an important role in chemotherapy resistance. circFARP was identified as a circRNA specifically upregulated in gemcitabine-resistant CAFs, and high circFARP expression was shown to be correlated with poor progression-free survival (Guan et al., 2021). When pancreatic cancer cells were cultured in conditioned medium derived from circFARP-overexpressing CAFs, they acquired gemcitabine resistance. Mechanistically, circFARP enhances leukemia inhibitory factor (LIF) expression by decoying miR-660-3p, and LIF secreted from CAFs induces gemcitabine resistance in pancreatic cancer cells by activating the LIF/ STAT3 pathway. Interestingly, circFARP inhibits CAV1 degradation by interacting directly with CAV1, which Roles of pathogenic circRNAs in pancreatic cancerPathogenic circRNAs in pancreatic cancer function as miRNA decoys and interact with proteins to stabilize mRNA, inhibit post-translational modifications (PTMs), and provide scaffolds for protein complex formation.
Frontiers in Cell and Developmental Biology frontiersin.org 02 (Continued on following page) Frontiers in Cell and Developmental Biology frontiersin.org 03 promotes caveola-mediated exocytosis of LIF. Thus, circFARP functions as both a miRNA decoy and protein decoy.
Recent studies have shown that circRNAs are enriched in exosomes and may mediate intercellular communication between cancer cells and the tumor microenvironment to promote cancer progression (Seimiya et al., 2020). For example, circPDE8A was identified as a circRNA enriched in exosomes secreted from liver metastatic pancreatic cancer cells (Li Z. et al., 2018). circPDE8A functions as a decoy for miR-338 to upregulate MACC1 and stimulates invasive growth via the MACC/MET/ERK or AKT pathway. Exosomes derived from circPDE8A-overexpressing cancer cells activate the MACC/ MET/ERK or AKT pathway and promote epithelial-mesenchymal transition. In clinical practice, circPDE8A was also detected in exosomes from the plasma of pancreatic cancer patients, and high exosomal circPDE8A expression was shown to be correlated with TNM stage and poor prognosis. circIARS was also enriched in exosomes from liver metastatic pancreatic cancer cells (Li J. et al., 2018). Exosomal circIARS decoyed miR-122 to increase RhoA expression, which in turn increased the permeability of human umbilical vein endothelial cells and facilitated invasion of pancreatic cancer cells. Another study showed that hypoxiainduced exosomal circZNF91 promoted chemoresistance in  (Zeng et al., 2021). circZNF91 was upregulated in exosomes derived from pancreatic cancer cells under hypoxic conditions compared with normoxic conditions. When hypoxia-induced exosomal circZNF91 was transmitted to normoxic tumor cells, circZNF91 bound to miR-23b-3p and upregulated SIRT1 expression. Consequently, SIRT1 stabilized HIF-1α protein, which enhanced glycolysis and promoted chemoresistance in recipient cells. These results suggest that exosomal circRNAs may function as decoys for miRNAs in recipient cells and play important roles in cancer progression by mediating signal transmission between pancreatic cancer cells and the tumor microenvironment.
Although there is accumulating evidence that circRNAs are involved in the progression of pancreatic cancer, caution is required when considering whether a given circRNA has a measurable effect on cellular functions by acting as an miRNA decoy. The most well-studied circRNA with miRNA decoy function is CDR1as, which has over 70 binding sites for miR-7 (Hansen et al., 2013). Another well-known circRNA has 16 miR-138 target sequences (Hansen et al., 2013). Compared with these well-known circRNAs, most circRNAs have few miRNA-binding sites and low expression levels (Guo et al., 2014). Therefore, when investigating the potential of a circRNA of interest to act as a miRNA decoy and contribute to the pathogenesis of pancreatic cancer, careful consideration of whether the expression of that circRNA and the number of miRNA-binding sites are sufficient to achieve a measurable effect is needed.

Inhibiting post-translational modifications
Some circRNAs interact directly with proteins and stabilize them by inhibiting ubiquitin-proteasomal degradation. For example, circFARP1, which is upregulated in CAFs, interacts with CAV1 and blocks the interaction between CAV1 and the E3 ubiquitin ligase ZNRF1 to inhibit CAV1 degradation (Hu et al., 2022). As CAV1 mediates caveola-mediated exocytosis, CAV1 stabilization enhances LIF exocytosis from CAFs and activates the JAK-STAT signaling pathway in pancreatic cancer cells. As another example, circRTN4 interacts with RAB11FIP, which plays an important role in epithelial-mesenchymal transition, and protects it from ubiquitin-proteasomal degradation (Wong et al., 2022). Prediction of the structure of the circRTN4-RAB11FIP1 complex revealed that circRTN4 blocked the Lys578 ubiquitination site of RAB11FIP1. These results suggest that circRNAs bind to proteins and enhance their stability by inhibiting ubiquitin-proteasomal degradation.
It has been suggested that circPTPN22 inhibits protein-protein interactions and promotes STAT3 acetylation (He et al., 2021). circPTPN22 is upregulated in pancreatic cancer, and it interacts directly with STAT3 to block the interaction between STAT3 and SIRT1. In a xenograft model, knockdown of circPTPN22 enhanced intratumoral T cell infiltration and inhibited tumor growth. These results suggest that circPTPN22 inhibits STAT3 deacetylation by SIRT1 and promotes immune evasion of pancreatic cancer.

Scaffolds for protein complexes
circRNAs also serve as scaffolds for mRNA-protein complex formation to enhance expression of the mRNA. circFOXK2 is highly expressed in pancreatic cancer and promotes cell proliferation, migration, and invasion (Wong et al., 2020). Pulldown of circRNAs followed by mass spectrometry revealed that circFOKX2 interacts with YBX1 and hnRNPK. The YBX1-hnRNPK complex is reportedly involved in cancer progression. To determine the direct targets of the YBX1-hnRNPK complex, a circFOKX2 RNA pulldown assay and RNA immunoprecipitation assay of YBX1 and hnRNPK were performed, and NUF2 and PDXK mRNA were identified as direct targets. Knockdown of circFOXK2 downregulated the expression of NUF2 and PDXK mRNA and inhibited the interactions of YBX1 and hnRNPK with NUF2 and PDXK mRNA. These results suggest that circFOXK2 provides a scaffold for the YBX1-hnRNPK complex to promote expression of NUF2 and PDXK. Another study showed that circATG7 interacted with HuR and increased the mRNA level of ATG7 (He et al., 2022). circATG7 exists in both the cytoplasm and nucleus; cytoplasmic circATG7 acts as a decoy for miR-766-5p to decrease the expression of its target gene, ATG7, while nuclear circATG7 interacts with HuR and increases the stability of ATG7 mRNA in a HuR-dependent manner.
Taken together, these observations suggest that pathogenic circRNAs in pancreatic cancers function as miRNA decoys and/ or interact with proteins. However, given that the exact mechanisms of action of many circRNAs remain unknown, that circRNAs acting as templates for protein translation have not been identified in pancreatic cancer, and that a single circRNA can have multiple functions, the impact of circRNAs on the progression of pancreatic cancer may be much greater than known at present. Further studies are needed to elucidate the overall impact of circRNAs on the pathogenesis of pancreatic cancer.

Pancreatic cancer
Although early diagnosis is important to improve the prognosis of pancreatic cancer, most pancreatic cancer cases are diagnosed at an advanced stage. One reason for this is that pancreatic cancer biomarkers are ineffective for early diagnosis.
Frontiers in Cell and Developmental Biology frontiersin.org 05 For example, serum pancreatic enzymes, such as amylase, are elevated in only 20-50% of patients with pancreatic cancer, and serum CA19-9, which is routinely used as a biomarker for pancreatic tumors, is elevated in 70-90% of cases (Sharma et al., 2011). However, the sensitivity of CA19-9 for detecting pancreatic cancer lesions ≤2 cm decreases to approximately 50% (Liu et al., 2012), which is insufficient for early diagnosis. Therefore, the development of novel diagnostic biomarkers is required.
circRNAs have a half-life of 18.8-23.7 h, which is significantly longer than that of their cognate mRNAs (4.0-7.4 h) (Enuka et al., 2016). In addition, circRNAs exhibit characteristic expression patterns in different types of cancer (Vo et al., 2019). Due to their remarkable stability and specificity, circRNAs are regarded as potential diagnostic biomarkers for cancers. To date, five studies have reported the utility of serum circRNAs as diagnostic biomarkers for pancreatic cancer (Table  2). That is, circLDLRAD3, circPDAC, hsa_circ_0013587, and circ_001569 were reported to be aberrantly expressed in the serum of pancreatic cancer patients (Yang et al., 2017;Xu K. et al., 2021;Shen X. et al., 2021;Seimiya et al., 2021), and another study suggested that the combination of hsa_circ_0006220 and hsa_circ_0001666 can improve diagnostic performance compared with each circRNA alone (Hong L. et al., 2022). These studies demonstrated that circRNAs have a sensitivity of 0.45-0.76 and specificity of 0.70-0.90 for diagnosis of pancreatic cancer.

Intraductal papillary mucinous neoplasm
It has been suggested that circRNAs may be useful for diagnosis of not only pancreatic cancer but also intraductal papillary mucinous neoplasm (IPMN), which is a precancerous lesion of pancreatic cancer (Seimiya et al., 2021). It was shown that circPDAC is highly expressed in pancreatic cancer, and that high circPDAC expression is associated with lymph node metastasis and cancer stage. This circRNA is enriched in exosomes and can be detected in the blood of patients with pancreatic cancer, suggesting that measurement of circPDAC levels in blood may be useful for the diagnosis of pancreatic cancer. Interestingly, circPDAC was also detected in the blood of patients with IPMN. As there are currently no effective biomarkers for diagnosis of IPMN, circPDAC may serve as a novel diagnostic biomarker for this disease. Recently, pancreatic surveillance of high-risk individuals with genetic and familial risk factors for pancreatic cancer was shown to lead to early diagnosis and better long-term survival (Dbouk et al., 2022). In the same way, pancreatic surveillance in patients with IPMN may also reduce pancreatic cancer mortality through early detection.
A number of issues remain to be resolved for the routine application of circRNAs as diagnostic biomarkers for pancreatic cancer or IPMN in real-world clinical practice. First, because of the relatively small number of cases in the studies discussed above, it will be necessary to evaluate the diagnostic performance in larger cohorts including patients with early-stage pancreatic

FIGURE 2
Effects of circRNAs on the tumor microenvironment in pancreatic cancercircFARP enhances LIF expression in cancerassociated fibroblasts. circNFIB1 enhances VEGF-C expression in pancreatic cancer cells. circZNF91 and circPDE8A are transmitted from pancreatic cancer cells to nearby cancer cells. circIARS is transmitted from pancreatic cancer cells to endothelial cells.
Frontiers in Cell and Developmental Biology frontiersin.org cancer. In addition, PCR is commonly used to measure the expression levels of circRNAs in blood, but PCR is laborintensive and requires multiple steps, including RNA extraction from blood, reverse transcription, and cDNA amplification. Therefore, it is necessary to develop a simpler and more sensitive measurement method. Future research will resolve these issues and provide hope for early diagnosis of pancreatic cancer.
4 Therapeutic applications of circular RNAs 4.1 circRNAs as therapeutic molecules As circRNAs have unique biological functions and molecular characteristics, they have potential applications as new nucleic acid therapeutic agents. One possible application is to use circRNAs as stable molecules for RNA interference therapeutics. For example, engineered circRNA molecules encoding miR-34a-3p and -5p sequences, called db34a RNA, significantly inhibited angiogenesis of pancreatic cancer cells (Gnanamony et al., 2021). This is the only study to date that has examined the potential application of circRNAs as therapeutic molecules in pancreatic cancer, but a variety of applications are being investigated in other diseases, which have been reviewed elsewhere (Liu and Chen, 2022). Briefly, based on the stability and biological functions of circRNAs, various applications of these RNAs are being investigated, including as decoys for miRNAs and proteins, stable antisense RNAs, boosters of innate immune responses, inhibitors of innate immune responses, and templates for protein translation. These various circRNA-based therapies may also be applicable in pancreatic cancer.

Targeting pathogenic circRNAs
As the roles of circRNAs in the pathogenesis of pancreatic cancer become clearer, new therapeutic approaches targeting pathogenic circRNAs are also expected. For example, circRTN4 is highly expressed in pancreatic cancer and is correlated with liver metastasis (Wong et al., 2022). Knockdown of circRTN4 in pancreatic cancer cell lines inhibits their proliferation, migration, and invasion. Furthermore, circRTN4 knockdown significantly suppressed tumor growth and metastasis to the liver in a mouse xenograft model. In addition, many pathogenic circRNAs have also been reported to be potential therapeutic targets.
Although many circRNAs are expected to be therapeutic targets, several challenges remain for clinical application of circRNA-targeted therapies. Antisense oligonucleotides, siRNAs, shRNAs, and CRISPR-Cas systems can be used to target RNAs. Clinical application of CRISPR-Cas systems is being intensively evaluated, and several oligonucleotide drugs have been approved by the FDA and are clinically available (Roberts et al., 2020;Modell et al., 2022). To apply these molecules to circRNAtargeted therapies, they should specifically recognize backsplice junctions. However, because circRNAs have identical sequences to those of their cognate linear mRNAs, in cases where the back-splice junction has a similar nucleotide sequence to that of a canonical splicing junction, it may not be possible to engineer therapeutic molecules that specifically recognize circRNAs. Even with proper design, care is required regarding off-target effects, especially those on cognate linear mRNAs. A number of processes are involved in the regulation of circRNA expression, including transcription, splicing, nuclear export, and degradation (Chen, 2020). Elucidating these molecular mechanisms may provide new therapeutic targets for regulating circRNA expression levels.

Concluding remarks
With the development of RNA sequencing methods for circRNAs, a great deal of progress in the research of these molecules has been made over the past decade. Accordingly, some circRNAs have been shown to play important roles in a variety of pathological conditions, including pancreatic cancer (Rong et al., 2021b;Chen Q. et al., 2022). However, the functional significance of many circRNAs is largely unknown. They may be meaningless byproducts generated during the splicing process, but they may have important biological functions that remain to be elucidated. It is hoped that the currently unknown pathological roles of circRNAs will be further elucidated, and new therapeutic strategies for pancreatic cancer using circRNAs will be developed in the future. Hepatitis from AMED (to MO, #JP20ck0106557 and #JP22fk0210092).

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.