The essential roles of mA RNA modification to stimulate ENO1-dependent glycolysis and tumorigenesis in lung adenocarcinoma
Authors: Wanxin Guo
Lifang Ma
Yongchun Yu
Yayou Miao
Susu Guo
Xin Xu
Yikun Wang
Fanyu Meng
Xiaoting Tian
Shiyu Qiu
Jiangtao Cui
You Li
Jiayi Wang
Xiangfei Xue
Xiao Zhang
Xiaoxin Liu
Keke Yu
Jinjing Xia
Journal: Journal of Experimental & Clinical Cancer Research (2022)
Discipline: CLINICAL MEDICINE
Keywords: ALKBH5; METTL3; RNA-protein interaction; YTHDF1; lung cancer
Affiliations: Department of Clinical Laboratory Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, No. 241 West Huaihai Road, 200030, Shanghai, China., Shanghai Institute of Thoracic Oncology, Shanghai Chest Hospital, Shanghai Jiao Tong University, No. 241 West Huaihai Road, 200030, Shanghai, China., Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital of Tongji University, 200072, Shanghai, China., Department of Bio-bank, Shanghai Chest Hospital, Shanghai Jiao Tong University, 200030, Shanghai, China., Nursing Department, Shanghai Chest Hospital, Shanghai Jiao Tong University, 200030, Shanghai, China., Department of Respiratory Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, No. 241 West Huaihai Road, 200030, Shanghai, China. ., Shanghai Institute of Thoracic Oncology, Shanghai Chest Hospital, Shanghai Jiao Tong University, No. 241 West Huaihai Road, 200030, Shanghai, China. ., Department of Clinical Laboratory Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, No. 241 West Huaihai Road, 200030, Shanghai, China. ., Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital of Tongji University, 200072, Shanghai, China. .

Abstract:  Background: Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. Patient prognosis is poor, and the existing therapeutic strategies for LUAD are far from satisfactory. Recently, targeting N6-methyladenosine (m6A) modification of RNA has been suggested as a potential strategy to impede tumor progression. However, the roles of m6A modification in LUAD tumorigenesis is unknown. Methods: Global m6A levels and expressions of m6A writers, erasers and readers were evaluated by RNA methylation assay, dot blot, immunoblotting, immunohistochemistry and ELISA in human LUAD, mouse models and cell lines. Cell viability, 3D-spheroid generation, in vivo LUAD formation, experiments in cell- and patient-derived xenograft mice and survival analysis were conducted to explore the impact of m6A on LUAD. The RNA-protein interactions, translation, putative m6A sites and glycolysis were explored in the investigation of the mechanism underlying how m6A stimulates tumorigenesis. Results: The elevation of global m6A level in most human LUAD specimens resulted from the combined upregulation of m6A writer methyltransferase 3 (METTL3) and downregulation of eraser alkB homolog 5 (ALKBH5). Elevated global m6A level was associated with a poor overall survival in LUAD patients. Reducing m6A levels by knocking out METTL3 and overexpressing ALKBH5 suppressed 3D-spheroid generation in LUAD cells and intra-pulmonary tumor formation in mice. Mechanistically, m6A-dependent stimulation of glycolysis and tumorigenesis occurred via enolase 1 (ENO1). ENO1 mRNA was m6A methylated at 359 A, which facilitated it's binding with the m6A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and resulted in enhanced translation of ENO1. ENO1 positively correlated with METTL3 and global m6A levels, and negatively correlated with ALKBH5 in human LUAD. In addition, m6A-dependent elevation of ENO1 was associated with LUAD progression. In preclinical models, tumors with a higher global m6A level showed a more sensitive response to the inhibition of pan-methylation, glycolysis and ENO activity in LUAD. Conclusions: The m6A-dependent stimulation of glycolysis and tumorigenesis in LUAD is at least partially orchestrated by the upregulation of METTL3, downregulation of ALKBH5, and stimulation of YTHDF1-mediated ENO1 translation. Blocking this mechanism may represent a potential treatment strategy for m6A-dependent LUAD. 

Retracted: No

PubPeer comments:
 We noticedu00a0Ma et al. reportedu00a0the role of m6A modification in lung cancer. Although theu00a0paper presents large amount of statistical data and figures, there are many doubts about the H E and IHC images associated with the mouse models. For example,u00a0the H E staining in Figure 2Cu00a0is very confusing. Actually, genetically engineered mouse models (KP)u00a0have been used inu00a0theu00a0basic research of lung cancers for decades. The KP lung tumor had some characteristics in the HE staining sections. For example, because immune cells and CAFs are involved in the progressive formation of tumors, the density of tumor cells should not be high and uniform. Furthermore, the AAV induction and the inflammatory response in tumor progression make it impossible for the tumor s surroundingu00a0boundaryu00a0to become too sharp. The authors claim that the four picturesu00a0in Figure 2Cu00a0are from genetically engineered mice with lung cancer. However, the densityu00a0of the tumor regionu00a0is high and too uniform. More importantly, the edges of the tumors seem too clear to meet the basic characteristics of AAV-induced genetic lung cancer. In fact, they look more like lung metastases caused by the injection of tumor cells into the tail vein. The same problem exists in Figure 7Q. The ENO1u00a0IHCu00a0stainingu00a0of KPEM tumors may also be misused. This image looks more like compressed alveolar tissue than tumor tissue. Since the author did not provide any other staining data in the paper, we cannot make a definite judgment at present. Since the authors draw very important evidences from these genetically engineered animal models for all subsequent mechanism studies in the paper, we suggest the authors provide the necessary data to clarify these possible issues. , 
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