SPAG6 hypermethylation silences a novel tumor suppressor and inhibits renal cell carcinoma progression via PI3K/AKT/mTOR pathway

Tianyu Wu; Xing Ji; Yongyang Yun; Xiaofei Wang; Ying Gan; Yu Fan; Qian Zhang

doi:10.1371/journal.pone.0333202

Peer Review History

Original SubmissionJuly 17, 2025
11 Aug 2025 Decision Letter - Yung-Hsiang Chen, Editor PONE-D-25-38827SPAG6 Hypermethylation Silences a Novel Tumor Suppressor and Inhibits Renal Cell Carcinoma Progression via PI3K/AKT/mTOR PathwayPLOS ONE Dear Dr. Zhang, 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. ============================== Thank you for submitting the following manuscript to PLOS ONE. Please revise the manuscript according to the reviewers' comments and upload the revised file. ============================== Please submit your revised manuscript by Sep 25 2025 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 . 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: Yes Reviewer #2: 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 ****** 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: Novel topic: The first systematic study on the epigenetic regulation mechanism of SPAG6 in RCC The research design system and methodology are solid, and the evidence chain is complete: starting with bioinformatics analysis of TCGA/EWAS databases, combining clinical samples, cell experiments, mouse models, and then transcriptome sequencing and functional enrichment analysis, multi-level data is integrated to ensure the scientific and credible nature of the conclusions. Comprehensive types of experiments, including CCK8, clone formation, scratch experiments Transwell、 Flow cytometry, Western blot, BGS, MSP, etc., with clear logic. In vivo experiments, it has been shown to have inhibitory effects on tumor growth and lung metastasis, enhancing clinical transferability. In depth mechanism: not only did we discover the anticancer effect of SPAG6, but we also elucidated its function by inhibiting the PI3K/AKT/mTOR pathway Clear clinical significance: providing new methylation markers for RCC diagnosis and potential targets for treatment Reviewer #2: The authors found that SPAG6 functions as a tumor suppressor in RCC, with its silencing driven by promoter hypermethylation. Through modulation of the PI3K/AKT/mTOR pathway, SPAG6 plays a vital role in restraining RCC initiation and progression, the design and results are good. And I only had one concern, the images of western blot should be improved in Figure 5G and 8C. ****** 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. 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Revision 1
24 Aug 2025 Author Response PONE-D-25-38827R1 SPAG6 Hypermethylation Silences a Novel Tumor Suppressor and Inhibits Renal Cell Carcinoma Progression via PI3K/AKT/mTOR Pathway Respected Dear Editor, Editor Comments, "Thank you for your response. Please provide additional information regarding the experiments involving animals and ensure you have included details on (methods of anesthesia and/or analgesia, and efforts to alleviate suffering." Reply: Respected Editor, we have revised the instructions and made changes in the manuscript. Hope it will be according to the editor's suggestion. The following is the changes made in the manuscript. 2.13 in vivo experiment To investigate whether SPAG6 suppresses tumor growth and metastasis in vivo, both subcutaneous xenograft and pulmonary metastasis mouse models were established. A total of 25 four-week-old male NDG mice (Biocytogen Co., Ltd., Beijing, China) were housed in a specific pathogen-free (SPF) facility at the Animal Experiment Center of Peking University First Hospital. For the tumorigenesis experiment, six mice per group (control and SPAG6 overexpression) were injected subcutaneously with 1.0×10⁷ OSRC-2 cells (transfected with either empty vector or SPAG6 overexpression vector) suspended in 150 μL serum-free medium containing 50% Matrigel. To ensure the welfare of the animals, after four weeks, all procedures were performed under anesthesia using, specific anesthetic, 2% isoflurane via inhalation with continuous monitoring of vital signs. Post-operative analgesia was provided using buprenorphine at 0.05 mg/kg subcutaneously every 12 hours for 48 hours. Humane endpoints were strictly enforced, including tumor size, weight loss, or signs of distress. Animals were euthanized using 99% CO₂ inhalation at a flow rate of 12.2 L/min, followed by cervical dislocation to ensure death. In accordance with institutional guidelines and approved protocols for animal welfare, and primary tumors were excised and weighed for evaluation. To assess the anti-metastatic effect of SPAG6 in vivo, a lung metastasis model was constructed. Six mice per group received tail vein injections of 1.0×10⁶ OSRC-2 cells (empty vector or SPAG6 overexpression), suspended in 150 μL sterile PBS. After four weeks, in vivo and ex vivo fluorescence imaging was performed to assess metastatic burden. All animal experiments in this study were approved by the institutional ethics committee and conducted in strict accordance with relevant guidelines and regulations governing the care and use of laboratory animals. All methods are reported by the ARRIVE guidelines. The Laboratory Animal Ethics Committee of Peking University First Hospital approved the animal studies in this study. PONE-D-25-38827 SPAG6 Hypermethylation Silences a Novel Tumor Suppressor and Inhibits Renal Cell Carcinoma Progression via PI3K/AKT/mTOR Pathway Respected Editor and Reviewer, I am writing to express my sincere gratitude for the thoughtful and constructive feedback provided by the reviewers on our manuscript titled “SPAG6 Hypermethylation Silences a Novel Tumor Suppressor and Inhibits Renal Cell Carcinoma Progression via PI3K/AKT/mTOR Pathway” with Manuscript ID: PONE-D-25-38827. The insights and suggestions were invaluable in improving the quality and clarity of our work. We have carefully addressed all the comments and concerns raised during the review process. The manuscript has been revised extensively, and we believe these changes have significantly strengthened the paper. A detailed point-by-point response to each comment is included in the accompanying document for your reference. I would like to extend my heartfelt thanks to the reviewers for their time, expertise, and dedication. Their feedback has been instrumental in shaping the final version of our manuscript. I am also grateful to you, Editor, for your guidance and support throughout this process. We are confident that the revised manuscript now meets the high standards of PLOS ONE and hope it will be favorably considered for publication. Please find the revised manuscript and all supplementary materials attached for your review. Thank you once again for the opportunity to contribute to PLOS ONE. I look forward to your positive response. 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 Respond: Respected Editor, we have revised the manuscript and made the changes according to the journal requirements. As given in the link. 2. To comply with PLOS One submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Respond: Respected Editor, we have revised and included the details in the “2.13 in vivo experiment” section. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Respond: Respected Editor, as a corresponding author, I have registered, and my ORCID ID is Qian Zhang , 0009-0003-0713-313X 4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. Respond: Respected Editor, the Ethical Statement is only written in the methods. 5. 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. Respond: Respected Editor, we have included the original uncropped and unadjusted images of Western blot in the supplementary image. 6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Respond: Respected Editor, we have included the captions for your Supporting Information files at the end of the manuscript. 7. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Respond: Respected Editor, the reviewer didn’t recommend any specific data or citation. 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 ________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: Yes Reviewer #2: 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 ________________________________________ 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: Novel topic: The first systematic study on the epigenetic regulation mechanism of SPAG6 in RCC. The research design system and methodology are solid, and the evidence chain is complete: starting with bioinformatics analysis of TCGA/EWAS databases, combining clinical samples, cell experiments, mouse models, and then transcriptome sequencing and functional enrichment analysis, multi-level data is integrated to ensure the scientific and credible nature of the conclusions. Comprehensive types of experiments, including CCK8, clone formation, scratch experiments Transwell, Flow cytometry, Western blot, BGS, MSP, etc., with clear logic. In vivo experiments, it has been shown to have inhibitory effects on tumor growth and lung metastasis, enhancing clinical transferability. In depth mechanism: not only did we discover the anticancer effect of SPAG6, but we also elucidated its function by inhibiting the PI3K/AKT/mTOR pathway. Clear clinical significance: providing new methylation markers for RCC diagnosis and potential targets for treatment Reviewer #2: The authors found that SPAG6 functions as a tumor suppressor in RCC, with its silencing driven by promoter hypermethylation. Through modulation of the PI3K/AKT/mTOR pathway, SPAG6 plays a vital role in restraining RCC initiation and progression, the design and results are good. And I only had one concern, the images of Western blot should be improved in Figure 5G and 8C. Respond: Respected Reviewer, as we have revised the western blot result and we have pasted the best ones and more significant results of the western blot. Also, we have included the full image of the Western blot in the supplementary file. Hopeful that after analysing the result, the reviewer will be convinced. ________________________________________ 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: Yes: Zhao Yang Attachments Attachment Submitted filename: Response to Editor-2.docx https://doi.org/10.1371/journal.pone.0333202.r002
11 Sep 2025 Decision Letter - Yung-Hsiang Chen, Editor SPAG6 Hypermethylation Silences a Novel Tumor Suppressor and Inhibits Renal Cell Carcinoma Progression via PI3K/AKT/mTOR Pathway PONE-D-25-38827R1 Dear Dr. Zhang, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . 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, Yung-Hsiang Chen, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Congratulations on the acceptance of your manuscript, and thank you for your interest in submitting your work to PLOS ONE. 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 #1: 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: (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 #1: No ******** https://doi.org/10.1371/journal.pone.0333202.r003
Formally Accepted
Acceptance Letter - Yung-Hsiang Chen, Editor PONE-D-25-38827R1 PLOS ONE Dear Dr. Zhang, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@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. Yung-Hsiang Chen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0333202.r004

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