Gene and protein expression of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and “normal” liver tissues

Marina Bortolami; Alessandra Comparato; Clara Benna; Andrea Errico; Isacco Maretto; Salvatore Pucciarelli; Umberto Cillo; Fabio Farinati

doi:10.1371/journal.pone.0244356

Peer Review History

Original SubmissionMarch 18, 2020
19 Aug 2020 Decision Letter - Vladimir Trajkovic, Editor PONE-D-20-07836 Gene and protein expression of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and "normal" liver tissues PLOS ONE Dear Dr. Bortolami, 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. ============================== Academic Editor: To make valid conclusions about the autophagy status, the authors should assess the levels of selective autophagy targets such as SQSTM1/p62 or NBR1, as well as the activation (phosphorylation) of key autophagy signalling molecules such as ULK1. Also, as the p values have already been shown in the figures, there is no need to repeat them in the text, it makes the manuscript difficult to follow. Reviewer 2 This is a well performed study showing the levels of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and "normal" liver tissues. Previous works show the important role that autophagy can have in liver diseases. In this study, the authors show the different levels of protein and mRNA expression in mTOR and LC3 that primary and secondary hepatic tumors present. I have no substantial objections to the experimental work, which is straightforward and presented in a clear manner. As such, these data support the conclusions. However, for completeness, there are a number of points that should be addressed by the authors. Minor points: • In Table 1 (Etiological characteristics of subject), in the HCC column it indicates that the number of patients is 57 but when separated by gender they add up to 56 (20 female and 36 male) • In line 268 of the document, the paragraph talks about Figure 1D but indicates Figure 1C • In the document I would add some information that helps your understanding: - In line 282 of the document the CIRR value is not indicated - In line 305 of the document the value of M is not indicated - At the end of the paragraph that begins to describe figure 2B I would add Fig 2B, on line 301. • In Figure 1A, we can see the difference in the errors of CH and CIRR, but in the document on line 254 it indicates that CH (53.3±30), CIRR (56.2±30). check • In Figure 4 A and B, we can see that the PHCC and HCC values are close to 0.2, but in line 320 of the document it indicates values of (0.4±0.5) or (0.39±0.5) respectively. Check • In Table 2 there are two questions that I would like to clarify: - First in the mRNA correlation column of mTOR and LC3 in CH HBV + there is no coefficient value but it does have p value. - Second, I do not understand the meaning of negative values greater than 1, such as the NM NA value of the same column as before. Spearman's correlation coefficient values are between 1 and -1. • As the authors explain that the M samples present the lowest levels of mRNA of all the samples in the mTOR and LC3 gene, but then they observe the highest expression values of both proteins in M. • Figure 3 presents 9 representative samples of all the samples studied that are included in the densitometers of figure 4. I believe that the films Western blots of all tumors can be displayed on platforms such as (Mendeley Data) for this to be consulted. (Justification Point 3) • The discussion talks about autophagy (line 519 of the document), but the document does not provide enough data to determine the state of autophagy, in my opinion it is difficult to determine the state of autophagy based on two genes or proteins only. It is more convenient to indicate the possible activation of autophagy, or something similar. • Finally, the quality of the images of all the figures is very low, which makes it difficult to understand the text. I think that they should be replaced by higher quality images that allow greater visibility. ============================== Please submit your revised manuscript by Oct 02 2020 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Vladimir Trajkovic Academic Editor 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 2.We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed: (1) https://clincancerres.aacrjournals.org/content/21/22/5037.full The text that needs to be addressed involves the Introduction section (lines 93-96) In your revision please ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. 3. Please provide additional details regarding participant consent. 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: 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: No Reviewer #2: No ****** 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: No Reviewer #2: 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: The manuscript of Bortolami et al. contains a series of data in the form of statistical comparisons which are difficult to follow. No experimental data are reported at least for some representative tests. Reviewer #2: This is a well performed study showing the levels of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and "normal" liver tissues. Previous works show the important role that autophagy can have in liver diseases. In this study, the authors show the different levels of protein and mRNA expression in mTOR and LC3 that primary and secondary hepatic tumors present. I have no substantial objections to the experimental work, which is straightforward and presented in a clear manner. As such, these data support the conclusions. However, for completeness, there are a number of points that should be addressed by the authors. Minor points: • In Table 1 (Etiological characteristics of subject), in the HCC column it indicates that the number of patients is 57 but when separated by gender they add up to 56 (20 female and 36 male) • In line 268 of the document, the paragraph talks about Figure 1D but indicates Figure 1C • In the document I would add some information that helps your understanding: - In line 282 of the document the CIRR value is not indicated - In line 305 of the document the value of M is not indicated - At the end of the paragraph that begins to describe figure 2B I would add Fig 2B, on line 301. • In Figure 1A, we can see the difference in the errors of CH and CIRR, but in the document on line 254 it indicates that CH (53.3±30), CIRR (56.2±30). check • In Figure 4 A and B, we can see that the PHCC and HCC values are close to 0.2, but in line 320 of the document it indicates values of (0.4±0.5) or (0.39±0.5) respectively. Check • In Table 2 there are two questions that I would like to clarify: - First in the mRNA correlation column of mTOR and LC3 in CH HBV + there is no coefficient value but it does have p value. - Second, I do not understand the meaning of negative values greater than 1, such as the NM NA value of the same column as before. Spearman's correlation coefficient values are between 1 and -1. • As the authors explain that the M samples present the lowest levels of mRNA of all the samples in the mTOR and LC3 gene, but then they observe the highest expression values of both proteins in M. • Figure 3 presents 9 representative samples of all the samples studied that are included in the densitometers of figure 4. I believe that the films Western blots of all tumors can be displayed on platforms such as (Mendeley Data) for this to be consulted. (Justification Point 3) • The discussion talks about autophagy (line 519 of the document), but the document does not provide enough data to determine the state of autophagy, in my opinion it is difficult to determine the state of autophagy based on two genes or proteins only. It is more convenient to indicate the possible activation of autophagy, or something similar. • Finally, the quality of the images of all the figures is very low, which makes it difficult to understand the text. I think that they should be replaced by higher quality images that allow greater visibility. ****** 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 [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. https://doi.org/10.1371/journal.pone.0244356.r001
Revision 1
29 Oct 2020 Author Response Response to Reviewers Dear Editor, we thank the Academic Editor and the Reviewers for the careful attention and the comments given to our manuscript PONE-D-20-07836 “Gene and protein expression of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and "normal" liver tissues”. We thoroughly followed their suggestions and we feel that the manuscript ultimately improved. We hope that the present revised version of our manuscript fully meets the criteria of Plos One and that will be considered for publication. Here a point by point answer to Academic Editor and the Reviewer #2 Academic Editor: To make valid conclusions about the autophagy status, the authors should assess the levels of selective autophagy targets such as SQSTM1/p62 or NBR1, as well as the activation (phosphorylation) of key autophagy signaling molecules such as ULK1. Answer: We thank the Academic Editor for the comments and for giving us the opportunity to resubmit a revised version of the manuscript. We agree with the suggestion, therefore, we assessed the protein expression of p62, ULK1 and Raptor in the studied tissues. We added the corresponding paragraphs in the results session and in the discussion section. All the inserted new paragraphs are written in blue. Also, as the p values have already been shown in the figures, there is no need to repeat them in the text, it makes the manuscript difficult to follow. Answer: done as requested. We showed the q-values in the text and deleted the P-values. Reviewer 2 This is a well performed study showing the levels of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and "normal" liver tissues. Previous works show the important role that autophagy can have in liver diseases. In this study, the authors show the different levels of protein and mRNA expression in mTOR and LC3 that primary and secondary hepatic tumors present. I have no substantial objections to the experimental work, which is straightforward and presented in a clear manner. As such, these data support the conclusions. We thank Reviewer 2 for the positive comments. However, for completeness, there are a number of points that should be addressed by the authors. Minor points: • In Table 1 (Etiological characteristics of subject), in the HCC column it indicates that the number of patients is 57 but when separated by gender they add up to 56 (20 female and 36 male) Answer: done as requested. We corrected the total number. • In line 268 of the document, the paragraph talks about Figure 1D but indicates Figure 1C Answer: done as requested. • In the document I would add some information that helps your understanding: - In line 282 of the document the CIRR value is not indicated Answer: done as requested. We apologize for the oversight. - In line 305 of the document the value of M is not indicated Answer: done as requested. We apologize for the oversight. - At the end of the paragraph that begins to describe figure 2B I would add Fig 2B, on line 301. Answer: done as requested. • In Figure 1A, we can see the difference in the errors of CH and CIRR, but in the document on line 254 it indicates that CH (53.3±30), CIRR (56.2±30). check Answer: done as requested. Figure 1 was redone. • In Figure 4 A and B, we can see that the PHCC and HCC values are close to 0.2, but in line 320 of the document it indicates values of (0.4±0.5) or (0.39±0.5) respectively. Check Answer: done as requested. Figure 4 was redone. • In Table 2 there are two questions that I would like to clarify: - First in the mRNA correlation column of mTOR and LC3 in CH HBV + there is no coefficient value but it does have p value. Answer: done as requested. We apologize for the oversight. - Second, I do not understand the meaning of negative values greater than 1, such as the NM NA value of the same column as before. Spearman's correlation coefficient values are between 1 and -1. Answer: done as requested. We apologize for the typos, all the values greater than 1 where missing the “0,” above. • As the authors explain that the M samples present the lowest levels of mRNA of all the samples in the mTOR and LC3 gene, but then they observe the highest expression values of both proteins in M. Answer: Exactly, we observed a decrease of mTOR and LC3 in gene expression with the progression of liver damage, but in order to evaluate the activity of the corresponding proteins we considered the difference between the phosphorylate form of the protein and the total quantity of the protein, independently of its total amount. • Figure 3 presents 9 representative samples of all the samples studied that are included in the densitometers of figure 4. I believe that the films Western blots of all tumors can be displayed on platforms such as (Mendeley Data) for this to be consulted. (Justification Point 3) Answer: we will follow this suggestion. • The discussion talks about autophagy (line 519 of the document), but the document does not provide enough data to determine the state of autophagy, in my opinion it is difficult to determine the state of autophagy based on two genes or proteins only. It is more convenient to indicate the possible activation of autophagy, or something similar. Answer: As suggested by the Academic Editor we assessed the protein expression of p62, ULK1 and Raptor in the studied tissues. We added the corresponding paragraphs in the results session and in the discussion section. All the inserted new paragraphs are written in blue. • Finally, the quality of the images of all the figures is very low, which makes it difficult to understand the text. I think that they should be replaced by higher quality images that allow greater visibility. Answer: done as requested. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0244356.r002
9 Dec 2020 Decision Letter - Vladimir Trajkovic, Editor Gene and protein expression of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and "normal" liver tissues PONE-D-20-07836R1 Dear Dr. Bortolami, 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. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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. Kind regards, Vladimir Trajkovic Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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 #2: (No Response) ******** 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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 #2: 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 #2: 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 #2: (No Response) ****** 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 #2: Yes:** Alicia Bort Bueno https://doi.org/10.1371/journal.pone.0244356.r003
Formally Accepted
11 Dec 2020 Acceptance Letter - Vladimir Trajkovic, Editor PONE-D-20-07836R1 Gene and protein expression of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and “normal” liver tissues Dear Dr. bortolami: 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 Prof. Vladimir Trajkovic Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244356.r004

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