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

Response: We have edited our manuscript strictly in accordance with the format requirements to ensure it meets the requirements of your journal.

2. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse.

Response: We have uploaded all the original R codes involved in the bioinformatics research of this study to the online system for inspection (https://figshare.com/s/5678c40e643bf55f363e).

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.

Response: We have uploaded all the original Western blot bands to the online database (https://figshare.com/s/5678c40e643bf55f363e).

4.We note that you have indicated that there are restrictions to data sharing for this study. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

Before we proceed with your manuscript, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., a Research Ethics Committee or Institutional Review Board, etc.). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see

https://journals.plos.org/plosone/s/recommended-repositories. You also have the option of uploading the data as Supporting Information files, but we would recommend depositing data directly to a data repository if possible.

We will update your Data Availability statement on your behalf to reflect the information you provide.

In the online submission form, you indicated that the dataset associated with this study can be obtained from the lead researcher if a justified request is made.

All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, Within the manuscript itself, or 3. Uploaded as supplementary information.

This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval.

Response: We have updated the data availability statement and stored the original data of this study on the online website (https://figshare.com/s/5678c40e643bf55f363e).

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.

Response: We have added the "Supporting Information" at the end of the article.

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.

Response: We have read the literatures recommended by the reviewers, which are highly relevant to our research. Therefore, we have cited these literatures as required by the reviewers. Among them, one has been cited before, one is newly added, and for another one, due to incomplete information provided by the reviewers, we haven't been able to locate the corresponding literature.

[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?

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: Partly

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: COX7A1-mediated mitochondrial dysfunction can induce ferroptosis in the endometrial

cancer cells

The study investigates the role of COX7A1 in mitochondrial dysfunction and ferroptosis in endometrial cancer. Bioinformatics analysis identified differentially expressed genes related to ferroptosis, and in vitro experiments confirmed that COX7A1 overexpression inhibits cancer cell proliferation and induces ferroptosis cell death. COX7A1 increased intracellular Fe2+ and MDA levels, reduced the GSH/GSSG ratio, and altered mitochondrial membrane potential. The findings of this study imply the significance of the COX7A1 gene as a driver of mitochondrial dysfunction and cellular stress in endometrial cancer cells. Although this gene has been investigated in other cancers, studying its role in endometrial cancer is also important. However, I have the following comments and concerns regarding the manuscript.

1) The abstract, conclusion, and discussion primarily focus on overexpression data obtained from Ishikawa cells. There should be a discussion on knockdown in AN3 CA cells and why this marker was highly expressed in these cells.

Response: Thank you for your valuable comments. We agree with your perspective. We have added the relevant changes in proteins and ferroptosis after AN3 CA knockdown in the abstract, conclusion, and discussion sections.

Main revisions in the abstract:

In vitro experiments have demonstrated that overexpression of COX7A1 inhibits the proliferation of endometrial cancer cells and induces ferroptosis by regulating intracellular iron metabolism and mitochondrial function. The specific mechanisms include increasing intracellular Fe²⁺ and malondialdehyde (MDA) levels, decreasing the GSH/GSSG ratio, and disrupting mitochondrial membrane potential, thereby leading to mitochondrial dysfunction. Furthermore, COX7A1 overexpression significantly reduces the expression of glutathione peroxidase 4 (GPX4) and SLC7A11, while upregulating acyl-coenzyme A synthetase long-chain family member 4 (ACSL4). In contrast, knockdown of COX7A1 promotes the proliferation of endometrial cancer cells and inhibits ferroptosis, exhibiting the opposite effects. These findings provide new insights into the molecular mechanisms of endometrial cancer�Lines 39 - 49�.

Main revisions in the discussion:

Notably, overexpression of COX7A1 significantly reduced the expression levels of GPX4 and SLC7A11 while increasing the expression of ACSL4. Conversely, knockdown of COX7A1 resulted in upregulation of GPX4 and SLC7A11 and decreased ACSL4 levels. These observations are consistent with the classic ferroptosis mechanism, which induces cell death through lipid peroxidation and disruption of the antioxidant system �Lines 434-439�.

Main revisions in the conclusion:

In contrast, low expression of COX7A1 exhibits the opposite effect, promoting tumor cell proliferation. These findings not only reveal a novel mechanism by which COX7A1 inhibits the progression of endometrial cancer through the ferroptosis pathway but also suggest its potential as a therapeutic target for ferroptosis-based therapies�Lines 481-486�.

We observed that almost all endometrial cancer cell lines expressed COX7A1, except for Ishikawa cells, suggesting a potential regulatory role of this gene in these cancers, which warrants further discussion. The results based on Ishikawa cells cannot be generalized to other pancreatic cancer cells unless data are available showing the same result of the overexpression in the cells, which already expresses COX7A1.

Response: We completely agree with your opinion. To elaborate our viewpoints more clearly and respond to your questions, we are providing more detailed background and explanations here, hoping to dispel your concerns:

One of the core and consistent findings in our study is that the basal expression of COX7A1 can be detected in the vast majority of endometrial cancer cell lines we examined. Significantly, compared with normal cells (HEEpiC - SV40), the expression of COX7A1 is lower in endometrial cancer cell lines, and this difference is statistically significant (Figure 3A).

Our central hypothesis is that COX7A1 is a tumor suppressor with down - regulated expression in cancer, and the inactivation or reduced expression of its function is conducive to tumor cells evading ferroptosis.

Therefore, in Ishikawa cells, we demonstrated that "introducing" COX7A1 can inhibit tumor growth.

Conversely, knocking down the basal - expressed COX7A1 in these cells promotes tumor proliferation (as described in the original text).

These two sets of experimental evidence, one positive and one negative, form a complete logical loop under multiple cell backgrounds, jointly supporting the conclusion that COX7A1 acts as a broad - spectrum regulatory factor. Its biological effects do not depend on whether a specific cell line expresses it "from scratch", but rather on its relative expression level and functional state.

2) The absence of COX7A1 in Ishikawa cells suggests that the absence of this gene contributes to the invasive nature of these cells. In contrast, why is the COX7A1 gene highly expressed in AN3 CA cells? Does this indicate that these cancer cells are less invasive and aggressive?

Response: Thank you for raising such profound questions. You've astutely observed a key phenomenon in our data: COX7A1 is absent in Ishikawa cells but highly expressed in AN3 CA cells, and you've logically questioned its impact on cell invasiveness. This is indeed a point worthy of in - depth exploration.

First of all, we fully agree with your observation. The differences in COX7A1 expression levels among different cell lines are likely to reflect the heterogeneity of endometrial cancer and its different molecular subtypes. Our core explanation for this phenomenon is that the absolute expression level of a single gene is not always in a simple linear negative correlation with the final cell phenotype (such as invasiveness).

Despite the differences in absolute levels, our research has revealed a more crucial common pattern:

Compared with normal tissues: As you know, we have clarified in the paper that in endometrial cancer tissue samples, the expression of COX7A1 is generally significantly lower than that in paired normal endometrial tissues. Even though the basal expression level of COX7A1 in AN3 CA cells is relatively high, our functional experimental data show that further knocking down COX7A1 in these cells can still significantly promote cell proliferation (Figure 4A). Conversely, overexpressing COX7A1 in Ishikawa cells can also lead to a further inhibition of cell proliferation.

However, the main focus of this study was on discussing the effects of COX7A1 on the proliferation, ferroptosis, and mitochondrial dysfunction of endometrial cancer. We acknowledge that the basal expression of COX7A1 may have varying degrees of impact on the invasion and migration of different endometrial cancer cells, but this still requires further investigation in our subsequent experiments. Once again, thank you for your valuable comments, which have given us a deeper understanding and insights into the research direction and cell line selection of this project.

3) Figure 6A: What is the quantified percent change in ROS? Please mention the percentage count of cells that expressed ROS.

Response: Thank you for raising this crucial question. We have re - composed the figures in terms of percentages as follows. We've noticed that in most papers, presenting the average fluorescence intensity of ROS and the positive ratio as output results are both quite common. If you need us to change it to the ROS positive ratio, please let us know. We will be happy to make the modification.

4) Figure 3 B: Data after transfection, the relative change was measured. Please add transfection efficiency data in the supplemental file. Additionally, please include the protein level expression of COX7A1 after transf

Attachments

Attachment

Submitted filename: Response to reviewers.pdf