Response Gene to Complement 32 promotes cell proliferation and tamoxifen resistance in breast cancer via elevated FoxM1 expression

Xinlei Li; Yan Liu; Zhiqian Wang; Xiaocui Bu; Yu Wang; Wei Zhang; Peng Zhao

doi:10.1371/journal.pone.0328698

Peer Review History

Original SubmissionJanuary 23, 2025
Decision Letter - Tudor C. Badea, Editor Dear Dr. Zhao, 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 Jun 08 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 . 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. 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Tudor C. Badea, M.D., M.A., Ph.D. 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. Please note that funding information should not appear in any section or other areas of your manuscript. 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Additional Editor Comments: The reviewers made suggestions for improving your manuscript, in particular related to the placing in context of your findings, i.e. introduction and discussion, and data processing of microarray data. This last point is particularly important, as availability of microarray data published in this study is mandatory before considering publication. Please deposit the microarray data in a public repository and describe the format, accession number and release date of the data in a separate paragraph in the data availability section. Also please expand on the performed DEG analysis in both methods and results section. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? 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 Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: This is an interesting manuscript investigating the role of RGC-32 in cell proliferation and tamoxifen resistance in breast cancer. The authors show that the RGC-32 mediated tamoxifen resistance is partially dependent on FoxM1 expression. Targeting RGC-32 can be an effective target in restoring sensitivity to Tamoxifen in breast cancer cells. Critique The introduction is very short. Adding more details on the role of RGC-32 in cancers will be helpful for the reader. The Discussion section will benefit from more details on how RGC_32 activate cell cycle and induce cell proliferation, especially concerning phosphorylation sites induced activated by CDK1, Akt and ERK1. Discussing the role of ERK1 in the context of cell proliferation will be also helpful. More details on the role of Fox M1 role in pathology and cancers will also be helpful. Reviewer #2: Strengths of the study: This is a very thorough translational and experimental research study that eloquently documents RGC-32 expression in ER+ breast cancer tissues, while also deciphering the mechanisms through which RGC-32 mediates tamoxifen resistance and promotes breast cancer cells cell proliferation. The methods used in this study are well documented and valid: immunohistochemistry, RNA interference, microarray gene analysis and the use of tumor xenograft model. The authors demonstrate here that RGC-32 can activate PI3K signaling pathway, consequently enhancing ERα activity. ERα activation is essential for RGC-32-mediated the expression of FoxM1, while Tamoxifen resistance mediated by RGC-32 seems partially dependent on FoxM1 expression. We believe that the data presented here, an elucidation of the molecular mechanisms of acquired drug resistance in breast cancer, represents a real progress in the field of translational oncology. Minor issues (limitations): 1. Abstract, Page 10: the acronym ERα is not detailed. 2. Neither in the DISCUSSION section, nor in the Introduction section you do not mention the connection with the complement system, namely with C5b-9, knowing that RGC-32 is a protein that promotes cell cycle progression in response to complement activation (Badea et al, J Biol Chem 2002). Please detail this connection and integrate it in harmony with the data presented here (your results). 3. In the Discussion section, when discussing the role played by RGC-32 in the proliferation of ER+ breast cancer cells, other immunohistochemistry data on RGC-32’s expression in cancer tissue ought to be mentioned: we have now shown that RGC-32 is expressed in precancerous states, and its expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages of colon cancer than in precancerous states or the initial stages of colon cancer. (S.I. Vlaicu et al. Experimental and Molecular Pathology 88 (2010) 67–76.) 4. The gene array data you performed in MCF-7 cells is insufficiently exploited in my view: what are other relevant pathways influenced by RGC-32 silencing, apart cell cycle control of chromosomal replication? And why is there no reference of microarray data that precedes your work: a gene array analysis to investigate the effect of RGC-32 knockdown on gene expression in the SW480 colon cancer cell line S.I. Vlaicu et al. Experimental and Molecular Pathology 88 (2010) 67–76.). A comparison the different RGC-32 dependent pathways found in these two studies should be present in your Discussion section. 5. You mention that “These results suggested a novel regulatory relationship between RGC-32 and PI3K/AKT-ERα crosstalk.” I would disagree that this relationship is a NEW finding, and here are the grounds for my disapproval: This relationship was previously documented in endothelial cells by Fosbrink et al. (Fosbrink et al., Exp Mol Pahol Vol 86, 2009) and in T-lymphocytes by Tegla et al. (Tegla CA, Cudrici CD, Nguyen V, Danoff J, Kruszewski AM, Boodhoo D, Mekala AP, Vlaicu SI, Chen C, Rus V, Badea TC, Rus H. RGC-32 is a novel regulator of the T-lymphocyte cell cycle. Exp Mol Pathol. 2015 Jun;98(3):328-37.) The aforementioned research showed that RGC-32 was involved in controlling the cell cycle of T cells in vivo; AKT connects PI3K to signaling pathways that promote cytokine transcription, survival, and cell-cycle activation. RGC-32 acts on Akt which phosphorylates FOXO1 protein, induces IL-2 transcription and cell cycle activation contributing to TCR mediated signaling. I would see it as optimal and necessary to integrate these previous findings into the Discussion chapter. My recommendation: Accepted for publication with minor revisions. ****** 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: Sonia Irina Vlaicu ******** [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.0328698.r001
Revision 1
8 May 2025 Author Response Reviewer #1: This is an interesting manuscript investigating the role of RGC-32 in cell proliferation and tamoxifen resistance in breast cancer. The authors show that the RGC-32 mediated tamoxifen resistance is partially dependent on FoxM1 expression. Targeting RGC-32 can be an effective target in restoring sensitivity to Tamoxifen in breast cancer cells. We would like to express our sincere thanks to the reviewers for the constructive and positive comments. Critique The introduction is very short. Adding more details on the role of RGC-32 in cancers will be helpful for the reader. Answer: We expand the introduction (page 40, paragraph 2) to include the more details on the role of RGC-32 in cancers. The Discussion section will benefit from more details on how RGC_32 activate cell cycle and induce cell proliferation, especially concerning phosphorylation sites induced activated by CDK1, Akt and ERK1. Answer: Several sentences have been added in the Discussion (page 45, paragraph 4) in the revised version to address this issue. Discussing the role of ERK1 in the context of cell proliferation will be also helpful. Answer: we added the role of ERK1 in cell proliferation in the Discussion (page 45, paragraph 4) in the revised version. More details on the role of Fox M1 role in pathology and cancers will also be helpful. Answer: Several sentences have been added in the Discussion (page 46, paragraph 2) in the revised version to address this issue. Reviewer #2: Strengths of the study: This is a very thorough translational and experimental research study that eloquently documents RGC-32 expression in ER+ breast cancer tissues, while also deciphering the mechanisms through which RGC-32 mediates tamoxifen resistance and promotes breast cancer cells cell proliferation. The methods used in this study are well documented and valid: immunohistochemistry, RNA interference, microarray gene analysis and the use of tumor xenograft model. The authors demonstrate here that RGC-32 can activate PI3K signaling pathway, consequently enhancing ERα activity. ERα activation is essential for RGC-32-mediated the expression of FoxM1, while Tamoxifen resistance mediated by RGC-32 seems partially dependent on FoxM1 expression. We believe that the data presented here, an elucidation of the molecular mechanisms of acquired drug resistance in breast cancer, represents a real progress in the field of translational oncology. We would like to express our sincere thanks to the reviewers for the constructive and positive comments. Minor issues (limitations): 1. Abstract, Page 10: the acronym ERα is not detailed.1. Answer: Correction has been made in the revised version. 2. Neither in the DISCUSSION section, nor in the Introduction section you do not mention the connection with the complement system, namely with C5b-9, knowing that RGC-32 is a protein that promotes cell cycle progression in response to complement activation (Badea et al, J Biol Chem 2002). Please detail this connection and integrate it in harmony with the data presented here (your results). Answer: Several sentences have been added in the Introduction (page 40, paragraph 2) in the revised version to address this issue. 3. In the Discussion section, when discussing the role played by RGC-32 in the proliferation of ER+ breast cancer cells, other immunohistochemistry data on RGC-32’s expression in cancer tissue ought to be mentioned: we have now shown that RGC-32 is expressed in precancerous states, and its expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages of colon cancer than in precancerous states or the initial stages of colon cancer. (S.I. Vlaicu et al. Experimental and Molecular Pathology 88 (2010) 67–76.) Answer: Several sentences have been added in the Discussion (page 46, paragraph 3) in the revised version to address this issue. 4. The gene array data you performed in MCF-7 cells is insufficiently exploited in my view: what are other relevant pathways influenced by RGC-32 silencing, apart cell cycle control of chromosomal replication? And why is there no reference of microarray data that precedes your work: a gene array analysis to investigate the effect of RGC-32 knockdown on gene expression in the SW480 colon cancer cell line S.I. Vlaicu et al. Experimental and Molecular Pathology 88 (2010) 67–76.). A comparison the different RGC-32 dependent pathways found in these two studies should be present in your Discussion section. Answer: Several sentences have been added in the Discussion (page 46, paragraph 3) in the revised version to address this issue. 5. You mention that “These results suggested a novel regulatory relationship between RGC-32 and PI3K/AKT-ERα crosstalk.” I would disagree that this relationship is a NEW finding, and here are the grounds for my disapproval: This relationship was previously documented in endothelial cells by Fosbrink et al. (Fosbrink et al., Exp Mol Pahol Vol 86, 2009) and in T-lymphocytes by Tegla et al. (Tegla CA, Cudrici CD, Nguyen V, Danoff J, Kruszewski AM, Boodhoo D, Mekala AP, Vlaicu SI, Chen C, Rus V, Badea TC, Rus H. RGC-32 is a novel regulator of the T-lymphocyte cell cycle. Exp Mol Pathol. 2015 Jun;98(3):328-37.) The aforementioned research showed that RGC-32 was involved in controlling the cell cycle of T cells in vivo; AKT connects PI3K to signaling pathways that promote cytokine transcription, survival, and cell-cycle activation. RGC-32 acts on Akt which phosphorylates FOXO1 protein, induces IL-2 transcription and cell cycle activation contributing to TCR mediated signaling. I would see it as optimal and necessary to integrate these previous findings into the Discussion chapter. Answer: we have integrated these previous findings into the Discussion chapter (page 46, paragraph 4). Attachments Attachment Submitted filename: response to reviewers(1).docx https://doi.org/10.1371/journal.pone.0328698.r002
Decision Letter - Tudor C. Badea, Editor Dear Dr. Zhao, 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 comply with data sharing requirements, by making microarray data publicly available as indicated. ============================== Please submit your revised manuscript by Jun 26 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 . 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. 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Tudor C. Badea, M.D., M.A., Ph.D. Academic Editor PLOS ONE Additional Editor Comments: The results of microarray experiments are not sufficiently presented. The raw counts should be submitted to a repository (e.g. gene expression omnibus), and the accession number provided and supplementary tables with differrentially expressed genes should be provided. Making your data available in this fashion is mandatory for publication. [Note: HTML markup is below. Please do not edit.] [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.0328698.r003
Revision 2
13 May 2025 Author Response Reviewer #1: This is an interesting manuscript investigating the role of RGC-32 in cell proliferation and tamoxifen resistance in breast cancer. The authors show that the RGC-32 mediated tamoxifen resistance is partially dependent on FoxM1 expression. Targeting RGC-32 can be an effective target in restoring sensitivity to Tamoxifen in breast cancer cells. We would like to express our sincere thanks to the reviewers for the constructive and positive comments. Critique The introduction is very short. Adding more details on the role of RGC-32 in cancers will be helpful for the reader. Answer: We expand the introduction (page 40, paragraph 2) to include the more details on the role of RGC-32 in cancers. The Discussion section will benefit from more details on how RGC_32 activate cell cycle and induce cell proliferation, especially concerning phosphorylation sites induced activated by CDK1, Akt and ERK1. Answer: Several sentences have been added in the Discussion (page 45, paragraph 4) in the revised version to address this issue. Discussing the role of ERK1 in the context of cell proliferation will be also helpful. Answer: we added the role of ERK1 in cell proliferation in the Discussion (page 45, paragraph 4) in the revised version. More details on the role of Fox M1 role in pathology and cancers will also be helpful. Answer: Several sentences have been added in the Discussion (page 46, paragraph 2) in the revised version to address this issue. Reviewer #2: Strengths of the study: This is a very thorough translational and experimental research study that eloquently documents RGC-32 expression in ER+ breast cancer tissues, while also deciphering the mechanisms through which RGC-32 mediates tamoxifen resistance and promotes breast cancer cells cell proliferation. The methods used in this study are well documented and valid: immunohistochemistry, RNA interference, microarray gene analysis and the use of tumor xenograft model. The authors demonstrate here that RGC-32 can activate PI3K signaling pathway, consequently enhancing ERα activity. ERα activation is essential for RGC-32-mediated the expression of FoxM1, while Tamoxifen resistance mediated by RGC-32 seems partially dependent on FoxM1 expression. We believe that the data presented here, an elucidation of the molecular mechanisms of acquired drug resistance in breast cancer, represents a real progress in the field of translational oncology. We would like to express our sincere thanks to the reviewers for the constructive and positive comments. Minor issues (limitations): 1. Abstract, Page 10: the acronym ERα is not detailed.1. Answer: Correction has been made in the revised version. 2. Neither in the DISCUSSION section, nor in the Introduction section you do not mention the connection with the complement system, namely with C5b-9, knowing that RGC-32 is a protein that promotes cell cycle progression in response to complement activation (Badea et al, J Biol Chem 2002). Please detail this connection and integrate it in harmony with the data presented here (your results). Answer: Several sentences have been added in the Introduction (page 40, paragraph 2) in the revised version to address this issue. 3. In the Discussion section, when discussing the role played by RGC-32 in the proliferation of ER+ breast cancer cells, other immunohistochemistry data on RGC-32’s expression in cancer tissue ought to be mentioned: we have now shown that RGC-32 is expressed in precancerous states, and its expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages of colon cancer than in precancerous states or the initial stages of colon cancer. (S.I. Vlaicu et al. Experimental and Molecular Pathology 88 (2010) 67–76.) Answer: Several sentences have been added in the Discussion (page 46, paragraph 3) in the revised version to address this issue. 4. The gene array data you performed in MCF-7 cells is insufficiently exploited in my view: what are other relevant pathways influenced by RGC-32 silencing, apart cell cycle control of chromosomal replication? And why is there no reference of microarray data that precedes your work: a gene array analysis to investigate the effect of RGC-32 knockdown on gene expression in the SW480 colon cancer cell line S.I. Vlaicu et al. Experimental and Molecular Pathology 88 (2010) 67–76.). A comparison the different RGC-32 dependent pathways found in these two studies should be present in your Discussion section. Answer: Several sentences have been added in the Discussion (page 46, paragraph 3) in the revised version to address this issue. 5. You mention that “These results suggested a novel regulatory relationship between RGC-32 and PI3K/AKT-ERα crosstalk.” I would disagree that this relationship is a NEW finding, and here are the grounds for my disapproval: This relationship was previously documented in endothelial cells by Fosbrink et al. (Fosbrink et al., Exp Mol Pahol Vol 86, 2009) and in T-lymphocytes by Tegla et al. (Tegla CA, Cudrici CD, Nguyen V, Danoff J, Kruszewski AM, Boodhoo D, Mekala AP, Vlaicu SI, Chen C, Rus V, Badea TC, Rus H. RGC-32 is a novel regulator of the T-lymphocyte cell cycle. Exp Mol Pathol. 2015 Jun;98(3):328-37.) The aforementioned research showed that RGC-32 was involved in controlling the cell cycle of T cells in vivo; AKT connects PI3K to signaling pathways that promote cytokine transcription, survival, and cell-cycle activation. RGC-32 acts on Akt which phosphorylates FOXO1 protein, induces IL-2 transcription and cell cycle activation contributing to TCR mediated signaling. I would see it as optimal and necessary to integrate these previous findings into the Discussion chapter. Answer: we have integrated these previous findings into the Discussion chapter (page 46, paragraph 4). Additional Editor Comments: The reviewers made suggestions for improving your manuscript, in particular related to the placing in context of your findings, i.e. introduction and discussion, and data processing of microarray data. This last point is particularly important, as availability of microarray data published in this study is mandatory before considering publication. Please deposit the microarray data in a public repository and describe the format, accession number and release date of the data in a separate paragraph in the data availability section. Also please expand on the performed DEG analysis in both methods and results section. Answer: The microarray data have been deposited in the ArrayExpress database (accession number E-MTAB-15130) on May 6, 2025. Detailed DEG analysis was provided in methods section. Attachments Attachment Submitted filename: response_to_reviewers(1)_auresp_2.docx https://doi.org/10.1371/journal.pone.0328698.r004
Decision Letter - Tudor C. Badea, Editor Dear Dr. Zhao, 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 Jul 19 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 . 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. 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Tudor C. Badea, M.D., M.A., Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] [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.0328698.r005
Revision 3
15 Jun 2025 Author Response Reviewer #1: This is an interesting manuscript investigating the role of RGC-32 in cell proliferation and tamoxifen resistance in breast cancer. The authors show that the RGC-32 mediated tamoxifen resistance is partially dependent on FoxM1 expression. Targeting RGC-32 can be an effective target in restoring sensitivity to Tamoxifen in breast cancer cells. We would like to express our sincere thanks to the reviewers for the constructive and positive comments. Critique The introduction is very short. Adding more details on the role of RGC-32 in cancers will be helpful for the reader. Answer: We expand the introduction (page 40, paragraph 2) to include the more details on the role of RGC-32 in cancers. The Discussion section will benefit from more details on how RGC_32 activate cell cycle and induce cell proliferation, especially concerning phosphorylation sites induced activated by CDK1, Akt and ERK1. Answer: Several sentences have been added in the Discussion (page 45, paragraph 4) in the revised version to address this issue. Discussing the role of ERK1 in the context of cell proliferation will be also helpful. Answer: we added the role of ERK1 in cell proliferation in the Discussion (page 45, paragraph 4) in the revised version. More details on the role of Fox M1 role in pathology and cancers will also be helpful. Answer: Several sentences have been added in the Discussion (page 46, paragraph 2) in the revised version to address this issue. Reviewer #2: Strengths of the study: This is a very thorough translational and experimental research study that eloquently documents RGC-32 expression in ER+ breast cancer tissues, while also deciphering the mechanisms through which RGC-32 mediates tamoxifen resistance and promotes breast cancer cells cell proliferation. The methods used in this study are well documented and valid: immunohistochemistry, RNA interference, microarray gene analysis and the use of tumor xenograft model. The authors demonstrate here that RGC-32 can activate PI3K signaling pathway, consequently enhancing ERα activity. ERα activation is essential for RGC-32-mediated the expression of FoxM1, while Tamoxifen resistance mediated by RGC-32 seems partially dependent on FoxM1 expression. We believe that the data presented here, an elucidation of the molecular mechanisms of acquired drug resistance in breast cancer, represents a real progress in the field of translational oncology. We would like to express our sincere thanks to the reviewers for the constructive and positive comments. Minor issues (limitations): 1. Abstract, Page 10: the acronym ERα is not detailed.1. Answer: Correction has been made in the revised version. 2. Neither in the DISCUSSION section, nor in the Introduction section you do not mention the connection with the complement system, namely with C5b-9, knowing that RGC-32 is a protein that promotes cell cycle progression in response to complement activation (Badea et al, J Biol Chem 2002). Please detail this connection and integrate it in harmony with the data presented here (your results). Answer: Several sentences have been added in the Introduction (page 40, paragraph 2) in the revised version to address this issue. 3. In the Discussion section, when discussing the role played by RGC-32 in the proliferation of ER+ breast cancer cells, other immunohistochemistry data on RGC-32’s expression in cancer tissue ought to be mentioned: we have now shown that RGC-32 is expressed in precancerous states, and its expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages of colon cancer than in precancerous states or the initial stages of colon cancer. (S.I. Vlaicu et al. Experimental and Molecular Pathology 88 (2010) 67–76.) Answer: Several sentences have been added in the Discussion (page 46, paragraph 3) in the revised version to address this issue. 4. The gene array data you performed in MCF-7 cells is insufficiently exploited in my view: what are other relevant pathways influenced by RGC-32 silencing, apart cell cycle control of chromosomal replication? And why is there no reference of microarray data that precedes your work: a gene array analysis to investigate the effect of RGC-32 knockdown on gene expression in the SW480 colon cancer cell line S.I. Vlaicu et al. Experimental and Molecular Pathology 88 (2010) 67–76.). A comparison the different RGC-32 dependent pathways found in these two studies should be present in your Discussion section. Answer: Several sentences have been added in the Discussion (page 46, paragraph 3) in the revised version to address this issue. 5. You mention that “These results suggested a novel regulatory relationship between RGC-32 and PI3K/AKT-ERα crosstalk.” I would disagree that this relationship is a NEW finding, and here are the grounds for my disapproval: This relationship was previously documented in endothelial cells by Fosbrink et al. (Fosbrink et al., Exp Mol Pahol Vol 86, 2009) and in T-lymphocytes by Tegla et al. (Tegla CA, Cudrici CD, Nguyen V, Danoff J, Kruszewski AM, Boodhoo D, Mekala AP, Vlaicu SI, Chen C, Rus V, Badea TC, Rus H. RGC-32 is a novel regulator of the T-lymphocyte cell cycle. Exp Mol Pathol. 2015 Jun;98(3):328-37.) The aforementioned research showed that RGC-32 was involved in controlling the cell cycle of T cells in vivo; AKT connects PI3K to signaling pathways that promote cytokine transcription, survival, and cell-cycle activation. RGC-32 acts on Akt which phosphorylates FOXO1 protein, induces IL-2 transcription and cell cycle activation contributing to TCR mediated signaling. I would see it as optimal and necessary to integrate these previous findings into the Discussion chapter. Answer: we have integrated these previous findings into the Discussion chapter (page 46, paragraph 4). Additional Editor Comments: The reviewers made suggestions for improving your manuscript, in particular related to the placing in context of your findings, i.e. introduction and discussion, and data processing of microarray data. This last point is particularly important, as availability of microarray data published in this study is mandatory before considering publication. Please deposit the microarray data in a public repository and describe the format, accession number and release date of the data in a separate paragraph in the data availability section. Also please expand on the performed DEG analysis in both methods and results section. Answer: The microarray data have been deposited in the ArrayExpress database (accession number E-MTAB-15130), made publicly available on June 6, 2025. Attachments Attachment Submitted filename: response to reviewers.docx https://doi.org/10.1371/journal.pone.0328698.r006
Decision Letter - Tudor C. Badea, Editor Response Gene to Complement 32 promotes cell proliferation and tamoxifen resistance in breast cancer via elevated FoxM1 expression PONE-D-25-01923R3 Dear Dr. Zhao, 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. If you have any questions relating to publication charges, 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, Tudor C. Badea, M.D., M.A., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0328698.r007
Formally Accepted
Acceptance Letter - Tudor C. Badea, Editor PONE-D-25-01923R3 PLOS ONE Dear Dr. Zhao, 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. 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. Tudor C. Badea Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0328698.r008

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