Introduction
Hepatocellular carcinoma (HCC) is the most common cancer and the leading cause of cancer-associated mortality worldwide. Surgical resection is the first choice treatment for HCC, but not for advanced HCC patients [
1]. For unresectable HCC, treatment options include transplantation, ablation, transarterial chemoembolization, targeted therapies and immunotherapies. Sorafenib, a multikinase inhibitor, is a first-line targeted drug approved by the US Food and Drug Administration for advanced HCC [
2]. According to the results of the Sorafenib Hepatocellular Carcinoma Assessment Randomized Protocol (SHARP) trial, sorafenib can prolong survival of HCC patients. However, drug resistance limits its efficacy. Although some mechanisms have been reported for sorafenib resistance, such as epithelial-mesenchymal transition, the proliferation of cancer stem cells, and metabolic reprogramming, the exact cause is still elusive [
3]. Therefore, understanding the underlying molecular basis of HCC sorafenib resistance and developing mechanism-based therapies are urgently needed.
Circular RNAs (circRNAs) have a circular configuration through a typical 5′ to 3′-phosphodiester bond and are recognized as a class of functional non-coding RNAs (ncRNAs). CircRNAs regulate biological processes by mediating alternative splicing (AS) of RNAs, cis-regulation of transcription, and by acting as competing endogenous RNAs (ceRNAs) [
4]. Importantly, many circRNAs are involved in cell proliferation, differentiation, apoptosis and invasion during tumor progression [
5,
6]. Recent studies show that the circRNA, CDR1as, can accelerate the proliferation and migration of HCC cells by promoting the expression of AFP via sponging miR-1270 [
7], while circRHOT1 can inhibit HCC development and progression via recruiting TIP60 to initiate NR2F6 expression [
8]. Circ_0003418 increases the sensitivity of HCC cells to cisplatin by inhibiting the Wnt/β-catenin pathway [
9]. However, the roles of circRNAs in HCC sorafenib resistance remain unknown.
N6-methyladenosine (m
6A) is the most prevalent internal modification associated with eukaryotic mRNAs and ncRNAs and it influences many steps of mRNA metabolism, including splicing, export, translation, and stability [
10]. The m
6A modification has been implicated in various cellular and physiological events, including carcinogenesis [
11]. In acute lymphoblastic leukemia (ALL), levels of METTL3 (a component of the methyltransferase complex that catalyzes adenosine methylation) increase in AML patients and it plays an oncogenic role by inhibiting cell differentiation and apoptosis, and promoting cell proliferation through increased c-MYC, BCL2, and PTEN translation [
12]. In breast cancer, mammalian hepatitis B X-interacting protein (HBXIP) enhances the expression of METTL3 by suppressing the tumor suppressor, let-7 g, and forms a positive feedback loop to enhance the level of HBXIP by facilitating m
6A modification of mRNA, which promotes cell proliferation [
13]. In HCC, m
6A modification also regulates the progress of oncogenesis. SIRT1 promotes HCC by increasing the overall m
6A modification level to attenuate the expression of the tumor suppressor, GNAO1 [
14]. However, the functions of m
6A modification in HCC sorafenib resistance remain elusive. Moreover, the roles of m
6A-modified circRNAs in HCC sorafenib resistance also need further investigation.
In the present study, we found that circRNA-SORE (also named circRNA_104,797 and circ_0087293) was up-regulated in sorafenib-resistant HCC cells, and was necessary for the maintenance of sorafenib resistance. By acting as a ceRNA and sequestering miR-103a-2-5p and miR-660-3p, circRNA-SORE competitively activates the Wnt/β-catenin pathway and promotes sorafenib resistance. The increased levels of circRNA-SORE in sorafenib-resistant HCC are due to its increased stability resulting from increased N6-methyladenosine (m6A) levels of a specific adenosine in circRNA-SORE. These results provide a novel mechanism for maintaining sorafenib resistance and demonstrate a proof-of-concept for targeting circRNA-SORE in sorafenib-treated HCC patients as a novel pharmaceutical intervention for advanced HCC.
Discussion
Many HCC patients are diagnosed too late for surgery because early stages of HCC present no clear symptoms. Other interventions, including molecular targeted therapy, are limited by a lack of efficacy. Unfortunately, patients who qualify for liver resection or transplantation have a high incidence of recurrence and metastases. Therefore, there is an urgent unmet medical need for the development of life-prolonging therapies. As the first US Food and Drug Administration-approved molecular targeted drug, sorafenib provided a 3 month prolongation of the median overall survival time [
25,
26] and transient and limited efficacy of sorafenib was frequently reported in HCC patients. Adverse effects, such as rash, diarrhea, high blood pressure, and hand-foot syndrome, also limit high-dosage use of sorafenib [
27]. Moreover, the STORM trial [
28] suggested that HCC patients who accepted radical treatment (resection or ablation) could not benefit from sorafenib treatment in the adjuvant setting, posing more challenges to its clinical application. Sorafenib resistance prompts the need for new therapies to overcome resistance [
29]. Multiple mechanisms underlying impaired sensitivity to sorafenib in HCC have been investigated, including Wnt/β-catenin, TGFβ, Ras/MEK/ERK, PI3K/Akt, TNFα/NF-κB, and JAK/STAT pathways, autophagy, epithelial-mesenchymal transition, cancer stem cells, tumor microenvironment, and epigenetic regulation (involving miR-222, miR-494, miR-21 and miR122) [
3]. To overcome sorafenib resistance and lower its onset concentration, efforts were made to develop combined therapies [
30‐
34]. Yet, the overall outcomes of liver cancer are still far from satisfactory. In the present study, sorafenib-resistant cell lines and animal models were developed to simulate the sorafenib resistance in HCC patients.
Non-coding RNAs are functional RNAs transcribed from the genome from which proteins cannot usually be translated. They mainly include miRNAs, long non-coding RNAs (lncRNAs) and circRNAs. Recent studies indicate that ncRNAs play vital roles in diverse biological and pathological processes, including cancer [
35]. First described in 1993, miRNAs are small noncoding RNA molecules that play crucial post-transcriptional regulatory roles. Importantly, miRNAs are involved in the development of sorafenib resistance through complementary base pairing with mRNAs, predominantly in the 3′-UTR. For example, miRNA-216a/217-induced epithelial-mesenchymal transition promotes sorafenib resistance and liver cancer recurrence by targeting PTEN and SMAD7 [
36]. However, its small linear structure has inherent limitations, including extensive but limited effect, poor specificity, instability, and off-target effects. In contrast to miRNAs, circRNAs have a covalently-closed loop structure with neither 5′ to 3′ polarity nor a polyadenylated tail, which makes them more stable than their linear counterparts and more resistant to RNase R degradation.
circRNAs were occasionally identified more than 20 years ago and were thought to be of low abundance and to result from alternative splicing errors during transcription. Using high-throughput sequencing and novel computational approaches, circRNAs derived from exons or introns were determined to be widespread and diverse endogenous eukaryotic ncRNAs participating in various normal and disease-related processes [
37,
38]. Recent findings indicate that circRNAs function through RNA interactions, protein interactions, or by serving as transcription or splicing regulators [
15]. For example, the circRNA, CDR1
as, harbors more than 60 conserved binding sites for miR-7 [
38]. Additionally, circRNAs may be associated with various miRNAs, as demonstrated for circHIPK3, which can bind to multiple miRNAs [
39]. Recently, particular functions of circRNAs have been revealed in HCC; circFBLIM1 and circ-FOXP1 can act as ceRNAs to promote HCC progression [
40,
41], while circMTO1 can act as a sponge of microRNA-9 to suppress HCC progression [
41]. Also, study of circ_0003418 showed that circRNAs can influence anti-tumor treatments such as cisplatin [
9]. Containing multiple binding sites for particular miRNAs, circRNAs have specific and efficient functionality. Furthermore, tissue- and stage-specific expression makes circRNAs potential targets for clinical intervention. Also, because of their RNA stability, circRNAs are valued as candidates for non-invasive biomarkers. circ_0005075, circ_0016788, ciRS7, circ_0128298, circ_0091579, and circ-CDYL have been identified as potential diagnostic biomarkers for HCC [
42]. However, the relationship between circRNAs and resistance of HCC to sorafenib has not been reported. Thus, we aimed to fully understand the circRNA landscape when HCC patients acquired sorafenib resistance during treatment. We identified consistently up-regulated circRNA-SORE in sorafenib resistant cell lines and CDX and PDX models that was critical for the maintenance of sorafenib resistance. Importantly, orthotopic in vitro developed sorafenib-resistant CDX model and subcutaneous in vivo developed sorafenib-resistant CDX model were applied in the present study, showing that circRNA-SORE silencing could effectively reverse the acquired sorafenib resistance and retard tumor progression. In particular, we found specific mechanisms by which circRNA-SORE could specifically bind miR-103a-2-5p and miR-660-3p to act as a miRNA sponge to competitively activate Wnt/β-catenin and induce sorafenib resistance, thereby identifying a potential biomarker for prediction of sorafenib resistance and a promising therapeutic target for HCC. Derived from the 7th and 8th exons of
TLE4, circRNA-SORE is generated into a circular form by back-splicing [
43]. It was recently reported that exon-derived circRNAs are predominantly located in the cytoplasm. In our study, both nucleocytoplasmic fractionation and circRNA-FISH assays revealed that circRNA-SORE located predominantly in the cytoplasm of HCC cells.
m6A modification was first discovered in the 1970s but the development of RNA-sequencing techniques and clarification of the proteins involved make a deeper understanding of the process possible. m
6A depends on m
6A writers (METTL3, METTL14, KIAA1429, WTAP, RBM15 and ZC3H13), erasers (FTO and ALKBH5) and readers (YTHDC1, YTHDC2, YTHDF1, YTHDF2 and HNRNPC) [
44]. m
6A modifications may play important roles in RNA production, stability and interactions in cancers [
45]. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a conserved lncRNA, is highly methylated with m
6A. Two of these m
6A residues can prevent RNA local secondary structure formation and enhance the recognition and binding of hnRNPC to a U5-tract in the MALAT1 hairpin through an “m6A switch” mechanism [
46]. hnRNPA2B1 can recognize pri-miRNAs with m
6A marks to promote interaction of DGR8 and pri-miRNAs and miRNA processing [
47]. However, in HCC, the roles of m
6A modification are intricate and controversial. Knock-down of YTHDF2 suppresses proliferation of HCC cells [
48]; however, YTHDF2 was also reported to suppress HCC tumors by targeting EGFR, IL11 and SRPINE2 [
49]. Indeed, the detailed roles of m
6A modification in HCC need further investigation. Recently, m
6A-modified circRNAs were found with cell-type-specific expression [
45]. m
6A can recruit YTHDF3 and initiation factor, eIF4G2, to regulate protein synthesis from circRNAs [
19]. The change of m
6A level by ALKBH5 and METTL3 affects circRNA biosynthesis in spermatogenesis by regulating enhanced splicing and promotes circRNA formation [
50]. YTHDF2–HRSP12–RNase P/MRP-mediated endoribonucleolytic cleavage is related to m
6A-containing circRNA decay [
51]. However, the functions of m
6A-modified circRNAs in HCC and sorafenib resistance were still elusive.
In this study, we identified a predicted m
6A site in circRNA-SORE by a series of experiments. Our results show that the m
6A level of circRNA-SORE is increased in sorafenib-resistant cells, and that the expression of circRNA-SORE is decreased when its m
6A modification was inhibited. Recently, lncRNA GAS5-AS was found to enhance GAS5 stability by interacting with ALKBH5 and regulating m
6A modifications of GAS5, which was dependent on ALKBH5 and YTHDF2 [
52]. In the present study, mechanistic dissection suggested that the m
6A modification can stabilize circRNA-SORE. However, no significant differences in the expression level of m
6A-related proteins were found between sorafenib-resistant and parental cells. The mechanism of increased circRNA-SORE m
6A levels in sorafenib-resistant cells requires further investigation.
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