Background
Intrahepatic cholangiocarcinoma (ICC), as the second most common primary hepatic malignancy, accounts for approximately 10–15% of all primary liver cancers [
1]. Owing to the increased prevalence of nonalcoholic steatohepatitis and hepatitis C, the incidence of ICC is increasing globally, with an average annual growth of 4.4% over the past 10 years [
2]. ICC is generally asymptomatic at the early stage. Most ICC patients are diagnosed at advanced stages, for which limited therapeutic options are available, resulting in poor clinical outcomes. Currently, curative resection remains the cornerstone for cure of ICC, however, 60% of patients who undergo surgery develop recurrent or metastatic disease [
3,
4]. Therefore, a better understanding of the molecular mechanism underlying ICC metastasis and identification of new therapeutic targets to suppress metastasis are urgently required for improving the survival outcomes of ICC patients.
Circular RNAs (circRNAs), characterized by single-stranded and covalently closed loop structures, are usually generated by the back-splicing of exons from pre-mRNAs [
5]. Previously, circRNAs had been considered as by-products of splicing errors with low abundance. However, through deep RNA sequencing and bioinformatics, circRNAs have been demonstrated as widespread and a substantial presence within transcriptomes [
6]. Their prominent features of higher stability than parental linear RNAs, highly conserved expression across species, and tissue- or developmental stage-specific expression suggest that circRNAs may possess multiple biological processes [
7,
8]. In addition, emerging studies have proved a vital role of circRNAs in tumor initiation and progression, including hepatocellular carcinoma [
9], colorectal cancer [
10], glioblastoma [
11], and cholangiocarcinoma [
12]. Mechanistically, some circRNAs exert function by sponging microRNAs or binding proteins to manipulate gene expression [
13,
14], some serve as platforms for protein interaction [
15], while some others can encode functional peptides [
16]. However, little is known about the contribution of circRNAs in ICC metastasis.
Mitogen-activated protein kinases (MAPK) consist of three major subfamilies: the extracellular-signal regulated kinases (ERK), the c-jun N-terminal kinase or stress-activated protein kinases (JNK or SAPK), and MAPK14 [
17]. The ERK signaling pathway, which includes the kinases RAS, RAF, MEK, and ERK, is thought to be a three-tiered or four-tiered phosphorylation cascade that relay upstream signals from membrane receptors to a series of downstream effector substrates [
18]. In detail, extracellular signal proteins bind to specific cell-surface receptors, such as cytokine receptors, receptor tyrosine kinases (RTKs) and G protein-coupled receptors, and activate a series of signaling cascades involving RAS, RAF and MEK [
19]. As dual-specificity kinases, activated MEK can phosphorylate the conserved threonine and tyrosine residues within the activation loop of ERK, which then regulates some other protein kinases and transcription factors involved in cell proliferation, cell survival, cell migration and cell differentiation [
20,
21]. Deregulated activation of, or an enhanced dependence on, RAS/RAF/MEK/ERK pathway is a common feature of many human cancers, including ICC [
22‐
24]. However, whether and how this signaling is regulated by circRNAs remain elusive.
In the current study, through an in-deep analysis of human ICC tissues, we confirmed a circRNA (cNFIB, circBase ID: hsa_circ_0086376) as a tumor suppressor involved in ICC metastasis. Loss of cNFIB favors invasion and metastasis of ICC cells both in vitro and in vivo by activating MEK1/ERK signaling and downstream target genes. Furthermore, cNFIB completely binds to MEK1, thereby impeding ERK phosphorylation and transcriptional activity. More importantly, exogenous overexpression of cNFIB also enhanced anti-tumor effects of trametinib (a specific MEK inhibitor), which implies its promising potential as a therapeutic molecule for combating ICC metastasis.
Materials and methods
Human tissues
A total of 222 patients with ICC who underwent curative surgery between October 2010 and December 2017 at West China hospital, Sichuan University (Chengdu, China) were included in this study. The patients were divided into two cohorts. Cohort 1 included 40 patients (20 patients who experienced extrahepatic metastases after surgery and 20 patients who did not experienced metastases after surgery). We chose 30 primary ICC tissues from patients in cohort1 (15 primary ICC tissues from patients with extrahepatic metastases after surgery and 15 primary ICC tissues from patients without postoperative metastases) to perform circRNA-seq. Then the 40 samples (cohort 1) were used for circRNAs validation. Cohort 2 including 182 patients was used for quantification of cNFIB and analysis of the relationship between the expression levels of cNFIB and prognosis of ICC patients. The follow-up period was defined as the interval between surgery and death or recurrence. Overall survival (OS) was defined as the interval from the time of surgery to death. Recurrence-free survival (RFS) was defined as the time from surgery until the detection of any types of recurrence. Patients alive or without recurrence at the time of last follow-up visit were censused. Patients were divided into high and low cNFIB expression groups according to a median cut-off value. All samples and related information from patients in this study were collected with informed consent, and this study was approved by the Institutional Ethics Committee of West China hospital.
Cell lines and cell culture
All human ICC cell lines (HuCCT1, HCCC9810, and RBE) were purchased from Cell Bank of the Shanghai Institute for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). The three cell lines were maintained in RPMI-1640 medium with 10% fetal bovine serum (HyClone, USA). They were all cultured in a humidified incubator at 37 °C with 5% CO2.
Statistical analysis
The statistical analysis was carried out using SPSS 23.0 and Prism version 7.0 software (GraphPad Software). Data are shown as mean ± standard deviation (S.D). For continuous variables, two-sided Student’s t test was used for two comparisons. One-way ANOVA with Tukey’s post hoc test was used for multiple comparisons. The relationships between cNFIB expression and clinicopathological features of ICC patients were calculated by χ2 test or Fisher’s exact test, while the correlation between cNFIB and p-ERK expression was analyzed by Pearson’s correlation test. Survival data were measured by the Kaplan-Meier method and analyzed by the Log-rank test. P values less than 0.05 were considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Discussion
Although numerous improved management strategies are available in the clinic, cancer-related death of ICC remains high, which is largely caused by metastasis. With the development of sequencing techniques, a significant portion of circRNAs that are closely related to tumor metastasis have been identified. For instance, upregulation of circASAP1 in HCC contributed to tumor cell proliferation, migration, invasion, and ultimately resulted in pulmonary metastasis and poor survival of patients [
9]. Loss of circCDR1as induces melanoma invasion and metastasis via interaction with IGF2BP3 [
40]. However, few studies have investigated the role of circRNAs in ICC metastasis. In the present study, we identified cNFIB as a tumor suppressor that inhibited ICC growth and metastasis. We found that cNFIB was frequently deleted in metastatic ICC tissues. Importantly, cNFIB levels were inversely related to tumor numbers, TNM stage, lymph node metastasis and tumor differentiation. Patients with decreased cNFIB expression exhibited unfavorable prognosis. Moreover, gain-of-function and loss-of-function experiments suggested cNFIB negatively modulated proliferation and metastasis of ICC cells. To our knowledge, this is the first time to delineate the effects of circRNA on the metastasis of ICC, supplementing the rationale of cNIFB as a prognostic indicator for ICC patients.
Overactivation of RTK pathway is a common event during ICC development. For instance, gene fusion of FGFR2 RTK was reported to occur in around 20% ICC patients [
41], and BRAF RTK mutations at the V600E locus have been identified in approximately 5% of ICC cases [
42]. Therefore, aberrant alterations of RTK signaling make it amenable for therapeutic interventions at multiple levels. As one of the main pathways triggered by RTK, dysregulation of RAF/MEK/ERK signaling pathway has been identified in up to 35% of ICC [
43]. Large scale studies have demonstrated that constitutive activation of ERK was associated with the pro-malignant functions of a wide number of cancers including ICC [
44]. Consistent with these data, ERK signaling was hyperactive during cNFIB knockdown-induced ICC proliferation and metastasis. Blocking ERK with phosphorylation inhibitor could abolish the tumor-promoting effects of cNFIB downregulation. Generally, ERK kinase can only be activated by the upstream kinase MEK, whose role was more likely to be a “kinase gatekeeper” for ERK [
31]. In our study, we demonstrated that instead of modulating the total or phosphorylation levels of MEK, cNFIB suppressed ERK phosphorylation through blocking the interaction between MEK and ERK. To sum up, our study revealed that cNFIB could serve as a key regulator of RAF/MEK/ERK signaling pathway in ICC.
Plenty of studies have been performed to understand how circRNAs exert their physiological or pathological functions. The ceRNA hypothesis is the most well-studied mechanism, proposing that circRNAs share the miRNA response elements, competitively bind to miRNAs and then regulate the expression of target genes. For example, circTP63 promoted FOXM1 expression by sponging miR-873-3p, which finally induced cell cycle progression [
45]. However, this sponging hypothesis has become controversial due to the low abundance of most circRNAs in mammals, making it less likely that they could effectively exert regulatory functions via binding to miRNAs [
46]. Although cNFIB was highly expressed in both ICC tissues and cell lines, it was incapable of serving as miRNA sponge because of the undetectable interaction between cNFIB and AGO2. On the other hand, mounting evidence suggests that some circRNAs can function as protein recruiters, scaffolds, and decoys in diverse biological contexts. An array of interaction patterns between circRNAs and proteins has been discovered based on more and more in-depth researches on circRNAs. After binding to proteins, circRNAs could cement or dissociate interaction between proteins, block protein from DNA or RNA, recruit proteins to chromatin, or alter protein distribution within cells [
46]. Herein, we provide a potent mechanism that cNFIB can inhibit the phosphorylation of ERK by preventing the interaction between MEK1 and ERK2. Mechanistically, we found that cNIFB directly bound to the NTD region of MEK1, which contained the domain responsible for the interaction between MEK1 and ERK2 [
34]. This effect impeded the binding of kinase (MEK1) to the substrate (ERK2), preventing the phosphorylation of ERK2, finally resulting in suppression of ICC proliferation and metastasis (Fig.
7G). Furthermore, administration of siRNA or inhibitor targeting MEK abrogated ERK activation and the tumor-promoting effects on ICC cells induced by cNFIB downregulation, indicating that MEK1 binding was essential for cNFIB-mediated ERK signaling regulation and ICC metastasis. An intriguing question that which binding region on cNFIB mediates the interaction between cNFIB and MEK1 requires further investigation.
ICC is an aggressive disease with limited therapeutic options. Despite advances in systemic management of patients, prognosis has not improved substantially during the past 10 years, with a 5-year survival of about 7–20% [
47]. Novel treatment strategies are urgently needed to improve outcomes for patients with ICC. Recently, genetic mutations in FGFR2, IDH1 and BRAF genes have been identified in ICC, making it possible for targeted treatment [
48]. For example, pemigatinib, a FGFR-specific tyrosine kinase inhibitor, has been approved by the US Food and Drug Administration (FDA) for advanced cholangiocarcinoma with FGFR2 fusions or rearrangements [
49]. In addition, for patients with BRAF
V600E-mutated biliary tract cancer, dual blockade of both BRAF (dabrafenib) and MEK (trametinib) have been considered as a promising treatment option from a phase 2, open-label, single-arm, multicenter basket trial [
39]. The combination of two inhibitors provided vertical suppression of the RAF/MEK/ERK pathway, resulting in synergistic effects and stronger tumor inhibition. Similarly, one recent study also proved the synergistic effects of dual blockage. Based on the murine model mimicking FGFR2 fusion (FF)-driven ICC pathogenesis, this study demonstrated that the FF oncogenic activity in ICC required the activation of a downstream effector called MEK. BGJ398 (FF inhibitor) plus trametinib (MEK inhibitor) combination treatment generated greater therapeutic efficacy than isolated inhibitor in vitro and in vivo [
38]. These results provide the notion that simultaneous inhibition of multiple molecules of an oncogenic pathway might induce stronger pathway blockage. In this regard, our data provide more evidences to support this notion. On the basis of MEK inhibition (trametinib), cNFIB competitively binds to MEK1, which results in the dissociation between MEK1 and ERK2, finally inducing more effective inhibition on ERK signaling and tumor invasion. On the other side, our finding that cNFIB is likely to generate synergistic effects on trametinib indicates that ICC cells with high levels of cNFIB might hold the potential to delay the trametinib resistance. Based on this part, we can foresee the therapeutic value of cNFIB for ICC treatment. Notably, recent advances in RNA-delivering techniques that the encapsulated circRNA SCAR was specifically delivered to the mitochondria via a nanoparticle platform for mitochondria-targeted therapy [
50], raised hopes for translational application of cNFIB to treat ICC.
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