A novel protein encoded by circMAPK1 inhibits progression of gastric cancer by suppressing activation of MAPK signaling

Gastric cancer (GC) is one of the most common malignancies with high morbidity and mortality worldwide, especially in East Asian countries such as China, Japan and Korea [1]. Gastric tumorigenesis is a multistage, slowly progressing and multifactorial pathological process [2]. The proven high-risk factors for GC include having Helicobacter pylori infection, excessive intake of salt and nitrates, obesity and blood group A [3]. In addition, genetic mutations, epigenetic changes, and aberrant molecular signaling pathways are also involved in the development and metastasis of GC [4]. Because early GC lacks specific symptoms, most GC patients are diagnosed at the intermediate or terminal stage and have poor prognosis [5]. It is urgent to identify the gene expression patterns and biomarkers in GC to advance GC research and improve patient survival.

Circular RNA (circRNA) is a special kind of noncoding RNA molecule that has recently become a hotspot in the field of noncoding RNA [6]. In contrast to traditional linear RNAs, circRNAs have a closed loop structure without a 5′ cap or a 3′ poly A tail [7]. The expression of circRNAs is more stable than linear RNAs, and the molecules are not as easily degradable. With the development of high-throughput sequencing technology and bioinformatic analysis, an increasing number of circRNAs have been found to regulate cellular proliferation, migration, invasion, apoptosis and differentiation [8, 9]. The great majority of circRNAs have been found to perform their biological functions by acting as microRNA (miRNA) sponges or binding to proteins [10]. A few studies have also suggested that circRNAs containing internal ribosome entry sites (IRESs) or extensive methyl modification sites have the potential to affect physiological behaviors by encoding proteins [11, 12]. For instance, circβ-catenin promotes liver cancer cell growth through activation of the Wnt pathway by encoding β-catenin-370aa [13]. SHPRH-146aa is encoded by circ-SHPRH, which can protect full-length SHPRH from degradation by the ubiquitin proteasome and inhibit glioma tumorigenesis [14]. Recently, the role of circRNAs as the sponge of miRNA and protein in GC has been well established [15‐17]. However, whether circRNAs could regulate the tumorigenesis and development of GC by encoding proteins remains unknown.

The mitogen-activated protein (MAP) kinase signaling cascade Ras-Raf-MEK-MAPK signal transduction pathway is an important evolutionarily conserved signaling pathway [18]. As the classic cellular phosphorylation cascade, it has been reported to be involved in a large variety of cellular and physiological processes essential to life [19, 20]. When extracellular stimulating factors such as cytokines, neurotransmitters, and hormones bind to transmembrane receptors, the inactive Ras-GDP in the plasma membrane is converted into active Ras-GTP. To activate downstream members of the MAPK pathway, Ras-GTP stimulates the formation of an active homodimer or heterodimer consisting of A-Raf, B-Raf, and C-Raf through a complex process [21]. The Raf enzymes are serine/threonine protein kinases that catalyze the phosphorylation and activation of dual specificity mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, where MEK activates MAPK [22]. MEK1/2 sequentially phosphorylates two sites on MAPK1/2: Y204/187 and T202/185 [23]. Once the two residues are phosphorylated, MAPK1/2 is activated to catalyze many cytoplasmic and nuclear substrates, including transcription factors and regulatory molecules [18, 24]. Overall, this signaling pathway can control cell growth, cell proliferation, cell survival, differentiation, immune response, metabolism, nervous system function and transcription through a series of phosphorylation reactions [25]. However, circRNAs related to the MAPK pathway are under-researched.

In this study, we identified a circRNA, named circMAPK1, derived from the MAPK1 gene by analyzing RNA sequencing data from various databases. The expression of circMAPK1 decreased in GC tissues and cells, which was related to a good prognosis in GC patients. Functional investigations showed that circMAPK1 suppressed GC cell growth in vitro and in vivo. Subsequent studies demonstrated that circMAPK1 encoded a novel 109-amino acid (aa) MAPK1 isoform. Functionally the opposite to MAPK1, MAPK1–109aa decreased MAPK1 phosphorylation by competing with MEK1, suppressed the activation of MAPK1 and its downstream factors, thereby inhibited the proliferation and invasion of GC cells. Our results indicate MAPK1–109aa, an inhibitor of the MAPK1 pathway, as an effective diagnostic tool and treatment target for GC.

MAPK cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiation [27]. The aberrant overactivation of MAPK cascades contributes to cancers and other human diseases [28‐31]. Recently, the application of compounds targeting components of the MAPK pathway (such as RAF and MEK inhibitors) has been proven to substantially improve the clinical outcome of metastatic melanoma and can also show surprising clinical effects in other tumors [32]. However, the response to the drugs varies greatly, and the clinical efficacy of these drugs is mainly limited by the development of drug resistance [33]. Acquired drug resistance often occurs within 1 year of therapy and has become an obstacle to the long-term survival of clinical patients [34]. It has been proposed that the combined use of selective MAPK1/2 inhibitors can expand the treatment scope to tumors driven by the MAPK pathway [35]. Ulixertinib, MK-8353 and GDC-0994 are orally effective and specific MAPK inhibitors that are being applied in early clinical trials for the treatment of numerous advanced/metastatic solid tumors [36]. Additional work will be required to determine whether MAPK inhibitors are clinically successful, and whether they can prevent the development of resistance through the MAPK pathway is also unknown. Therefore, more innovative methods are needed to target this pathway.

As a new type of RNA, circRNA has a unique circular structure that is derived from the back-splicing biogenesis process [37]. Recently, with the rapid development of high-throughput sequencing technology and bioinformatics analysis, a great number of circRNAs have been found in mammalian cells [38]. Abnormal expression of circRNAs has been reported in numerous human diseases, including cancer, neurologic diseases, endocrine diseases, and cardiovascular diseases [39]. Owing to their abundance, high stability, tissue-specific expression pattern, and wide distribution in various body fluids, circRNAs have great potential as biomarkers of human diseases. Here, we focused on circRNAs arisen from the MAPK pathway. We identified the circRNA hsa_circ_0004872, termed circMAPK1 in this article, through the analysis and screening of the circBase database. The expression of circMAPK1 was relatively downregulated in GC. Further survival analysis also showed that higher expression of circMAPK1 was associated with longer survival time of GC patients, which indicated that circMAPK1 had a potential role in the occurrence and development of GC and that it might be a therapeutic target.

Since unlimited cell proliferation, migration and metastasis are the hallmarks of malignant tumors, we next explored the role of circMAPK1 in the progression of GC through gain- and loss-of-function experiments. A series of in vitro and in vivo functional experiments showed that circMAPK1 inhibited the proliferation, migration and metastasis of GC. We then focused our research on the mechanism by which circMAPK1 exerts its tumor suppressor function.

In recent years, research on circRNAs related to GC has received increasing attention [40]. A considerable number of articles have confirmed that circRNAs can affect the malignant biological behavior of GC by acting as miRNA sponges or interacting with related proteins [41]. For example, circHuR inhibits the expression of HuR and the progression of GC by suppressing CNBP transactivation [42]. CircHECTD1 promotes glutamine breakdown by targeting miR-1256 and activating β-catenin/c-Myc signaling to promote the progression of GC [43]. A small number of studies have found that circRNAs can affect cell proliferation, differentiation, metastasis and other processes by encoding proteins [12], challenging their traditional role as noncoding RNA molecules. Our research showed that circMAPK1 encodes a 109-aa-long protein (MAPK1–109aa). Because the aa sequence of the ORF includes the cyclization site of circMAPK1, MAPK1–109aa shares most of its aa sequence with MAPK1, except for the unique aa sequence LCL in the C-terminus. Subsequent functional experiments also confirmed that MAPK1–109aa mediates the inhibitory effect of circMAPK1.

To explore the mechanism of action of MAPK1–109aa, coimmunoprecipitation followed by MS was conducted using Flag-tagged MAPK1–109aa as bait. Among the identified precipitated proteins, MAPKK1 (MEK1) was the most abundant, which prompted us to further investigate the regulatory mechanism between MAPK1–109aa and MEK1. MEK1 is a bispecific protein kinase belonging to the MAP kinase kinase (MKK) family and an important component of the MAPK signal transduction pathway [44]. MEK1 plays a key role in the regulation of gene expression and cytoplasmic function by transmitting upstream signals to downstream response molecules [45]. MKKs phosphorylate a threonine and a tyrosine in a TXY motif in the MAPK activation loop [46]. In principle, the residues in and around this motif are also involved in the recognition and phosphorylation of the MAPK family by MKKs. That is, MEK1/2 could recognize the sequence FLTEY in MAPK1/2, which may be important for the mutual recognition of MEK1/2 and MAPK1/2. In addition, we found that MAPK1–109aa contains the phosphorylation sites and surrounding sequences of MAPK1, which suggests that MEK1 also has the potential to recognize and phosphorylate MAPK1–109aa. The binding of MAPK1–109aa to MEK1 was confirmed by subsequent immunoprecipitation and Western blot experiments.

Studies focused on the encoding abilities of circRNA have demonstrated that some circRNAs encoding proteins play the inconsistent role with their host gene products. For instance, it has been reported that AKT3-174aa inhibits glioma tumorigenicity by decreasing activated AKT [47]. In contrast, some products encoded by circRNAs are involved in the functions of their host genes. SHPRH-146aa encoded by circSHPPH protects full-length SHPRH from degradation by the ubiquitin proteasome [14]. Here, we found that the product MAPK1–109aa encoded by circMAPK1 interfered with the function of its host gene MAPK1. A coimmunoprecipitation experiment was conducted to confirm that MAPK1–109aa competitively bound to MEK1 and inhibited the phosphorylation of MAPK1, but we did not detect the presence of phosphorylated MAPK1–109aa at a molecular weight of 13 kDa, indicating that MEK1 is unable to phosphorylate this short peptide, even if it contains the sequence of the MAPK1 activation fragment, which was consistent with a previous research [23]. CircRNAs are generated by cells through specific mechanisms, while some circRNAs can be translated into specific short peptides to regulate the function of their parental genes. This may represent an efficient feedback mechanism in cells and deserves further study. Although the sequence of the peptide translated by the circRNA was similar to that of the protein translated by the parent gene, the former has several specific aa differences from the latter. Whether these specific aa have specific functions needs further exploration. Simultaneously, we also proved that MAPK1–109aa inhibited the activation of tumor-promoting factors downstream of the MAPK1 pathway. This further showed that the short peptide MAPK1–109aa encoded by circMAPK1 can negatively regulate the cancer-promoting effect of the MAPK pathway. Besides, what’s more refreshing is that MAPK1–109aa may solve the problem of acquired drug resistance in clinical applications of MAPK pathway inhibitors since it can cooperate with MAPK pathway inhibitors to exert a tumor suppressor effect. Taken together, attributing to the suppressing effects on MAPK pathway activation, the short peptides MAPK1–109aa may be a promising inhibitor, which needs further studies on its clinical application.

In conclusion, we identified a novel downregulated circRNA derived from MAPK1 in gastric cancer. MAPK1–109aa, encoded by circMAPK1, exerts an anticancer effect by competing with MAPK1 to bind to the upstream kinase MEK1, suppressing the phosphorylation of MAPK1 and the downstream oncogenic factors. Our findings suggested that circMAPK1 may be used as a therapeutic target in GC and it also provides new treatment ideas for diseases caused by the activation of the MAPK pathway.