Unraveling the independent role of METTL3 in m6A modification and tumor progression in esophageal squamous cell carcinoma

METTL3 and METTL14 are traditionally posited to assemble the m6A methyltransferase complex in a stoichiometric 1:1 ratio, modulating mRNA fate via m6A modifications. Nevertheless, recent

investigations reveal inconsistent expression levels and prognostic significance of METTL3 and METTL14 across various tumor types, challenging their consistent functional engagement in

neoplastic contexts. A pan-cancer analysis leveraging The Cancer Genome Atlas (TCGA) data has identified pronounced disparities in the expression patterns, functional roles, and correlations

with tumor burden between METTL3 and METTL14, particularly in esophageal squamous cell carcinoma (ESCC). Knockdown experiments of METTL3 in EC109 cells markedly suppress cell proliferation

both in vitro and in vivo, whereas METTL14 knockdown shows a comparatively muted effect on proliferation and does not significantly alter METTL3 protein levels. mRNA sequencing indicates

that METTL3 singularly governs the expression of 1615 genes, with only 776 genes co-regulated with METTL14. Additionally, immunofluorescence co-localization studies suggest discrepancies in

cellular localization between METTL3 and METTL14. High-performance liquid chromatography–mass spectrometry (HPLC–MS) analyses demonstrate that METTL3 uniquely associates with the

Nop56p-linked pre-rRNA complex and mRNA splicing machinery, independent of METTL14. Preliminary bioinformatics and multi-omics investigations reveal that METTL3’s autonomous role in

modulating tumor cell proliferation and its involvement in mRNA splicing are potentially pivotal molecular mechanisms. Our study lays both experimental and theoretical groundwork for a

deeper understanding of the m6A methyltransferase complex and the development of targeted tumor therapies focusing on METTL3.

METTL3 serves as the catalytic subunit of the N6-adenosine methyltransferase complex and, in conjunction with METTL14, comprises the “writer” complex essential for catalyzing m6A

modifications on mRNA1. The presence of m6A modifications on mRNA facilitates their recognition by “reader” proteins, thereby enhancing the processes of mRNA translation or degradation2. m6A

modifications can also be reversed by “eraser” enzymes. The METTL3–METTL14-mediated m6A modification plays a pivotal role in regulating cellular proliferation, differentiation, and

responses to various stresses. In recent years, mRNA m6A methylation mediated by the METTL3–METTL14 complex has offered a novel paradigm for elucidating the mechanisms underlying cancer

progression. Numerous studies have demonstrated that the upregulation of METTL3, frequently observed in tumors, promotes tumor cell survival, proliferation, self-renewal, metastasis, and

drug resistance by modulating mRNA metabolism3.

Elevated expression of METTL3/METTL14 has been significantly correlated with the advancement and adverse prognosis of various cancers4. In acute myeloid leukemia, the overexpression of

METTL3/METTL14 enhances the stability and translation efficiency of c-myc via m6A modifications, thereby accelerating cancer progression5. In breast cancer, the METTL3-METTL14 complex

upregulates the expression of genes like Bcl-2 and CXCR4 through m6A-dependent mechanisms, thereby fostering breast cancer growth and metastasis6. Nevertheless, in certain cancers, METTL3

and METTL14 demonstrate divergent effects on tumor progression. Knockdown of METTL3 has been observed to alter the malignant phenotype across various tumor types, whereas multiple studies

have indicated that elevated expression of METTL14 inhibits tumor proliferation and migration via m6A-dependent mechanisms. These contrasting prognostic impacts have been documented in

colorectal, bladder, lung, gastric, and other cancers7,8.

Additional research has demonstrated that the regulatory effect of METTL3 on downstream genes may operate independently of METTL14. For instance, in ovarian cancer, METTL3 shows a

significant association with prognosis; however, the knockdown of METTL14 does not inhibit the clonogenic formation capacity of TOV-112D cells. Moreover, the expression of oncogenes such as

EIF3C, AXL, CSF1, and FZD10 is solely modulated by METTL3, underscoring its unique regulatory role9. Similarly, in esophageal squamous cell carcinoma (ESCC), METTL3 and METTL14 demonstrate

distinct functional roles. Specifically, the modulation of miR-99a-5p by METTL14 occurs independently of METTL3, highlighting differential regulatory pathways within this cancer type10,11.

The findings from the referenced studies elucidate that while METTL3 and METTL14, integral components of the m6A writer complex, collaboratively influence m6A modifications in cells, they

display marked disparities in their influence on downstream gene regulation in tumor cells, either directly or indirectly. The distinct mechanisms by which METTL14 regulates genes

independently of METTL3 pose critical questions for future m6A research. In our current study, employing a pan-cancer analytical approach, we developed METTL3 and METTL14 knockdown EC109

esophageal cancer cell lines to explore the impact of gene silencing on cellular proliferation. Additionally, comprehensive transcriptomic and proteomic analyses were conducted to delineate

the intricate molecular mechanisms involved.

We retrieved RNA-sequencing expression profiles at level 3 and associated clinical data for different cancer types from the Cancer Genome Atlas (TCGA) database, accessible via the website

https://portal.gdc.com. Subsequent statistical analysis was carried out using R version 4.0.3 software, provided by the R Foundation for Statistical Computing, based in Vienna, Austria, with

a significance threshold set at P-value