MYEOV overexpression induced by demethylation of its promoter contributes to pancreatic cancer progression via activation of the folate cycle/c-Myc/mTORC1 pathway

With the clinical introduction of novel therapies such as molecular targeted drugs and immune checkpoint inhibitors for many malignancies, the overall mortality rate of cancer patients is decreasing, but pancreatic cancer is still considered one of the malignancies with the poorest prognosis [1]. Pancreatic cancer is also predicted to become one of the major causes of cancer-related death in the near future because it can metastasize to other organs by the time the diagnosis is confirmed and due to the lack of treatment options. In recent years, therapies such as FOLFIRINOX have been applied and have shown good efficacy and survival improvement compared to gemcitabine monotherapy. Immune checkpoint inhibitors have also been approved for use in some pancreatic cancers, but effective molecular targeted therapy has yet to be developed. To identify new molecular markers or candidate therapeutic targets in pancreatic cancer, we searched for genes whose expression was significantly correlated with shorter disease-specific survival in the expression profiles of RNA-Seq datasets of pancreatic cancer samples in The Cancer Genome Atlas (TCGA) because we have enough experience to perform such analysis [2, 3]. Gene screenings using overall survival information have been reported by other groups. Therefore, we newly performed screening using disease-specific survival information because overall survival information considers patients who died from causes other than the disease being studied. Generally, disease-specific survival can characterize outcomes best among various survival metrics. This screening revealed that patients with pancreatic cancer overexpressing MYEOV, named the myeloma overexpression gene [4], had significantly shorter disease-specific survival than those with low levels of MYEOV. MYEOV is known to be overexpressed not only in myeloma but also in breast, oral, esophageal, lung and colorectal cancers [5‐9]. In recent years, similar results have been reported in pancreatic cancer as in other malignant tumors [10‐12]. The protein encoded by the MYEOV gene contains a hydrophobic region and a characteristic C-terminal poly-leucine/isoleucine sequence [4], suggesting that the protein localizes to the cell membrane. However, the biological role of MYEOV is poorly understood. This may be due in part to the fact that MYEOV is an acquired gene in primates [13], making it difficult to conduct carcinogenesis in mouse models.

In this study, we report that MYEOV expression is significantly upregulated at the transcriptional level in pancreatic cancer tissues and that the expression of MYEOV is a prognostic indicator that correlates with shorter disease-specific survival independent of lymph node metastasis. Furthermore, knockdown of MYEOV induced the downregulation of the c-Myc and mTORC1 pathways, as well as the folic acid metabolism containing their target genes. These data emphasize the potential role of MYEOV as a therapeutic target.

In this report, we show that the expression of MYEOV is upregulated in pancreatic cancer and that upregulation of MYEOV is significantly correlated with shorter disease-specific survival. Recently, two groups reported that pancreatic cancer patients with higher MYEOV expression showed shorter overall survival [10, 11]. In particular, 20 candidate genes for prognostic indicators have been listed by Tang et al. Our study suggests that the expression level of MYEOV may be useful as a prognostic marker for survival. However, the candidate prognostic factors shown in Table 2, except for MYEOV, did not overlap with the marker candidates shown by Tang et al. This discrepancy is due to the difference in input data. We assessed disease-specific survival instead of overall survival, which includes death events due to factors other than tumors. Therefore, our gene list may indicate more appropriate candidate prognostic factors in pancreatic cancers. The qPCR results showed that the expression of MYEOV was upregulated in some pancreatic cancer cell lines compared to normal pancreatic cells (Fig. 3 A). On the other hand, in MiaPaCa-2 cells, MYEOV was silenced by methylation of the CpG sites in the promoter region (Fig. 3 B). These results suggest that the decrease in methylation level and the increase in MYEOV expression do not occur in all pancreatic cancers. As shown in Fig. 2, this is consistent with the results of the analysis showing that MYEOV promoter methylation is maintained in some pancreatic cancer specimens at levels comparable to those in normal pancreatic tissues. The correlation between MYEOV expression and DNA demethylation of transcriptional regulatory regions was also confirmed in other tumors (Fig. S3).

GSEA showed that many genes whose expression was altered by knockdown of MYEOV were targets of the c-Myc or mTORC1 complex (Fig. 4 A). Fang et al. showed that the expression of genes with c-Myc-binding motifs in their transcriptional regulatory regions was reduced upon knockdown of MYEOV using the non-small-cell lung cancer cell line A549 [8]. Our results confirmed similar results in pancreatic cancer. Recently, Tang et al. also reported the relationship between MYEOV expression and the c-Myc/mTORC1 signaling pathway [10]. These results suggest that high expression of MYEOV in pancreatic cancer tissues is accompanied by increased activity of c-Myc and mTORC1. In both datasets of our and previous study, [12] GSEA analysis showed that the significant enrichment of c-Myc target genes and other genes was commonly observed. However, the mTORC signaling pathway did not show statistically significant reduction in the previous dataset. This may be due to the difference of cell lines used in the experiments and the different knockdown efficiencies in the two data sets, as shown in Fig. S4A. The log-transformed fold change of MYEOV expression after its knockdown was − 2.51 in our SUIT-2 RNA-Seq dataset, whereas − 0.86 in the Panc-1 dataset of previous study (Table S4 and Table S5). The low efficiency of MYEOV knockdown may be the reason why the Panc-1 RNA-Seq dataset did not show reduced expression of genes in the folate metabolic pathway. Actually, our experiments using Panc-1 cells with high efficacy of MYEOV knockdown show the significant reduction of these genes (Fig. 5).

Several metabolic pathways are altered in tumors, which contribute to survival and growth. The relationship between the glycolytic pathway and MYEOV has already been reported by other groups [10], but we found that the expression of a gene cluster in one-carbon metabolic pathways was reduced by MYEOV knockdown. Folate and methionine are two pathways by which cells produce one-carbon units, which are used for nucleic acid synthesis and methylation reactions. The enzymes involved in the folate cycle are distributed in the mitochondria and cytoplasm. Of these, the MTHFD2 gene is known to be expressed during embryonic development and is rarely expressed in adult tissues [28]. MTHFD2 is overexpressed in some tumors and has been shown to contribute to the development of cancer stem cells and is considered a poor prognostic factor in several tumors, including pancreatic cancer [24, 29]. Strikingly, the expression of MTHFD2 is induced by both c-Myc and mTORC1. Since our analysis suggests that knockdown of MYEOV decreases the activity of c-Myc and mTORC1, it is likely that these events lead to a decrease in the expression of several folate metabolism-related genes, including MTHFD2. The expression of MYEOV is positively correlated with that of a group of genes that comprise the folate pathway (Table S6). Based on these results, the expression of folate metabolism-related genes may be suppressed by MYEOV knockdown in other tumors. Our data suggest that the suppression of MYEOV expression is a new factor leading to a poor prognosis in pancreatic cancer patients through activation of c-Myc and mTORC1 and metabolic remapping, such as enhancement of the one-carbon metabolic pathway.

Notably, our RNA-Seq results showed that the expression of MXD4 and CASTOR2 was markedly upregulated by MYEOV knockdown (Fig. 5 B and Table S4). The Mad4 protein represses the transcriptional activity of c-Myc by forming a protein complex with Max [27]. Therefore, repression of MXD4 expression by MYEOV may facilitate the malignant transformation of pancreatic cancer through activation of c-Myc and other proteins. CASTOR2 protein forms a heterodimeric complex with CASTOR1 and binds to GATOR2, which induces mTORC1 activation by inhibiting GATOR1 [26]. Our study showed that knockdown of MYEOV increased CASTOR2 expression in several cell lines, suggesting that MYEOV contributes to mTORC1 activation by regulating CASTOR2 expression. In TCGA-PAAD, the expression levels of MXD4 and CASTOR2 were inversely correlated with the expression level of MYEOV (Table S6). An inverse correlation between MYEOV and MXD4 expression levels was also observed in lung adenocarcinomas. In gastric cancer, both CASTOR2 and MXD4 expression levels were inversely correlated with MYEOV expression levels (Table S6). In TCGA dataset of pancreatic cancers, the absolute value of correlation coefficients between MYEOV and MXD or CASTOR4 expression are around − 0.2. Although the value is not necessarily high, we consider that this correlation supports our results because the value is above the 80th percentile of all measured correlation coefficients among all genes and is above the 90th percentile of measured correlation coefficients among inversely correlated genes (Fig. S7).

However, MYEOV knockdown in Panc-1 RNA-seq dataset from GEO database did not show the significant upregulation of MXD4. This result may also be due to the low efficacy of MYEOV knockdown (Fig. S4A), or may suggest that the upregulation of MXD4 is not essential for the downregulation of MYC target genes induced by the downregulation of MYEOV.

Although some groups report that MYEOV contributes to the progression of not only pancreatic cancer but also several other tumors, the detailed functions of the protein encoded by this gene are limited, except for one report stating that it binds to HES1 and promotes the transcriptional activation of SOX9 [11]. This may be due in part to the fact that the MYEOV protein has no known functional domain. A comparative study of multiple animal genomes reported that the MYEOV gene is unique among primates only in Catarrhini. The report also notes that MYEOV has a very unusual feature as a cancer-related gene: the amino acid sequence significantly differs between humans and apes [13]. Analysis of MYEOV expression levels and promoter methylation status in several cancers in the TCGA showed that the promoter region was hypermethylated in normal tissues, while some tumors showed hypomethylation and overexpression. This is a comparatively rare event in cancers because most tumor suppressor genes are generally inactivated by DNA methylation, and this mechanisms is similar to the regulation of long interspersed element-1 (LINE-1), which shows that DNA demethylation and increased expression levels correlate with a poor prognosis in cancer [30]. According to lung cancer cell line experiments, Fang et al. proposed that MYEOV functions as a competing endogenous RNA (ceRNA [31]) that inhibits the binding of miRNAs to their targets, thereby contributing to the invasive and metastatic potential of tumors [8].

The outline of our research is shown in Fig. 6. In normal cells, MYEOV expression is low. Thus, MXD4 and CASTOR2 appropriately suppress c-Myc and mTORC1. Conversely, in pancreatic cancer cells, MYEOV expression is upregulated and promotes the downregulation of MXD4 and CASTOR2, resulting in the activation of c-Myc and mTORC1 and tumor progression.

We sought prognostic markers to identify groups of pancreatic cancer patients with an unfavorable prognosis. In conclusion, we showed that MYEOV suppression is a new mechanism that contributes to a poor prognosis in pancreatic cancer patients through the activation of the folate cycle and the c-Myc and mTORC1 pathways (Fig. 6). These studies suggest that MYEOV could serve as an ‘actionable’ therapeutic target in several human cancers, including pancreatic cancer. Further research is needed to understand how the MYEOV protein as well as the MYEOV transcript itself contribute to tumor progression.