YTHDF2 promotes the liver cancer stem cell phenotype and cancer metastasis by regulating OCT4 expression via m6A RNA methylation
Authors: Hongkai Zhuang
Shiye Ruan
Shanzhou Huang
Kaijun Huang
Zixuan Zhou
Zuyi Ma
Xiaoshun He
Chuanzhao Zhang
Baohua Hou
Fei Ji
Journal: Oncogene (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: N6-methyladenosine; YTHDF2; cancer stem cell; hepatocellular carcinoma; OCT4
Affiliations: Department of General Surgery, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, School of Medicine, South China University of Technology, Guangzhou, 510080, China. ., Department of General Surgery, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, School of Medicine, South China University of Technology, Guangzhou, 510080, China., Shantou University of Medical College, Shantou, 515000, Guangdong Province, China., Organ Transplant Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China. .

Abstract:  N6-methyladenosine (m6A) RNA methylation contributes to the cancer stem cell (CSC) phenotype through regulating gene expression. YTHDF2, an m6A reader, was shown to be associated with hepatocellular carcinoma (HCC) patient prognosis. However, the effect of YTHDF2 on liver CSC and cancer metastasis and the molecular mechanism of this effect have not been documented. Here, we show that YTHDF2 expression is negatively correlated with HCC patient survival in both data from the Cancer Genome Atlas (TCGA) database and clinical data from our center. By detecting CD133+ cells and carrying out sphere culture assays, we found that knockdown of YTHDF2 led to impaired stemness in Hep3B and Huh7 cells. In contrast, overexpression of YTHDF2 increased the CSC phenotype. Mechanistically, the knockdown and overexpression of YTHDF2 in liver cancer cells resulted in decreased and increased m6A levels in the 5'-untranslated region (UTR) of OCT4 mRNA, respectively, leading to decreased and increased OCT4 protein expression, respectively. A luciferase activity assay showed that mutation of the corresponding m6A methylation sequence in the 5'-UTR of OCT4 mRNA caused significantly decreased gene expression, suggesting a role for YTHDF2-dependent m6A methylation in protein translation. Polysome profiling results also indicated the knockdown and overexpression of YTHDF2 could decrease and increase OCT4 translation, respectively. In particular, overexpression of OCT4 rescued the impaired stemness caused by YTHDF2 depletion, which confirmed the effect of YTHDF2 on CSC phenotype is dependent on OCT4. In vivo, the loss of YTHDF2 reduced tumor burden and inhibited lung metastasis following orthotopic transplantation in nude mice. Last, we demonstrated that YTHDF2 expression is positively correlated with OCT4 expression and m6A levels in the 5'-UTR of OCT4 mRNA in clinical HCC specimens. In conclusion, YTHDF2 promotes the CSC liver phenotype and cancer metastasis by modulating the m6A methylation of OCT4 mRNA. 

Retracted: No

PubPeer comments:
 As a tumor statistician, when I see the survival statistical curves of figure 1C and figure 1D, it is totally incomprehensible. Figure 1C and figure 1D are from the OS and RFS of HCC patients in their own institution. A total of 100 patients with HCC were analyzed. What the authors try to show is that the expression of YTHDF2 can distinguish between OS and RFS. But the author s figure is incomprehensible, that is, the survival of RFS is consistent with that of OS. Let s go on as follows: ![file] First, we use the software to merge the survival curves from the author s two figures about OS and RFS, that is, let them be displayed in the same coordinate system. It can be clearly observed that these curves representing different clinical significance basically coincide. ![file] Then, as the author said, the OS and RFS of the same gene YTHDF2 in TCGA database were analyzed by using the bioinformatics website GEPIA. Also let them be displayed in the same coordinate system. It can be clearly observed that the curve of RFS is significantly lower than that of OS. ![file] Furthermore, the OS and RFS of the same gene YTHDF2 in TCGA database were analyzed by KM-PLOTTER in TCGA database. Also let them be displayed in the same coordinate system. It can be clearly observed that the curve of RFs is significantly lower than that of OS. ![file] Finally, we can clearly see that the survival curve from the author s own data is contradictory and unreasonable. Because OS represents the overall survival time of patients, while RFS represents the time of tumor progression. The patients with tumor progression can still be in alive, so the survival time of RFS is certainly shorter than that of OS. In other words, in the statistical analysis chart, the curve of RFS will always be lower than the curve of OS of the same group of patients. ![file] While the author s RFS and OS curves are coincident, almost without any difference. As we all know that, HCC is a kind of tumor with high degree of malignancy, and a relatively high number of patients will have tumor progression. Therefore, as a tumor statistician, I am seriously concerned about the author s own survival data and hope that the author can explain it. thank you! , 
 Figure 5A-B, Measure OD at 260nm was probably not suitable in this step of Hep3B cell lysate preparation for polysome profiling, according to the reported protocol was cited by the authors, 254nm might be better recommended. ![file] ![file] , 
 Also, the absorbance curve looks strange and abnormal at several discontinuous points. ![file] , 
 This is Chuanzhao Zhang, fist author, and I would like to response as follow: (PS: I tried to signed log in but failed, I have contacted contact@pubpeer.com but have no response yet) Comment #1: for the survival curve, in the dataset of 100 HCC patients, we had 33 cases of recurrence before death. And 24 cases were not found to have recurrence before death. Based on these we generated the survival curve. The data is from one single center and sure will be different from public dataset. Comment #2 and #3: I donu2019t see any problem for using OD260. The figure for absorbance are not lines, but many dots. ,