Identification of Lipid Droplet-Associated Genes in Breast Cancer Patients

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper by Dogan et al. aimed at identify lipid droplets-related DEPs in breast cancers to be used as prognostic marker for the early detection of the tumor growth. Thanks to a supervised and an unsupervised bioinformatic approach the authors identify some DEPs and linked this notion to the differences in lipid content from breast cancer cell lines. The latter were also co-cultured with primary human adipose tissue as source of fat to compare LDs vs not co-cultured breast cancer cell types.

The authors identified FABP7 as a good prognostic marker since i) it was found significantly downregulated in breast cancer samples compared to healthy ones, ii) it shows the lowest overall survival rate compare with the other analyzed genes, and iii) it is significantly changed between tumor stages.  The first two point as already published by Chang et al. in 2022 (Chang and Xing Lipids in Health and Disease (2022) 21:43, Identification of a novel lipid metabolism-related gene signature within the tumor immune microenvironment for breast cancer), with their Kaplan-Meyer curves almost perfectly superimposing (Fig. 3A of the Chang’s paper) with the one obtained from the present authors (Fig. 3C of the manuscript).

Regarding the in vitro part of the study, it is very poor and seems not perfectly connected with the in silico findings. The authors should perform a HPTCL analysis or a MS analysis on the cells lines after co-cultivation to check for any differences in lipids profile. In addition the authors should perform knock-down, knock-out of knock-in (or provide a recombinant protein) experiments with FABP7 or other targets resulted of interest from this study to check for changes in LD numerosity and size both in co-cultivation with primary adipose tissue or alone to provide a direct rationale and link for the identified target and LDs in breast cancer cell lines.

Minor comments:

1. in some figures Y-axis title is missing.

2. in dot-blot graphs is always reported BRCA on the X-axis.

Author Response

Dear Reviewer,

We have done all your comments points by points. Please check our letter.

Thank you so much for your precious time.

Best Regards

Dr. Senol Dogan

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The work is structured as predominantly bioinformatics research for the identification of specific prognostic markers in breast cancer associated with the formation and metabolism of LDs.

As said by the authors themselves, no gene is directly involved in the formation of lipid droplets but the genes that code for enzymes and proteins involved in the more general lipid metabolism and mechanism of formation of LDs were therefore analyzed. Some in vitro experiments are added as preliminary data.

The research topic is of interest given the need to identify prognostic biomarkers for use in the clinic. The paper is well structured and just minor revisions have to be performed:

The rationale for the lipid droplet co-culture and analysis experiment is unclear in this form. It is appropriate to integrate in the introduction or the result section information regarding the relevance of the tumor microenvironment (with specific reference to adipose tissue) in the context of breast cancer and its aggressiveness.

Some typos have to be checked and corrected.

Comments on the Quality of English Language

The quality of English is adequate

Author Response

Dear Reviewer,

We have done all your comments points by points. Please check our letter.

Thank you so much for your precious time.

Best Regards

Dr. Senol Dogan

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors Dogan et al., have submitted an original manuscript entitled "Identification of lipid droplet-associated genes in breast cancer patients". The manuscript presents a significant amount of work done in characterizing the details. However, there are a few points of concern:

In lines 47 - 48, the authors should describe the specific details provided on www.cancer.gov similar to the details they provide from WHO.

In section 3.2, the DEGs can be grouped and described in text. The authors could carry out Gene Ontology (GO) analysis to generate the groups in which these DEGs are present. This will make the analysis more compact and also demonstrate specific relationships.

All figures do not appear to be of the quality requested by the journal. The authors should consider revising them.

The manuscript lacks a discussion section. The authors are encouraged to make the manuscript per the guidelines.

Comments on the Quality of English Language

None

Author Response

Dear Reviewer,

We have done all your comments points by points. Please check our letter.

Thank you so much for your precious time.

Best Regards

Dr. Senol Dogan

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors Dogan et al., have submitted a revised manuscript entitled "Identification of lipid droplet-associated genes in breast cancer patients". While the authors have improved the manuscript from the previous version, significant areas of improvement remain. Here are the comments for improving the manuscript:

In lines 62-63, while the scope of the statement is accurate in the context of the study, a more helpful and engaging description would be to use studies like 10.1038/s41598-021-04176-w and 10.1186/s12014-022-09362-0 that have proposed a few candidates as protein biomarkers for breast cancer from serum and demonstrate why these candidates don't fit the criteria for early-stage breast cancer identification. 

The authors also need to comment on why it is important to search for new protein biomarkers for early-stage breast cancer detection given that there are studies proposing the use of cell-free DNA from the serum as early-stage breast cancer biomarkers (10.1186/s13058-016-0788-z).

In lines 64-67, the mention of the relevance of animal studies in cancer is unwarranted as most of the biomarker studies utilize human samples. The authors could remove these statements.

Lines 69-76 giving a general overview of lipids are irrelevant to the scope of the study. The authors could remove these lines and Figure 1.

In lines 80-83, the authors should mention "breast" cancer instead of just cancer to emphasize the relevance of these findings to their study and set a premise for their results.

The legend of Figure 1 is misleading. The title of the legend mentions that the figure describes important lipid classes in breast cancer research. In contrast, the following text mentions that these are the most abundant lipid classes in all mammals (including humans). The authors should maintain uniform relevance in the legend and the places where the Figure is cited in the text. If needed please revise the figure accordingly.

In lines 104-106, the cited article doesn't mention free fatty acids acting as detergents and destabilizing membrane structures. The authors should revise the cited article or their statement.

Lines 106-108 can be combined with lines 93-95 as the functions of LD are complementary in both statements.

In lines 108-110, the statement's initial part contradicts the latter part. The authors should revise the statement to relate to the point they are trying to make.

In lines 110-111, the authors mention that the scope of the study was to identify the expression of LD-associated proteins as prognostic factors by examining their expression during the metastatic transition. This scope contradicts the initial part of the introduction, where the authors discuss using different omic technologies to identify blood-based biomarkers for early breast cancer detection. The authors need to revise the introduction to fit the scope of their study.

In lines 112-114, the authors mention using the lipid composition of the cancer cells as prognostic markers. The authors should justify why they utilized whole-cell lipid composition for lipid analyses while focusing on LD for the protein analyses.

The references for the genes listed in Supplement 1 are not always the articles that demonstrate the association of the respective genes to LD. Including the articles that the authors used as a basis for including each gene in generating their set of genes would be more appropriate.

In lines 143-144, the words in apostrophes appear like phrases instead of keywords. The authors should mention the keyword(s) they used to generate their dataset.

In line 145, the authors should clarify whether they selected articles with all human cancers or human breast cancers.

In lines 147-149, it would be helpful to include the studies that describe the factors for their roles (as mentioned by the authors) in the supplementary for robustness of this study.

In sub-sections 3.1 -3.4, the authors need to discuss the significance of their results as there is no separate discussion section.

For sub-section 3.2, it would be helpful if the authors could include the results of their analysis on the GEPIA platform for the 143 genes as a supplementary table.

In lines 309-314, it would be helpful if the authors also included the common names of the genes apart from their HGNC-approved symbols. This will help the reader to relate to the results. Also, the authors could classify the genes based on criteria like most significantly altered vs less significant changes in expression and include these details as a separate figure.

In supplement 2, the authors should make it easy for readers to arrive at the same genes listed in sub-section 3.2. Also, the authors haven't discussed the details of the "Expression" column in materials and methods.

In Table 1, the authors have included genes with p-values = 0.05 meanwhile in the materials and methods, they mention using genes with p-values < 0.05 for their analysis. The authors should revise the methods or the table. It is also unclear how the authors arrived at the values of OS HR as this data is not available for the entire set of 143 DEGs.

 

 

Comments on the Quality of English Language

The article needs to be proofread to improve the overall quality of the language.

Author Response

Dear Reviewer,

Thank you very much for your careful and constructive criticism. We answered all your criticisms one by one. You will find the answers to all your questions in the attached letter. All changes were shown in the manual with a yellow background. Since I could only send one file from here, we showed in which lines the critical points were made. Thank you very much for improving the quality of the manual.

Best Regards

Dr. Senol Dogan

Author Response File: Author Response.pdf