Expression of Demethylase Genes, FTO and ALKBH1, Is Associated with Prognosis of Gastric Cancer

Gastric cancer (GC) is one of the most common malignancies worldwide [1] and the second biggest health burden in China [2]. The basis of treatment is surgical removal of the tumor mass. The 5-year survival rate for R0 surgical resection ranges from 30 to 50% for patients with stage II disease and from 10 to 25% for patients with stage III disease [3]. However, these patients have a high likelihood of local or systemic relapse. At present, there are no solid biomarkers for GC, except the tissue expression level of human epidermal growth factor receptor 2 (HER2) and regular clinicopathological parameters, such as TNM stage, which predict prognosis and response to therapy [4]. Thus, there is an urgent need to locate and identify robust prognostic biomarkers for GC.

In recent years, another form of gene regulation at the RNA level, RNA epitranscriptomics, has attracted the attention and interest of the research community. N⁶-methyladenosine (m⁶A), the most abundant internal modification in eukaryotic messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs), is known to play critical roles in various bioprocesses such as tissue development, stem cell self-renewal and differentiation, heat-shock or DNA damage response, and maternal-to-zygotic transition [5]. The m⁶A patterns involve a series of proteins identified as “writers,” “erasers,” and “readers” of m⁶A. Methyltransferase-like 3 and methyltransferase-like 14 (METTL3 and METTL14) and their cofactor, Wilms’ tumor 1-associated protein (WTAP), compose the m⁶A methyltransferase complex (MTC), which catalyzes m⁶A modification [6]. Fat mass and obesity-associated protein (FTO), alkB homolog 1 (ALKBH1), and ALKBH5 have been shown to act as m⁶A demethylases, oxidizing the N-methyl group of m⁶A site to a hydroxymethyl group [7, 8]. Similar to DNA methylation [9], information contained in m⁶A codes needs to be deciphered by “readers” such as YTHDF1, YTHDF2, and YTHDF3, directly or indirectly [10‐12]. All of the above m⁶A-related genes were reported to regulate vital biological processes associated with a range of malignancies, such as hepatocellular carcinoma [13‐15], glioblastoma [16, 17], acute myeloid leukemia [18], breast cancer [19, 20], lung cancer [21], and GC [22]. However, the association between m⁶A-related genes and GC has not been studied in detail. The present study evaluated the prognostic role of mRNA expression of nine m⁶A-related genes using the Kaplan–Meier (KM) plotter database and The Cancer Genome Atlas (TCGA) database. Next, genes stated as being significantly associated with prognosis in both databases were further detected in 450 paired GC and corresponding adjacent non-tumor tissues using tissue microarray (TMA)-based immunohistochemistry (IHC) for independent validation (Fig. 1).

m⁶A, which is the most abundant modification in RNAs, is linked to human diseases, but its function in mammalian development is poorly understood. In addition, the role of m⁶A-related genes has been established in several malignancies, but the link between m⁶A expression and GC has not been elucidated [13‐22]. Here, we evaluated the prognostic role of mRNA expression of nine m⁶A-related genes in GC.

Thousands of mRNA and lncRNAs show conserved m⁶A patterns, including transcripts encoding core pluripotency transcription factors. Thus, m⁶A can be taken as a mark of transcriptome flexibility required for stem cells to differentiate into specific lineages. FTO, also known as ALKBH9, belongs to the non-heme Fe II/α-KG-dependent dioxygenase AlkB family of proteins that also contains ALKBH1 to ALKBH8 [7]. It was reported that FTO was overexpressed at both protein and mRNA levels in GC tissues compared to corresponding adjacent non-tumor tissues, where high FTO expression was significantly associated with poor prognosis in GC patients [22]. By contrast, via analysis of RNAseq data from KM plotter and TCGA databases, we demonstrated that high mRNA expression of demethylase genes, FTO and ALKBH1, predicted a worse prognosis. However, TMA-IHC staining indicated that FTO and ALKBH1 were significantly downregulated at the protein level in GC tissue specimens, suggesting that high FTO protein expression is indicative of a better OS for GC patients.

Notably, results of several studies are in total or partial agreement with our findings. Pi et al. believed that high level of FTO expression inhibited GC cell line MGC-803 proliferation, migration, and invasion [25]. Liu et al. demonstrated a noticeable increase in proliferation in ALKBH1 knockdown HeLa cells caused by elevated methylation of transfer RNA (tRNA). This dynamic process was used by human cells in response to glucose deprivation [26]. Zhou et al. suggested that a lack of ALKBH1 restricted its binding with the promoter of ATF4, an osteoblast-enriched transcriptional factor indispensable for osteogenic differentiation, and led to transcriptional silencing of ATF4 with increasing m⁶A levels [27]. Gao et al. reported that ALKBH2, another member of the dioxygenase AlkB family, was downregulated in GC tissues and cell lines as determined by real-time PCR and confirmed by IHC and Western blot. Overexpression of ALKBH2 significantly inhibited GC cell proliferation and induced G1 arrest of the cell cycle in GC cell lines [28]. The above-mentioned observations reveal the suppressive role of FTO and ALKBH1 in GC and are substantiated by findings of the current study. However, it has been established that a high level of transcription does not necessarily guarantee a high level of translation that produces proteins that orchestrate complex cellular processes. These discrepant findings between mRNA and protein levels may be caused, at least in part, by various post-transcriptional regulation mechanisms, such as 5′ capping of mRNA, polyadenylation, alternative splicing, and multiple covalent RNA modifications [29]. The specific mechanisms underlying these discrepancies may have to be elucidated via more sophisticated experiments in the future.

In the last few years, modification in mRNAs or non-coding RNAs has been reported to play a critical role in virtually all major normal bioprocesses. Modification of mRNAs is now being pushed to the forefront of biology due to the discovery of the “writers,” “erasers,” and “readers” that add, remove, or preferentially bind to the m⁶A site [30]. RNA m⁶A patterns are involved in various aspects of mRNA metabolism including mRNA export, translation, and decay and perform an important function in post-transcriptional regulation of gene expression and protein translation [12]. In the non-coding RNA field, m⁶A modification acts as a barrier to tRNA accommodation and translation elongation [31]. Reversible demethylation of tRNA mediated by ALKBH1 results in attenuated translation initiation and tRNA decreased use in protein synthesis [26]. Moreover, researchers expanded the concept of the RNA epitranscriptome to circular RNAs (circRNAs), providing evidence that m⁶A modifications in circRNAs are written and read by the same machinery used for mRNAs, but often at different locations [32].

We acknowledge several limitations in our study. Firstly, mRNA expression levels of candidate genes were not substantiated in GC and non-tumor tissues via experiments. Secondly, the effect of FTO and ALKBH1 expression on the biological function of GC cells remains to be elucidated. Thirdly, the reason for the contradictory trends between mRNA and protein expression levels of FTO and ALKBH1 in GC remains to be further investigated.

In conclusion, the aberrantly high mRNA expression of demethylase genes, FTO and ALKBH1, was markedly associated with poor OS in light of the in silico analysis, while FTO and ALKBH1 expression was significantly downregulated in GC tissues at the protein level, according to the TMA-IHC staining results. Additionally, lower ALKBH1 protein expression level was closely correlated with larger tumor size (≥ 5 cm) and more advanced TNM stages (III/IV). Lower FTO protein expression was markedly associated with a shorter OS in GC patients.