m6A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-mediated YAP expression and inhibiting miR-107/LATS2‚Äìmediated YAP activity in NSCLC
Authors: Juanjuan Dai
Yan Wu
Kaikai Gong
Jing Du
Lijuan Yang
Dan Jin
Jiwei Guo
Weiwei Chen
Hongliang Sun
Shuang Miao
Xuelin Li
Xiaohong Wang
Journal: Molecular Cancer (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: ALKBH5; HuR; LATS2; YAP; YTHDFs
Affiliations: Clinical Medical Laboratory, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Cancer research institute, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China. ., Cancer research institute, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of Thyroid and Breast Surgery, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China., Department of reproductive medicine, Binzhou Medical University Hospital, Binzhou, 256603, People's Republic of China.

Abstract:  Background: The importance of mRNA methylation erased by ALKBH5 in mRNA biogenesis, decay, and translation control is an emerging research focus. Ectopically activated YAP is associated with the development of many human cancers. However, the mechanism whereby ALKBH5 regulates YAP expression and activity to inhibit NSCLC tumor growth and metastasis is not clear. Methods: Protein and transcript interactions were analyzed in normal lung cell and NSCLC cells. Gene expression was evaluated by qPCR and reporter assays. Protein levels were determined by immunochemical approaches. Nucleic acid interactions and status were analyzed by immunoprecipitation. Cell behavior was analyzed by standard biochemical tests. The m6A modification was analyzed by MeRIP. Results: Our results show that YAP expression is negatively correlated with ALKBH5 expression and plays an opposite role in the regulation of cellular proliferation, invasion, migration, and EMT of NSCLC cells. ALKBH5 reduced m6A modification of YAP. YTHDF3 combined YAP pre-mRNA depending on m6A modification. YTHDF1 and YTHDF2 competitively interacted with YTHDF3 in an m6A-independent manner to regulate YAP expression. YTHDF2 facilitated YAP mRNA decay via the AGO2 system, whereas YTHDF1 promoted YAP mRNA translation by interacting with eIF3a; both these activities are regulated by m6A modification. Furthermore, ALKBH5 decreased YAP activity by regulating miR-107/LATS2 axis in an HuR-dependent manner. Further, ALKBH5 inhibited tumor growth and metastasis in vivo by reducing the expression and activity of YAP. Conclusions: The presented findings suggest m6A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-mediated YAP expression and inhibiting miR-107/LATS2-mediated YAP activity in NSCLC. Moreover, effective inhibition of m6A modification of ALKBH5 might constitute a potential treatment strategy for lung cancer. 

Retracted: No

PubPeer comments:
 Could the authors comment on the apparent repeated band in Figure 2K? ***  , 
 Dear Dr Biskrensis: I appreciate your interest in our work and your comment on our article in the article. I have carefully re-examined our manuscript and experimental records. The quality for some WB bands in Figure2K are low, leading to some confusion about the results. In fact, theses bands are the different bands with difference as indicated in the Figure2K. Thank you again for reading my article carefully, and I appreciates your work very much agian.  , 
 #2 Thank you for your reply. Could you please provide the original uncropped scans? As you are probably aware, slight differences can appear even in identical images after image compression and processing. It certainly is possible that I am mistaken, and the original scans will alleviate my concerns. , 
 Thank you for your reply. Here are the original uncropped scans.  , 
 #4 Thank you for your reply. I agree there appear to be some differences and I may be mistaken, but the images in the paper appear unexpectedly similar. The images you have provided here do show more differences but they appear to be already cropped, and perhaps printed on paper (?). An image of the uncropped gel from which these came could better address my concerns. I appreciate your patience and cooperation. , 
 #4 It looks to me that the  original uncropped scan you provided has some apparent differences from the one appeared in paper which #1 pointed out. In the following image, the bottom one is a brightness-contrast adjusted version of your scan to illustrate my points.  , 
 #6 The quality for some WB bands in Figure2K are low, so slight differences can appear after image compression and processing. , 
 #7 Thank you again for your reply. I agree that u201c slight differences can appear after image compression and processing,u201d which is why I remain unconvinced by the reply in #2. Original data would be appreciated. I urge you again to post the **original uncropped scans** of the gels to address these concerns. I would anticipate that they appear similar to one shown below:  , 
 Dear Actinopolyspora Biskrensis. when we performed western blot, the PDVF membrane was cut to the indecated size instructed by the protein size and number inorder to incubate with antibdoy. The images were obtained by the ChemiDoc TMTM XRS+ (Bio-Rad) instead of the developing the film to get the original uncropped scans. , 
 Response from the journal: u201cI am just writing to let you know that the investigation has concluded that there is no manipulation and so no editorial action will be taken. We take all allegations seriously and investigate them all following COPE guidelines.u201d , 
 Figure 4A also appears to have some repeating bands (boxes) and possible splices (arrows). Shown with original 4A (top) and false color image (bottom). ***  , 
 Could the authors comment on the apparent repeated band in Figure 1a and 1d? In additionuff0cthe original uncropped scans would be appreciated.  , 
 From the journal: u201cDear reader, I am writing to provide an update on Molecular Canceru2019s investigation into the concerns raised over the following article: Jin, D., Guo, J., Wu, Y. et al. m6A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-mediated YAP expression and inhibiting miR-107 LATS2u2013mediated YAP activity in NSCLC. Mol Cancer 19, 40 (2020). https: doi.org 10.1186 s12943-020-01161-1 The concerns were raised on Pubpeer here: https: pubpeer.com publications 05FADC2E9A81B9FE46EEAEF7D6B71A The Journal asked the authors to provide an explanation to the observations, and to provide all original data and blots. The EIC assessed the materials provided and is satisfied that the western blots are not repeated panels. Best wishesu201d , 
 There appears to be two images which overlap in Figure 1b. ***  ,