USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2

Yuanyuan Chen; Yunfei Guo; Mei Yuan; Song Guo; Shuaishuai Cui; Dahu Chen

doi:10.1371/journal.pone.0290688

Peer Review History

Original SubmissionJuly 2, 2023
18 Jul 2023 Decision Letter - Kishor Pant, Editor PONE-D-23-20608USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2PLOS ONE Dear Dr. Chen, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Sep 01 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this article, Yuanyuan et al. investigate the unique relationship between USP4 and PKM2 as well as how it controls the way cancer cells use glucose. According to their findings, USP4 controls PKM2 levels through the process of de-ubiquitylation, and its inhibition lowers PKM2 stability. They look at the effects on glucose uptake and metabolism in cancer cells of both over- and under-expressing USP4 and PKM2. They conclude that USP4 modulates PKM2's stability by deubiquitinating, which helps to increase the Warburg effect and support cancer surveillance. While this paper addresses an essential subject—namely, how USP4 regulates PKM2 levels and their connection to cancer cell metabolism—there are significant concerns that still need to be resolved. These are listed below. By doing immunoprecipitation with mutants of PKM2 at ubiquitination sites, the findings in Figure 1 may also be significantly reinforced. The available information generally supports the interaction between PKM2 and USP4, although it is not specific to PKM2's ubiquitin state. Additionally, authors can show interaction and localization experiments involving additional cells in Figure 1E. The authors should use varied expression levels of USP4 and its catalytic mutant to test the stability of PKM2 in a dose-dependent manner. The authors need to discuss how cells survive when PKM2 levels are significantly reduced (Figure 2C). When USP4 is overexpressed, the authors should check the levels of ubiquitinated PKM2. Western blot analysis is necessary to display the PKM2 and USP4 levels in Figures 3 and 4. Reviewer #2: The manuscript entitled “USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2” provides new insights. However, the manuscript needs some minor editing to make the suitable for publication. 1. It would be help full to readers if authors could include a graphical abstract. 2. Does authors have checked the basal protein levels of PKM2 or USP4 in experimental cells lines? 3. Does knockdown of UPS4 in non-cancerous cell line effects its growth and proliferation? 4. Authors are recommended to also confirm protein expression of Glut1 or 4. Reviewer #3: The manuscript was well drafted but some questions need to addressed to improve the manuscript 1.Please include the data for reverse Immunoprecipitation assay to check the interaction. 2.Have the authors have done mass spec analysis to check the interaction. 3.Have you made the knockdown cells and then checked the interaction. Please include data 4.Also include the mouse model experiments ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: Yes: jasvinder singh ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. 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Revision 1
2 Aug 2023 Author Response Dear editor and reviewers, We thank PLOS ONE for the interest in our findings and the reviewers for their highly careful, thorough reviews and insightful comments. We appreciate that you encouraged us to submit a revised version of our manuscript. In response to the specific comments, we have made all necessary changes that were requested by the reviewers. Revisions in the manuscript are highlighted in YELLOW to make them easy for reviewing. What follows is a point-by-point response to the reviewers (under “RE:”). We believe that we have carefully addressed all the concerns. We hope that these changes adequately address the reviewers' comments, and that the manuscript is now suitable for publication in PLOS ONE. Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf RE: We have carefully checked our manuscript and are confident that it meets PLOS ONE’s requirements. 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. RE: We do not have any data that is stored in any repository. All data supporting the findings of this study are available within the article. We have changed our Data Availability Statement in our re-submission cover letter to reflect the fact that all data supporting the findings of this study are available within the article. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. RE: We have provided original, uncropped and unadjusted images for each blot or gel data that appears in the submission figures in the supporting information file. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. RE: We have removed this phrase from the manuscript (Page 10, Line 176) as suggested. 5. We notice that your supplementary figures are uploaded with the file type 'Figure'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. RE: We have amended the file type to ‘Supporting Information’ as instructed. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this article, Yuanyuan et al. investigate the unique relationship between USP4 and PKM2 as well as how it controls the way cancer cells use glucose. According to their findings, USP4 controls PKM2 levels through the process of de-ubiquitylation, and its inhibition lowers PKM2 stability. They look at the effects on glucose uptake and metabolism in cancer cells of both over- and under-expressing USP4 and PKM2. They conclude that USP4 modulates PKM2's stability by deubiquitinating, which helps to increase the Warburg effect and support cancer surveillance. While this paper addresses an essential subject—namely, how USP4 regulates PKM2 levels and their connection to cancer cell metabolism—there are significant concerns that still need to be resolved. These are listed below. • By doing immunoprecipitation with mutants of PKM2 at ubiquitination sites, the findings in Figure 1 may also be significantly reinforced. The available information generally supports the interaction between PKM2 and USP4, although it is not specific to PKM2's ubiquitin state. Additionally, authors can show interaction and localization experiments involving additional cells in Figure 1E. RE: We agree that the immunoprecipitation experiments using PKM2 mutants at ubiquitination sites would further reinforce our conclusion. However, we have not yet identified the specific ubiquitination sites on PKM2 by mediated by USP4. Therefore, we are unable to generate the suggested PKM2 mutants at this stage. This is an area we are actively investigating and hope to publish future studies on the precise ubiquitination sites. We demonstrated the interaction between USP4 and PKM2 by co-expressing these proteins in 293T cells. Additionally, we provided evidence for an endogenous interaction between USP2 and PKM2 in two different gastric cancer cell lines. Our results showing the interaction using both overexpressed and endogenous proteins across multiple cell lines provide strong evidence for a direct physical interaction between USP4 and PKM2. • The authors should use varied expression levels of USP4 and its catalytic mutant to test the stability of PKM2 in a dose-dependent manner. RE: For exogenous expression of a gene in cells, it is difficult to precisely control the expression level. This is because we cannot control how many copies of the DNA integrate into the chromosomes. Even when transfecting different amounts of DNA into cells, the resulting expression levels are unpredictable. While we acknowledge that this experiment could help strengthen the conclusions, we have encountered challenges in achieving dose-dependent exogenous expression using standard transfection methods. We would welcome any specific suggestions from the reviewer regarding approaches to achieve dose-dependent expression. • The authors need to discuss how cells survive when PKM2 levels are significantly reduced (Figure 2C). RE: We have added brief explanations to the Results section (Page 15, Lines 283-287) to address the reviewer's concern. • When USP4 is overexpressed, the authors should check the levels of ubiquitinated PKM2. RE: When USP4 is overexpressed, ubiquitinated PKM2 levels are greatly reduced, as we demonstrated in the top panel of Figure 2 (middle lane). • Western blot analysis is necessary to display the PKM2 and USP4 levels in Figures 3 and 4. RE: We utilized stable cell lines to perform cell proliferation, glucose uptake, and lactate production assays. The expression levels of USP4 and PKM2 in these stable cell lines are presented in Figures 2A-D. Reviewer #2: The manuscript entitled “USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2” provides new insights. However, the manuscript needs some minor editing to make the suitable for publication. 1. It would be help full to readers if authors could include a graphical abstract. RE: Thank you for the feedback on including a graphical abstract. We have updated the manuscript to include a graphical abstract as Figure 5, as you suggested. We believe this addition helps summarize the main findings of our study and makes the work more accessible to readers. We have added Figure 5 to the discussion part. If you feel the position of Figure 5 is inappropriate, please let us know. We appreciate you taking the time to provide constructive comments to improve our paper. 2. Does authors have checked the basal protein levels of PKM2 or USP4 in experimental cells lines? RE: Thank you for asking about the basal protein levels of PKM2 and USP4 in our cell lines. You raise a good point - we should have more clearly presented this data. The endogenous expression of both proteins can be found in Figures 2A-D, as you noted. However, we agree this is easy to overlook without specific mention in the text. To address this, we have added a sentence in the Results section mentioning the detectable basal levels of PKM2 (Page 12, Lines 224-225). As for the basal expression of USP4, we mentioned it on Page 13, Lines 231-232: ‘while the endogenous of USP4 was knocked down...’. We appreciate you identifying this omission. 3. Does knockdown of UPS4 in non-cancerous cell line effects its growth and proliferation? RE: Thank you for your insightful question about the effect of USP4 knockdown on non-cancerous cell growth. As you suspected, we did perform experiments examining USP4 knockdown in 293T cells. We found that USP4 knockdown inhibited 293T proliferation, similar to what we observed in gastric cancer cell lines. We did not include this result in our manuscript, but we would be happy to add it if you feel it is necessary. 4. Authors are recommended to also confirm protein expression of Glut1 or 4. RE: We did attempt to analyze Glut1 protein expression, trying both a rabbit polyclonal antibody and a mouse monoclonal antibody. Unfortunately, neither antibody produced robust results for us. Therefore, since the available antibodies did not work well in our hands, we opted to present the Glut1 mRNA levels by qPCR instead. Reviewer #3: The manuscript was well drafted but some questions need to addressed to improve the manuscript 1.Please include the data for reverse Immunoprecipitation assay to check the interaction. RE: We did perform a reverse immunoprecipitation assay to check the interaction. Flag-USP4 and Myc-PKM2 were co-transfected into cells, and then either immunoprecipitated with Myc and blotted with Flag, or immunoprecipitated with Flag and blotted with Myc. The results are shown in Figure 1A. 2.Have the authors have done mass spec analysis to check the interaction. RE: No, we have not performed mass spectrometry analysis to check the interaction. We were limited by time and resources, but we may consider performing this analysis in the future. We understand that mass spectrometry analysis is a valuable tool for confirming protein interactions, and we appreciate the suggestion. 3.Have you made the knockdown cells and then checked the interaction. Please include data RE: Our Co-IP assay results showed that Flag-USP4 and Myc-PKM2 co-immunoprecipitated with each other. We also identified an interaction region between these two proteins in both USP4 and PKM2. We feel that this evidence is sufficient to conclude that there is a physical interaction between these two proteins. We do not feel that it is necessary to make knockdown cells to confirm this interaction. 4.Also include the mouse model experiments RE: We agree that mouse model experiments would be a valuable addition to our manuscript. We are currently working on a mouse model of gastric cancer that expresses USP4. We hope to generate data from this model that will support our hypothesis that USP4 plays a role in gastric cancer development. We understand that the requirement from this commenter is beyond the scope of our current study, but we hope to be able to present this data in a future paper. 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: Yes: jasvinder singh [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0290688.r002
15 Aug 2023 Decision Letter - Kishor Pant, Editor USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2 PONE-D-23-20608R1 Dear Dr. Chen, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Dear Dr. Dahu Chen, I applaud you on a well-written paper. My main worry is that this work may overstate the effects of PKM2 ubiquitinoylation and interaction with USP4 on readers because these effects are not well established. It would be a good idea, in my opinion, to include some discussion. Regards Srinivasu Karri Reviewer #2: After revision MS looks better. Authors have addressed the concerns raised during the revision. So, I would recommend to accept in current form. Reviewer #3: Authors have addressed all the comments that was raised and manuscript should be accepted for publication. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: Yes: jasvinder singh ******** https://doi.org/10.1371/journal.pone.0290688.r003
Formally Accepted
18 Aug 2023 Acceptance Letter - Kishor Pant, Editor PONE-D-23-20608R1 USP4 promotes the proliferation and glucose metabolism of gastric cancer cells by upregulating PKM2 Dear Dr. Chen: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Kishor Pant Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0290688.r004

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