Oncoproteins of High-Risk HPV and EBV Cooperate to Enhance Cell Motility and Invasion of Human Breast Cancer Cells via Erk1/Erk2 and β-Catenin Signaling Pathways
Authors: Hamda Al-Thawadi
Ayesha Jabeen
Semir Vranic
Ala-Eddin Al Moustafa
Ishita Gupta
Journal: Frontiers in Oncology (2021)
Discipline: CLINICAL MEDICINE
Keywords: EBV; HPV; breast cancer; gene deregulation; in-vitro
Affiliations: College of Medicine, Qatar University Health, Qatar University, Doha, Qatar., Biomedical and Pharmaceutical Research Unit, Qatar University Health, Qatar University, Doha, Qatar., Biomedical Research Centre, Qatar University, Doha, Qatar.

Abstract:  Breast cancer is a leading cause of death in women around the world. Most breast cancer-related deaths are a result of complications from the metastatic spread. Several recent studies reported that high-risk human papillomaviruses (HPVs) and Epstein-Barr virus (EBV) are co-presented in different types of human carcinomas including breast; however, the cooperative effects between high-risk HPVs and EBV oncoproteins in human breast cancer have not been investigated yet. Thus, we herein explored the cooperation outcome between E6/E7 and latent membrane protein 1 (LMP1) oncoproteins of high-risk HPV type 16 and EBV, respectively, in two human breast cancer cell lines, MCF7 and MDA-MB-231. Our data revealed that the cooperation of E6/E7 and LMP1 oncoproteins stimulates cell proliferation and deregulates cell cycle progression of human breast cancer and normal mammary cells; in parallel, we noted that E6/E7/LMP1 incite colony formation of both breast cancer cell lines but not normal cells. More significantly, our results point out that the co-expression of E6/E7 and LMP1 oncoproteins enhances cell motility and invasion of MCF7 and MDA-MB-231 cell lines; this is accompanied by deregulation of epithelial-mesenchymal transition biomarkers including E-cadherin, β-catenin, fascin, and vimentin. The molecular pathway analysis of HPV and EBV oncoproteins cooperation shows that it can enhance the phosphorylation of extracellular signal-regulated kinases (Erk1/Erk2) in addition to β-catenin, which could be behind the effect of this cooperation in our cell models. The study clearly suggests that high-risk HPV and EBV coinfection can play an important role in breast cancer progression via Erk1/Erk2 and β-catenin signaling pathways. 

Retracted: Yes

PubPeer comments:
 Fig.1 GAPDH is identical for MCF7 and MDA-MB-231: ![file] Fig.7 contains a WB image with potentially spliced bands. Could the authors please provide the uncropped original images: ![file] , 
 Dear Mr. Heterochasta We would like to thank you for pointing out the errors and sincerely apologize for the unintentional errors. For Figure 1, the GAPDH was run for MDA-MB-231; however, there was an error while pasting the GAPDH and apologize for the duplication. The correct image has been provided and will be submitted to the journal for the same.![file] Moreover, for the Figure 8, for beta-cactenin and vimentin, prior to the revision, the cropped blots were used in the original version. As suggested by the reviewers, the blots for proteins had to be replaced; however, after revisions, by mistake the old image was uploaded. The correct blot image for vimentin and beta-catenin has been attached ![file] and will now submit the journal the updated image. The original blot images will be provided to the journal as Supplementary Data. We are really sorry and sincerely apologize for the unintentional error. , 
 Dear authors, Thank you for your response. Could you please explain, why neither the **original** b-catenin nor the vimentin blot look like the ones you published? , 
 #3 Dear Mr. Heterochasta Thank you for the comment. The original blots were used in the untouched form. However, as one of the reviewers suggested to change the figures with better quality ones, apparently, we cropped the clearer bands from the same experiments that we normally conduct three times for confirmation, to provide a clear figure. Hope this clarifies your doubt. Thank you for your concern. , 
 I am sorry to ask again, but I do not see any similarity between the original blots you posted here and the published ones: ![file] If I understood correctly, you generated the Figure by putting together bands from different blots, and you did so, because one of the reviewers asked you to do that? That sounds, to be honest, quite  unusual , 
 Dear Mr. Heterochasta Like mentioned previously, these were initially submitted to the journal. However one of the reviewers had asked to change the blot images as they were fuzzy. Hence, as mentioned, the experiment was conducted was three times and we cropped the clearer bands from the same experiments, to provide clearer figures; unlike the belief that the reviewer asked us to crop the figures. , 
 #6 Thank you for engaging in this discussion. Please can you provide the original gel image for Figure 1? Please see marked image for MCF7 and HNME, after adjusting brightness to 75% (no other modification). It will be instructive to check if these abnormalities are seen in the original image too. ![file] , 
 Dear Miss Antarctica Thank you for joining the discussion in between The original images have been attached as per your request. HNME (E6 E7)![file] HNME (LMP1) ![file] HNME (GAPDH) ![file] MCF7 (E6 E7) ![file] MCF7 (LMP1) ![file] MCF7 (GAPDH) ![file] As from the original image for E6 E7, it can be noted that there was a very faint expression of LMP1 in MCF7 cells, but was negligible and hence was eliminated. When experiments are performed, there can be negligible artifacts and I believe that this is a part of research which is commonly found in papers. We believe, if we had to manipulate data, we would not have attached the figures. By this figure we only wanted to show that the transfection was successful. The paper aims to show the underlying pathways involved in EMT when breast cancer cells co-express HPV and EBV. , 
 Dear authors, Thank you for providing the original gel pictures. If I may quote from #8: * As from the original image for E6 E7, it can be noted that there was a very faint expression of LMP1 in MCF7 cells, but was negligible and hence was **eliminated**. * This **IS** by definition manipulation of original data, regardless if you think that you  eliminated artifacts or not. It is not up to you to modify images to better support the message of your data or even just to make them look nicer. I am sorry, but this is not acceptable and to be honest pointless to be discussed further. ![file] , 
 #8 Please can you describe how you did the elimination step regarding your statement - As from the original image for E6 E7, it can be noted that there was a very faint expression of LMP1 in MCF7 cells, but was negligible and hence was **eliminated** Are you referring to the LMP1 band that can be see in the original image but not in the published image? ![file] Also, please can you check if the HNME original images you posted were actually the published images? They look completely different to me! I am assisting you by attaching this image below, where I indicated why I think they are different from each other. ![file] , 
 I agree with comment #10. The provided originals do not resemble the published images for Figure 1. In addition, the b-actin band in Figure 7A appears to four lanes that all look remarkably similar. Note the presence and location of a white dot above all four bands in this enhanced-contrast image, as well as sharp vertical background transitions. ![file] , 
 An additional finding for Figure 7B. * Pink boxes: Lanes in the E-Cadherin panel look remarkably similar to both the B-Catenin and p-B-Catenin panels in Figure 7A of another paper from the same research group, i.e. Ayesha Jabeen et al., Molecules (2020), DOI: 10.3390 molecules25184240 - https: pubpeer.com publications A6572009D520A16743FF0FB8B38187 ![file] , 
 **-- RETRACTED --** https: www.frontiersin.org articles 10.3389 fonc.2021.759578 full Following publication, concerns were raised regarding the integrity of the images in the published figures. The authors failed to provide a satisfactory explanation during the investigation, which was conducted in accordance with Frontiersu2019 policies. This retraction was approved by the Chief Editors of Frontiers in Oncology and the Chief Executive Editor of Frontiers. The authors agree to this retraction. ,