FTO promotes cell proliferation and migration in esophageal squamous cell carcinoma through up-regulation of MMP13

https://doi.org/10.1016/j.yexcr.2020.111894Get rights and content

Highlights

  • FTO upregulation is found in ESCC tissues.

  • FTO promotes ESCC cell growth and migration.

  • MMP13 may act as a downstream effector of FTO and mediate the effect of cell growth and migration induced by FTO.

Abstract

FTO, a demethylase for N6-methyladenosine (m6A) modification, has been implicated in multiple tumors. However, its roles in esophageal squamous cell carcinoma (ESCC) remain uncovered. This study aims to evaluate the clinical relevance and functional roles in this disease. Through immunohistochemistry, qRT-PCR and Western blot analyses, we found FTO expression in ESCC tissues was stronger than that in adjacent normal tissues, and the survival curves displayed high FTO expression had a trend toward poor prognosis. Functionally, silencing of FTO inhibited ESCC cell growth and migration in CCK8, EdU, colony formation and transwell assays and FTO overexpression showed the opposite results. Furthermore, FTO was also required for the tumorigenicity of ESCC cells in nude mice. The data from RNA-seq analysis revealed that MMP13 expression was significantly affected by FTO knockdown. qRT-PCR and Western blot assays confirmed that MMP13 was positively regulated by FTO in both mRNA and protein levels. Additionally, the functional link between FTO and MMP13 was explored by CCK8 and transwell chamber approaches. These findings suggest that FTO is up-regulated and plays oncogenic roles in ESCC. MMP13 may function as a downstream target of FTO.

Introduction

Cancer has become a major global menace to human health, and esophageal cancer (EC) is reported to be the seventh leading cause of cancer incidence (572,000 new cases) and the sixth main reason of cancer death (509,000 deaths) according to GLOBOCAN 2018 [1]. The two common histologic subtypes of EC are squamous cell carcinoma (SCC) and adenocarcinoma (AC). There are significant differences between the two types of risk factors, affecting their global distribution[2] In China, esophageal squamous cell carcinoma (ESCC) is the dominate subtype of EC[3,4]. Because ESCC is often accompanied by extensive lymphatic diffusion and vascular invasion, the patients are generally diagnosed at advanced stage[5,6]. Even though lots of studies and experiments have made significant progress in the therapy of ESCC, the patients’ survival rates remain low[2] and our knowledge on ESCC is still very limited. Therefore, it is urgently required to investigate new drug targets and update therapeutic strategies for improving the overall survival of patients with ESCC.

In recent years, more and more evidences have revealed that N6-methyladenosine (m6A) has a large impact on the genesis and development in many cancer types[7]. m6A is an abundant chemical modification of eukaryotic messenger RNA (mRNA). The metabolisms and functions of m6A are controlled by the m6A “writers”, “erasers” and “readers” proteins[[7], [8], [9]]. As methyltransferase, “writers” is mainly composed of METTL3, METTL14 and WTAP, which is responsible for the methylation of m6A[10]. The m6A demethylases FTO and ALKBH5 are known as “erasers”. “Readers” are a class of RNA-binding proteins that interact with, or, are repelled by m6A, such as the YT521-B homology (YTH) domain family proteins that specifically recognize m6A[11,12]. Furthermore, “readers” is a class of important proteins to identify the m6A sites and decides its reversible regulation by YT521-B homology (YTH) domain family[7,13]. Of all the proteins, FTO has been reported to contribute to carcinogenesis in many cancers, including gastric cancer, acute myeloid leukemia and cervical squamous cell carcinoma[[14], [15], [16], [17]]. However, it's unclear whether FTO exerts a tumorigenic role in ESCC as mentioned above.

The aim of this study was to explore the function and expression of FTO in ESCC. The final results demonstrated that FTO expression was increased in ESCC tissues and knockdown of FTO significantly restrained the proliferation and migration of ESCC cells. The next-generation sequencing of the transcriptome indicated that MMP13, an extracellular matrix metalloproteinase, may be a possible downstream target of FTO. These observations will provide new promising therapeutic targets for ESCC.

Section snippets

Cell lines and cultures

Human ESCC cell lines KYSE150, Eca-109 and TE-1 were obtained from cell bank of Chinese academy of Sciences (Shanghai, China). Cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified incubator with 5% CO2.

Human tissue microarray (TMA) and immunohistochemistry (IHC)

The human ESCC tissue microarray (#HEsoS160CS01) was constructed by Shanghai Outdo biotech Co. Ltd, China, which consisting of 80 ESCC tissues and paired

FTO expression is up-regulated in ESCC tissues

In order to explore the expression of FTO in ESCC, a tissue microarray consisting of 80 ESCC tissues and corresponding para-cancerous normal esophagus tissues was subject to immunohistochemistry (IHC) staining. The representative images of IHC were shown in Fig. 1A, and the IHC results revealed that the percentage of FTO positive expression in caner samples was higher than that in adjacent normal tissues (Fig. 1B). Similarly, the data from Gene Expression Profiling Interactive Analysis (GEPIA)

Discussion

m6A modification has acquired substantial progress in the past few years and has been found to be closely connected with human diseases, including cancer. Previous studies have reported that elevated expression of m6A's demethylase FTO was discovered in several types of cancer by immunohistochemical analysis, including breast tumor, lung squamous cell carcinoma, leukemia and gastric cancer[14,15,[18], [19], [20]]. However, the effect of FTO in ESCC is not clarified. In the present work, we

CRediT authorship contribution statement

Shenxiang Liu: Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review & editing. Mei Huang: Methodology, Writing - review & editing. Zhiqin Chen: Formal analysis, Methodology, Writing - review & editing. Jingde Chen: Formal analysis, Writing - review & editing. Qian Chao: Writing - review & editing. Xudong Yin: Investigation, Supervision, Writing - review & editing. Ming Quan: Methodology, Writing - original draft, Writing - review & editing.

Declaration of competing interest

The authors have declared that they have no competing interests.

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (No. 81972280), Natural Science Foundation of Shanghai (No. 19ZR1441800), Key Disciplines Group Construction Project of Pudong Health Bureau of Shanghai (No. PWZxq2017-13), and Outstanding Clinical Discipline Project of Shanghai Pudong (No. PWYgy2018-02).

References (28)

  • R. Pan et al.

    Cancer incidence and mortality: a cohort study in China, 2008-2013

    Int. J. Canc.

    (2017)
  • Y. Zhao et al.

    BRCA1-Associated protein increases invasiveness of esophageal squamous cell carcinoma

    Gastroenterology

    (2017)
  • K. Wu et al.

    Silencing of CXCR2 and CXCR7 protects against esophageal cancer

    Am. J. Trans. Res.

    (2016)
  • N. Liu et al.

    N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

    Nature

    (2015)
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    Shenxiang Liu, Mei Huang and Zhiqin Chen contributed equally to this work.

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