RETRACTED: The antineoplastic drug metformin downregulates YAP by interfering with IRF-1 binding to the YAP promoter in NSCLC
Authors: Shuying Tan
Deqiang Wang
Yan Wu
Xin Xu
Yong Gao
Dan Jin
Jiwei Guo
Cuijie Shao
Xiaohong Wang
Journal: EBioMedicine (2018)
Discipline: CLINICAL MEDICINE
Keywords: EMT; IRF-1; Lung cancer; Metformin; Verteporfin
Affiliations: Department of Pain, Binzhou Medical University Hospital, Binzhou 256603, PR China., Cancer research institute, Binzhou Medical University Hospital, Binzhou 256603, PR China. Electronic address: ., Cancer research institute, Binzhou Medical University Hospital, Binzhou 256603, PR China., Department of Thyroid and Breast Surgery, Binzhou Medical University Hospital, Binzhou 256603, PR China.

Abstract:  Background: Activation of the oncogene YAP has been shown to be related to lung cancer progression and associates with poor prognosis and metastasis. Metformin is a drug commonly used in the treatment of diabetes and with anticancer activity. However, the mechanism through which metformin inhibits tumorigenesis via YAP is poorly understood. Methods: The mRNA and protein expressions were analyzed by RT-PCR and western blot. The cellular proliferation was detected by CCK8 and MTT. The cell migration and invasion growth were analyzed by wound healing assay and transwell assay. The activities of promoter were analyzed by luciferase reporter assay. Chromatin immunoprecipitation detected the combining ability of IRF-1 and 5'UTR-YAP. Findings: Our immunohistochemistry staining and RT-PCR assays showed that the expression of YAP was higher in lung carcinoma samples. Interestingly, metformin was able to downregulate YAP mRNA and protein expression in lung cancer cells. Mechanistically, we found that metformin depressed YAP promoter by competing with the binding of the transcription factor IRF-1 in lung cancer cells. Moreover, combination of metformin and verteporfin synergistically inhibits cell proliferation, promotes apoptosis and suppresses cell migration/invasion by downregulating YAP, therefore reduces the side effects caused by their single use and improve the quality of life for patients with lung cancer. Interpretation: we concluded that metformin depresses YAP promoter by interfering with the binding of the transcription factor IRF-1. Importantly, verteporfin sensitizes metformin-induced the depression of YAP and inhibition of cell growth and invasion in lung cancer cells. FUND: This work was supported by National Natural Science Foundation of China (No.31801085), the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067 and ZR2017MH125), and Research Foundation of Binzhou Medical University (BY2015KYQD29 and BY2015KJ14). 

Retracted: Yes

PubPeer comments:
 Another paper with image concerns by this group. Highlighted here is Figure 1B. In addition to similar and repeated features in the bands, please note the apparent repeated areas in the background. Notably, the bands do not always appear identical in the YAP blot. If the authors are able, please post here the original uncropped scans of the gels. *** Contrast enhanced ![file] , 
 Dear Actinopolyspora Biskrensis, thank you very much for reminding us the mistake. When the completion image in Fig.1B, the image was exported incorrectly by software. We will submit the right image to the journal to make a correction. Fortunately, this correction does not affect the results, discussions and conclusions of this paper. We apologize for such an inadvertent error and thank you again for the comments. , 
 Dear Actinopolyspora Biskrensis, Sorry for the late reply. Thank you very much for your comments on our research. It will be helpful for our future work. The similar shaped bands seem to be appeared in the proteins of YAP and Tubulin in Fig 1B. One possible reason is that when the image of Fig 1B was exported, the band was compressed whereby leading to generate the low-quality image along with the possible similar shaped bands for YAP and Tubulin proteins. It is important to emphasize that the possible similar shaped bands are different bands for YAP and Tubulin proteins in the Fig 1B. To alleviate this confusion, we provided another independent repeated result as included below. To remove the confusion, we have contacted the editorial office of EBioMedicine for correction and the corrected data will be published soon. Sincerely, JW Guo The published and the independent repeation Fig1B is included below: ![file] , 
 #3 Dr. Guo: I am pleased that you located the original images. Would you be so kind to upload here the highest resolution possible image of the gels marked with the red box below? Thank you for your assistance. *** ![file] , 
 #4 Frankly, after further examining the images provided by the author, there is no need for the authors to post a higher resolution image. I have compared below the Tubulin band from the author-provided  Uncropped original data (outlined with green box) to the Tubulin band from the published manuscript (red box). I adjusted the contrast of the two images so that they are similar. Comparing the smears in the  original to its corresponding smear in the  published band, readers will note that there are substantial differences, most notably in the background. While the vertical cropping lines do not appear  original , nether do the dark areas under bands 2,4,6,8,10, and 12 that appear in the  published image; see magenta box for illustration. Likewise, under bands 1,3,5,7,9,11 in the  published image there is no dark area, yet in the  original there sometimes are dark areas; see yellow box for illustration. Readers can compare by eye the YAP bands which have similar issues. These discrepancies between the  original and  published image suggest to me that the  original is not the source of the  published image. *** ![file] , 
 Perhaps the authors can also be persuaded to look into why it seems that all the images in the top row depicting the scratch assay in figure 6 appear to be the same scratch? I realize that this is the  before condition , but these samples should, nevertheless, not be the same. ![file] , 
 There seem to be duplicate images in Figure 2 that overlap with Figure 1 of another article of the same author that is published in the same journal in the same issue [] ![file] , 
 There are more duplicated images. There seem to be duplicate images in Figure 2 that overlap with Figure 1 of another article of the same author that is published in the same journal in the same issue [] ![file] , 
 Thanks for the comments. For Fig 1B, as indicated in the response #3, the differences between original and published band come from the compressed band with the following reasons: 1) the published Fig 1B was compressed when it was exported from photoshop; 2) the completed published Figure 1 was compressed when it was exported from illustrator; 3) anther possible reason is the procession of the contrast of the two images, which is not certainly known for sure. In addition, before correction and response here, our institution had organized an independent investigation and draw the following conclusions: the provided original data were reliable, the experimental process was rigorous, and the results were not manipulated. The unintentional error has no bearing on the work s scientific conclusions. Furthermore, we have sent the detailed eventual report and adjudication to the editorial office of EBioMedicine. For Fig 6F, we have checked our original data of images and found that this error was occured during figure preparation. The graduated student conducted multiple samples together and mistakenly mixed up the data for Fig 6F. We show the corrected the Fig 6F as following: ![file] For the 2K, we have checked our original data of images and found that this error was occured during figure preparation. Owing to simultaneously prepare the data (two papers) for publishing in the EBioMedicine using the same name folder, the graduated student mistakenly mixed up the transwell data for the two papers. We show the corrected the Fig 2K as following: ![file] In a conclusion, the unintentional error has no bearing on the work s scientific conclusions. we have contacted the editorial office of EBioMedicine for correction. , 
 There are more duplications. Here is one from Figure 8. ![file] , 
 There are more duplications in the colony forming assays as well. ![file] , 
 Thanks for the comments. For Fig 8D, we have carefully checked our original data and found this error. During organizing the results, the IHC image of Vimentin was mistakenly duplicated into the IHC image of E-cadherin. The corrected version of Fig 8D is included below. For Fig 5C and 6D, we have checked our original data of images and found that this error was occurred during figure preparation. The graduated student conducted multiple samples together for clone formation assay and mistakenly mixed up the data for Fig 5C. We show the corrected the Fig 5C as following. In a conclusion, the unintentional error has no bearing on the work s scientific conclusions. ![file] , 
 #12 Dear Dr. Jiwei Guo, Could you elborate on what the rationale for rotating and mirroring the images of the colony forming assays in preparing the images might be? Are certain angles better suited for the reader perhaps? In the name of expedience I will also list the next set of duplications. These tumors appear to have been copied, rescaled and rotated in the image preparation process. For perfectly inoccuous reasons, I am sure. ![file] , 
 Just out of curiosity, were there any aspects of this paper which were **not** conducted by the error-prone graduate student? , 
 There are more duplicated images between this publication and doi: 10.1016 j.ebiom.2018.08.001. Perhaps, the  error-prone graduate student needs some coaching. In addition of the duplication in the colony forming assays highlighted by #11, there are more duplication between Figure 6 and Figure S3 of another article of the same author that is published in the same journal of the same issue https: pubpeer.com publications 30C4B226FBDA768041AF53AB1F391A ![file] , 
 This article has been retracted at the request of the Authors. After publication of the EBioMedicine article [1], several concerns were raised with respect to the veracity of the data. Evidence of image duplication was found within figures relating to the transwell assays, scratch assays, immunohistochemistry images, clone formation assays, and xenografted tumours. Discrepancies were also detected between the published Western blots shown in Figure 1B and the original blots. The corresponding author Jiwei Guo and all co-authors would like to withdraw this article to maintain the integrity of the scientific record. The authors sincerely apologise to the editors and journal readership for these oversights and inconvenience that this may have caused. ,