METTL3 facilitates tumor progression via an m6A-IGF2BP2-dependent mechanism in colorectal carcinoma

Ting Li,Demeng Chen,Pei-Shan Hu,Qi-Nian Wu,Zhao-Lei Zeng,Dan Xie,Zhixiang Zuo,Dongxin Lin,Jin-Fei Lin,Bo Li,Feng Wang,Rui-Hua Xu,Tie-Bang Kang,Zhan-Hong Chen,Jian Zheng,Huai-Qiang Ju,Xingyang Li

doi:10.1186/s12943-019-1038-7

Abstract

BackgroundColorectal carcinoma (CRC) is one of the most common malignant tumors, and its main cause of death is tumor metastasis. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and methyltransferase-like 3 (METTL3) participates in tumor progression in several cancer types. However, its role in CRC remains unexplored.MethodsWestern blot, quantitative real-time PCR (RT-qPCR) and immunohistochemical (IHC) were used to detect METTL3 expression in cell lines and patient tissues. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3. The biological functions of METTL3 were investigated in vitro and in vivo. RNA pull-down and RNA immunoprecipitation assays were conducted to explore the specific binding of target genes. RNA stability assay was used to detect the half-lives of the downstream genes of METTL3.ResultsUsing TCGA database, higher METTL3 expression was found in CRC metastatic tissues and was associated with a poor prognosis. MeRIP-seq revealed that SRY (sex determining region Y)-box 2 (SOX2) was the downstream gene of METTL3. METTL3 knockdown in CRC cells drastically inhibited cell self-renewal, stem cell frequency and migration in vitro and suppressed CRC tumorigenesis and metastasis in both cell-based models and PDX models. Mechanistically, methylated SOX2 transcripts, specifically the coding sequence (CDS) regions, were subsequently recognized by the specific m6A “reader”, insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), to prevent SOX2 mRNA degradation. Further, SOX2 expression positively correlated with METTL3 and IGF2BP2 in CRC tissues. The combined IHC panel, including “writer”, “reader”, and “target”, exhibited a better prognostic value for CRC patients than any of these components individually.ConclusionsOverall, our study revealed that METTL3, acting as an oncogene, maintained SOX2 expression through an m6A-IGF2BP2-dependent mechanism in CRC cells, and indicated a potential biomarker panel for prognostic prediction in CRC.

Highlights

Colorectal carcinoma (CRC) is one of the most common malignant tumors, and its main cause of death is tumor metastasis
methyltransferase-like 3 (METTL3) is highly expressed in metastatic CRC and associated with poor prognosis To evaluate the expression profile of m6A WERs in CRC, we analyzed The Cancer Genome Atlas (TCGA) database, and the results showed that several m6A WERs were dysregulated in colon adenocarcinoma (COAD) (Fig. 1a)
We verified that METTL3, YTH N6methyladenosine RNA binding protein 1 (YTHDF1), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1), and IGF2BP2 were significantly increased in CRC tumors tissues from Sun Yat-sen University Cancer Center (SYSUCC), while the other WERs showed no significant differences (Fig. 1b and Additional file 4: Figure S1a)

Summary

Introduction

Colorectal carcinoma (CRC) is one of the most common malignant tumors, and its main cause of death is tumor metastasis. Exploring the factors that drive tumor initiation and establishing a more accurate model for prognostic prediction in CRC are urgently needed Epigenetic regulatory mechanisms, such as DNA methylation, or N6-methyladenosine (m6A), are emerging research frontiers in tumor biology [6,7,8,9]. Our previous study demonstrated that one of the RNA demethylases, fat-mass and obesity-associated protein (FTO), plays a critical role in cell transformation in leukemia cells [14]. These findings have indicated that m6A has a broad impact on embryonic development, circadian clock control, and the DNA damage response, as well as on tumor progression [10, 14,15,16,17]. The functions of m6A modification and the underlying connection among the m6A “writers”, “readers”, and “targets” are still unexplored in CRC

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