(In)Distinctive Role of Long Non-Coding RNAs in Common and Rare Ovarian Cancers

Round 1

Reviewer 1 Report

The review manuscript titled “(In)distinctive Role of Long Non-Coding RNAs in Common and Rare Ovarian Cancers” by Dr. Sabol and colleagues provides a detailed overview on the role/currently known molecular mechanisms of long non-coding RNAs in the pathogenesis of both common and rare Ovarian Cancer subtypes. The current state of the art about the use of these RNA molecules as predictive biomarkers alongside a detailed description of the current strategies for targeting lncRNAs as a treatment for OCs is also provided.

The review is interesting, well written and easy to follow. Overall, this topic is quite important and interesting. This review can help to better understand the role of ncRNAs dysregulation in Ovarian Cancer. While I recommend the article for publication, I have some minor observations to be reviewed by authors before such publication:

Introduction
1.    “Ovarian malignancies mostly share the same problem, which is late diagnosis.” ---> this sentence, which underlies an important point for the purpose of the review, is lacking in supporting reference, for instance PMID: 20230172
2.    “diagnostic, prognostic and predictive biomarkers” diagnostic and predictive are quite redundant, as expressing the same notion
LncRNA Classification and Mechanisms of Action
1.    A figure describing the lncRNA biogenesis would be helpful for the reader. A very detailed description/representation of lncRNA biogenesis can be found here PMID: 26332907
2.    “Even though lncRNAs were originally defined as RNA which do not encode proteins, recent reports suggest that some lncRNAs contain open reading frames (ORF) and may encode peptides.” It has also been reported that lncRNAs are capable to encode microRNAs. For instance, lncRNA H19 generates miR-675 (PMID: 26417995). Please include this information/ref

The Role of lncRNAs in Various Biological Processes Related to Ovarian Carcino-genesis
1.    LncRNAs also play a pivotal role in osteogenic differentiation (PMID: 33898434). Please include this information/ref
2.    “LncRNAs can change this process either directly by attacking the binding sites [36] or interacting with associated enzymes [37].” Please describe which lncRNAs present these functions 
3.    “Dysregulated phosphorylation and proteins activated through..”

Dysregulated histone phosphorylation? Or proteins/enzymes in general?
H19
1.    “This lncRNA acts as a molecular scaffold, and binds the Poly-comb Repressive Complex 2 (PRC2) and the lysine-specific histone demethylase 1A (LSD1), bringing them in close proximity and enabling trimethylation of the H3K27 his-tone and consequent gene silencing” – The role of LSD1 in this process is unclear as this enzyme removes mono-/di-methylation marks on lysine 9 in histone H3. 

HOTAIR
1.    “HOTAIR expression is also associated with chemoresistance, and its knockdown can lead to increased sensitivity to cisplatin and carboplatin [63,72–74].” In vitro? In vivo? Both? 
2.    “The potential use of this lncRNA as a biomarker has led to development of various methods for detection of this lncRNA from samples derived from patients with OC, primarily plasma [75,76].” I would move this sentence at the end of the paragraph as the last sentence is describing an additional functional role for H19 as the previous ones in the paragraph

MALAT1
1.    “significantly overexpressed in various cancers,” Please include supporting references. For instance PMID: 31186750, PMID: 25481511

MEG3 
It is unclear why MEG3 is considered a tumor suppressor, as (i) it is downregulated in EOC and (ii) its overexpression is related with a better progression-free survival and overall survival in HGSOC, while, as described, it also regulates tumor suppressor genes such as TP86 and RB1. Is this regulation positive or negative?

NEAT1
“Based on all this, NEAT1 has been proposed as a considerable biomarker in…” better prognostic biomarker. In addition, studies underling its role as diagnostic biomarker should be, at least briefly, mentioned. As an example, PMID: 33917553 for prostate cancer

UCA1
“This is an important factor in OC metastasis [121].” Who? UCA1, miR-485-5p or MMP14?

Author Response

We would like to thank both editor and reviewers for putting an effort to read our manuscript very thoroughly and for providing valuable comments and suggestions. We sincerely believe that we have significantly improved this review paper. We have re-written, expanded, and added new parts of the manuscript, because of which reading it with turned on track changes would be impossible. Therefore, we have highlighted the new or changed text in yellow.

Here we are providing our answers and responses to the reviewers’ comments and suggestions:

 

Reviewer 1

 

The review manuscript titled “(In)distinctive Role of Long Non-Coding RNAs in Common and Rare Ovarian Cancers” by Dr. Sabol and colleagues provides a detailed overview on the role/currently known molecular mechanisms of long non-coding RNAs in the pathogenesis of both common and rare Ovarian Cancer subtypes. The current state of the art about the use of these RNA molecules as predictive biomarkers alongside a detailed description of the current strategies for targeting lncRNAs as a treatment for OCs is also provided.

 

The review is interesting, well written and easy to follow. Overall, this topic is quite important and interesting. This review can help to better understand the role of ncRNAs dysregulation in Ovarian Cancer. While I recommend the article for publication, I have some minor observations to be reviewed by authors before such publication:

Introduction

1. “Ovarian malignancies mostly share the same problem, which is late diagnosis.” ---> this sentence, which underlies an important point for the purpose of the review, is lacking in supporting reference, for instance PMID: 20230172

We added the reference you suggested.

2. “diagnostic, prognostic and predictive biomarkers” diagnostic and predictive are quite redundant, as expressing the same notion

We changed the phrase to “diagnostic and prognostic”, omitting predictive.

LncRNA Classification and Mechanisms of Action

1. A figure describing the lncRNA biogenesis would be helpful for the reader. A very detailed description/representation of lncRNA biogenesis can be found here PMID: 26332907

New Figure 1 has been made which better describe the lncRNA biogenesis. However, we did not go to much into the details but put reference you have mentioned.

2. “Even though lncRNAs were originally defined as RNA which do not encode proteins, recent reports suggest that some lncRNAs contain open reading frames (ORF) and may encode peptides.” It has also been reported that lncRNAs are capable to encode microRNAs. For instance, lncRNA H19 generates miR-675 (PMID: 26417995). Please include this information/ref

Thank you for the suggestion, this was added to the text.

 

The Role of lncRNAs in Various Biological Processes Related to Ovarian Carcino-genesis

1. LncRNAs also play a pivotal role in osteogenic differentiation (PMID: 33898434). Please include this information/ref

This information was elaborated in the new paragraph added to section 5 (former section 4).

2. “LncRNAs can change this process either directly by attacking the binding sites [36] or interacting with associated enzymes [37].” Please describe which lncRNAs present these functions

This sentence was re-written to be more accurate and to provide the examples of these lncRNA. It now states: LncRNAs can change this process either by interrupting the histone acetylation process, such as lncPRESS1 binding to SIRT6 and preventing its binding to histones [38], or by acting as scaffolds regulating histone acetylation and methylation, such as GClnc1 which acts as a scaffold for WDR5 and KAT2A proteins [39].

3. “Dysregulated phosphorylation and proteins activated through..”

Dysregulated histone phosphorylation? Or proteins/enzymes in general?

This refers to protein phosphorylation in general. The sentence was re-written to clarify this. It is well-known that phosphorylation of proteins is one of the most common mechanisms that regulates protein activity, and phosphorylation is often dysregulated in cancer.

 

H19

1. “This lncRNA acts as a molecular scaffold, and binds the Poly-comb Repressive Complex 2 (PRC2) and the lysine-specific histone demethylase 1A (LSD1), bringing them in close proximity and enabling trimethylation of the H3K27 his-tone and consequent gene silencing” – The role of LSD1 in this process is unclear as this enzyme removes mono-/di-methylation marks on lysine 9 in histone H3.

We have changed the sentence for a more detailed description and added an additional reference for this process (Tsai et al 2010, Science). This lncRNA acts as a molecular scaffold, and binds the Polycomb Repressive Complex 2 (PRC2) on its 5’ domain and the lysine-specific histone demethylase 1A (LSD1)/CoREST/REST complex on its 3’ domain, bringing them in close proximity and methylating lysine 27 and demethylating lysine 4 of histone H3, leading to consequent gene silencing.

 

HOTAIR

1. “HOTAIR expression is also associated with chemoresistance, and its knockdown can lead to increased sensitivity to cisplatin and carboplatin [63,72–74].” In vitro? In vivo? Both?

The references provided here cover in vitro experiments on cell lines, tumor xenograft models in nude mice and clinical samples from carboplatin-treated patients compared to non-treated patients. It now reads: HOTAIR expression is also associated with chemoresistance in clinical samples and in vitro models, and its knockdown can lead to increased sensitivity to cisplatin and car-boplatin both in vitro and on a mouse xenograft model.

 

2. “The potential use of this lncRNA as a biomarker has led to development of various methods for detection of this lncRNA from samples derived from patients with OC, primarily plasma [75,76].” I would move this sentence at the end of the paragraph as the last sentence is describing an additional functional role for H19 as the previous ones in the paragraph

Thank you for the suggestion, it makes more sense putting it at the end.

 

MALAT1

1. “significantly overexpressed in various cancers,” Please include supporting references. For instance PMID: 31186750, PMID: 25481511

We have added the suggested references.

 

MEG3

It is unclear why MEG3 is considered a tumor suppressor, as (i) it is downregulated in EOC and (ii) its overexpression is related with a better progression-free survival and overall survival in HGSOC, while, as described, it also regulates tumor suppressor genes such as TP86 and RB1. Is this regulation positive or negative?

Thank you for noticing this discrepancy, it is now corrected. The regulation of TP53 is positive, resulting in TP53 upregulation and increased stability. The paper describing better survival of MEG3-high expressing patients also demonstrated on cell lines and xenograft models that MEG3 upregulation leads to inhibition of tumor growth. This may be due to the fact that the HGSOC patients already underwent platinum therapy before sampling, but as the authors do not elaborate on this fact we decided only to present the facts as they were shown in the paper.

 

NEAT1

“Based on all this, NEAT1 has been proposed as a considerable biomarker in…” better prognostic biomarker. In addition, studies underling its role as diagnostic biomarker should be, at least briefly, mentioned. As an example, PMID: 33917553 for prostate cancer

Thank you for the suggestion. We added your suggested reference, along with two more, one referring to breast cancer and the other to EOC serum biomarker panel for early detection of EOC.

 

UCA1

“This is an important factor in OC metastasis [121].” Who? UCA1, miR-485-5p or MMP14?

The statement refers to MMP14, and to strengthen the point we have added an additional reference showing the role of MMP14 in metastasis of OC.

 

Reviewer 2

This manuscript by Sabol et al. reviews the current state of the role of long non-coding RNAs (lncRNAs) in ovarian cancer (OC) with a special focus on the rare ovarian cancer (ROC) types. They describe the regulatory mechanisms related to lncRNAs function and general roles of lncRNAs in cancer. Then, an extensive survey of the specific roles of lncRNAs in OC and ROC is provided. Finally, they provide a general account of targeting lncRNAs using different strategies. The topic of the review is important as it tries to look into the similarities and differences in lncRNA expression and function between common OCs and ROCs. This review will encourage researchers in the field to look for specific lncRNA biomarkers for ROCs. That said, the review misses a mark in being more ROC-centric and also more OC-centric. There is a lot of general information about lncRNA mechanisms, role of lncRNAs in cancer, and lncRNA targeting that have been extensively reviewed elsewhere. The review could describe these topics more in relation to OC and ROC. Especially the differences and similarities between OC and ROC in relation to lncRNA function need to be more clearly described. Also, the review is missing important references related to the topics presented. The suggestions to improve the review are provided below.

Major changes:

1. A separate section describing the general differences between ROCs and common OCs would be great to have in the beginning and will set the stage for later sections. The authors can describe the differences in relation to gene expression patterns, pathways, mutations, structural variations, and epigenetics. The authors can add a table describing different types of ROCs and OCs (especially the subtypes and cell lines described in the review).and features relevant to these subtypes. That way it will be easier for the readers to find this information. Also, the authors can describe the problems related to studying ROCs like availability of pre-clinical models.

As suggested, we added a new section (now section 2) describing the genetic and clinical features of ROCs and common OCs. A summary table (now Table 1) was also added as suggested. We also added new subsection 2.1 in which we described problems with using available cell lines as models for different OCs. However, we did not go too much into details because we plan to conduct a comprehensive literature and database survey, as well as bioinformatic analyses on ovarian cancer cell lines and publish results in a separate paper.

1. In Section 4, the authors can describe the various functions of lncRNAs more in relation to OC, ROC, and other gynecological cancers.

As suggested, section 5 (former section 4) has been expanded with more cellular processes related to OCs in which lncRNAs have known roles. However, in general we did not want to involve other gynecological cancers besides OCs in this review.

1. In Section 5, the authors can clearly point out the similarities and differences between common OC and ROC in relation to all the lncRNAs described in this section. There can be more emphasis on that as that is the main topic of the review. It is not always immediately clear which studies are for common OCs and which ones are for ROCs. The authors can try to add all the original references instead of citing reviews in Section 5.

We believe that now, after adding new section 2 and new Table 1, as well as after providing more detailed explanation what Tables 2 and 3 present, readers, when they come to section 6 (former section 5), would comprehend which part of text (lncRNAs) are related to which type of OC.

1. In Section 5.1, the authors can check the sentence related to ectopic expression and silencing of H19 that comes from reference 47 to see if that is correct or not.

Thank you for noticing this, it was indeed an error. We corrected the sentence according to the data presented in the paper.

1. In Table 1, the authors can check if all the OC and ROC types described in Section 5 are included or not. It can be made clearer which OCs are common and which ones are rare types in Table 1.

Not all references are included in the tables, but only those with the described miRNA interactors and mechanisms of action. Many studies only reported the effect of overexpression or silencing of a certain lncRNA without going into mechanisms. If the table was expanded to include those papers, then all the data in the text and tables would be overlapping and redundant. By limiting our tables to only those papers with known miRNA targets we focus the attention only on the work that demonstrates actual lncRNA-miRNA interactions and their consequences in OCs. Here is an example how the table rows would look like if all the references were added, and you can see many of the fields would be “undefined”.

	undefined	HGSOC	undefined	Increases cisplatin resistance	Oncogene	[53]
	undefined	EC	undefined	Increases cisplatin resistance through downregulation of EZH2	Oncogene	[54]
	undefined	EC	undefined	Increases cisplatin resistance through regulation of glutathione metabolism	Oncogene	[55]

We added the abbreviations of rare and common OC types in the headings of Tables 1 and 2 (now Table 2 and 3) so it can be easily distinguishable which are common, and which are rare subtypes.

1. Section 7 is quite general and the authors should add more examples of lncRNA targeting in OC or other gynecological cancers.

Thank you for your observation and suggestion, we expanded section 7 (now section 8) with examples of targeting lncRNAs related to OC. However, in general we did not want to involve other gynecological cancers besides OCs in this review.

1. The authors can cite all the articles mentioned in the reference list below and look at instructions provided for some of the references.

Minor changes:

1. In the Introduction section, information about 5-year survival rates or other clinical outcome metrics can be added.

Thank you for suggestion, this information about 5-year overall survival rates has been provided in new section 2.

1. In the second paragraph of the Introduction section, the authors cite only one review (reference 7) in relation to lncRNAs and OC. There are other recent reviews published on this topic and the authors should cite them as well.

As suggested, much more recent references on lncRNAs in OCs have been cited.

1. For Section 2, the authors should cite more recent reviews on lncRNAs in addition to references 10 and 11. Also, the authors should check if all lncRNAs are polyadenylated or not and modify the sentence accordingly. They can check this recent review on lncRNA and cite this and other recent reviews and update the section 2: Statello L, Guo CJ, Chen LL, Huarte M. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol. 2021.

We have added the suggested information about the percentage of polyadenylated lncRNA and cited the suggested reference, as well as added more recent references.

1. The authors should try to cite the original references wherever possible and not just cite the review article.

We have implemented this suggestion.

References that need to be included:

1. Tripathi MK et al., Role of lncRNAs in ovarian cancer: defining new biomarkers for therapeutic purposes. Drug Discov Today. 2018 Sep;23(9):1635-1643. PMID: 29698834.

Reference has been included in section 10.

1. Arun G, Diermeier SD, Spector DL. Therapeutic Targeting of Long Non-Coding RNAs in Cancer. Trends Mol Med. 2018 Mar;24(3):257-277. PMID: 29449148.

Reference and information from it have been added to section 8.

1. Schmitt AM, Chang HY. Long Noncoding RNAs in Cancer Pathways. Cancer Cell. 2016 Apr 11;29(4):452-463. PMID: 27070700.

Reference and information from it have been added to section 5.

1. Liu SJ et al., Long noncoding RNAs in cancer metastasis. Nat Rev Cancer. 2021 Jul;21(7):446-460. PMID: 33953369.

Reference and information from it have been added to section 5.

1. Carlevaro-Fita J et al., Cancer LncRNA Census reveals evidence for deep functional conservation of long noncoding RNAs in tumorigenesis. Commun Biol. 2020 Feb 5;3(1):56. PMID: 32024996. (This paper needs to be discussed in relation to Ovarian cancer.)

Reference and information from it have been added to section 5.

1. Papp E et al. Integrated Genomic, Epigenomic, and Expression Analyses of Ovarian Cancer Cell Lines. Cell Rep. 2018 Nov 27;25(9):2617-2633. PMID: 30485824. (The authors can check what information is available for common and rare OC cell lines in this paper and summarize that in the review.)

Reference and information from it have been included in the new section 2.

1. Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature. 2011 Jun 29;474(7353):609-15. PMID: 21720365

This reference has been added to the new section 2. However, this paper contains only the data on HGSOC available from the TCGA, and no data on ROC.

1. Jiang MC et al., Emerging roles of lncRNA in cancer and therapeutic opportunities. Am J Cancer Res. 2019 Jul 1;9(7):1354-1366. PMID: 31392074.

Reference and information from it have been added to section 8.

1. Yan X et al., Comprehensive Genomic Characterization of Long Non-coding RNAs across Human Cancers. Cancer Cell. 2015 Oct 12;28(4):529-540. PMID: 26461095. (The authors should briefly describe the lncRNAs related to OC from this paper in the text and in the table if not already included.)

This reference has been added to the new section 2. However, this paper contains only the data on HGSOC available from the TCGA, and no data on ROC.

1. Akrami R et al., Comprehensive analysis of long non-coding RNAs in ovarian cancer reveals global patterns and targeted DNA amplification. PLoS One. 2013 Nov 12;8(11):e80306. PMID: 24265805.

This reference has been added to section 6 but we had to remove one of the cited references describing the role of MALAT1 in epithelial OC as the paper has been retracted.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript by Sabol et al. reviews the current state of the role of long non-coding RNAs (lncRNAs) in ovarian cancer (OC) with a special focus on the rare ovarian cancer (ROC) types. They describe the regulatory mechanisms related to lncRNAs function and general roles of lncRNAs in cancer. Then, an extensive survey of the specific roles of lncRNAs in OC and ROC is provided. Finally, they provide a general account of targeting lncRNAs using different strategies. The topic of the review is important as it tries to look into the similarities and differences in lncRNA expression and function between common OCs and ROCs. This review will encourage researchers in the field to look for specific lncRNA biomarkers for ROCs. That said, the review misses a mark in being more ROC-centric and also more OC-centric. There is a lot of general information about lncRNA mechanisms, role of lncRNAs in cancer, and lncRNA targeting that have been extensively reviewed elsewhere. The review could describe these topics more in relation to OC and ROC. Especially the differences and similarities between OC and ROC in relation to lncRNA function need to be more clearly described. Also, the review is missing important references related to the topics presented. The suggestions to improve the review are provided below.

Major changes:

1. A separate section describing the general differences between ROCs and common OCs would be great to have in the beginning and will set the stage for later sections. The authors can describe the differences in relation to gene expression patterns, pathways, mutations, structural variations, and epigenetics. The authors can add a table describing different types of ROCs and OCs (especially the subtypes and cell lines described in the review).and features relevant to these subtypes. That way it will be easier for the readers to find this information. Also, the authors can describe the problems related to studying ROCs like availability of pre-clinical models.
2. In Section 4, the authors can describe the various functions of lncRNAs more in relation to OC, ROC, and other gynecological cancers.
3. In Section 5, the authors can clearly point out the similarities and differences between common OC and ROC in relation to all the lncRNAs described in this section. There can be more emphasis on that as that is the main topic of the review. It is not always immediately clear which studies are for common OCs and which ones are for ROCs. The authors can try to add all the original references instead of citing reviews in Section 5.
4. In Section 5.1, the authors can check the sentence related to ectopic expression and silencing of H19 that comes from reference 47 to see if that is correct or not.
5. In Table 1, the authors can check if all the OC and ROC types described in Section 5 are included or not. It can be made clearer which OCs are common and which ones are rare types in Table 1.
6. Section 7 is quite general and the authors should add more examples of lncRNA targeting in OC or other gynecological cancers.
7. The authors can cite all the articles mentioned in the reference list below and look at instructions provided for some of the references.

Minor changes:

1. In the Introduction section, information about 5-year survival rates or other clinical outcome metrics can be added.
2. In the second paragraph of the Introduction section, the authors cite only one review (reference 7) in relation to lncRNAs and OC. There are other recent reviews published on this topic and the authors should cite them as well.
3. For Section 2, the authors should cite more recent reviews on lncRNAs in addition to references 10 and 11. Also, the authors should check if all lncRNAs are polyadenylated or not and modify the sentence accordingly. They can check this recent review on lncRNA and cite this and other recent reviews and update the section 2: Statello L, Guo CJ, Chen LL, Huarte M. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol. 2021.
4. The authors should try to cite the original references wherever possible and not just cite the review article.

References that need to be included:

1. Tripathi MK et al., Role of lncRNAs in ovarian cancer: defining new biomarkers for therapeutic purposes. Drug Discov Today. 2018 Sep;23(9):1635-1643. PMID: 29698834.
2. Arun G, Diermeier SD, Spector DL. Therapeutic Targeting of Long Non-Coding RNAs in Cancer. Trends Mol Med. 2018 Mar;24(3):257-277. PMID: 29449148.
3. Schmitt AM, Chang HY. Long Noncoding RNAs in Cancer Pathways. Cancer Cell. 2016 Apr 11;29(4):452-463. PMID: 27070700.
4. Liu SJ et al., Long noncoding RNAs in cancer metastasis. Nat Rev Cancer. 2021 Jul;21(7):446-460. PMID: 33953369.
5. Carlevaro-Fita J et al., Cancer LncRNA Census reveals evidence for deep functional conservation of long noncoding RNAs in tumorigenesis. Commun Biol. 2020 Feb 5;3(1):56. PMID: 32024996. (This paper needs to be discussed in relation to Ovarian cancer.)
6. Papp E et al. Integrated Genomic, Epigenomic, and Expression Analyses of Ovarian Cancer Cell Lines. Cell Rep. 2018 Nov 27;25(9):2617-2633. PMID: 30485824. (The authors can check what information is available for common and rare OC cell lines in this paper and summarize that in the review.)
7. Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature. 2011 Jun 29;474(7353):609-15. PMID: 21720365
8. Jiang MC et al., Emerging roles of lncRNA in cancer and therapeutic opportunities. Am J Cancer Res. 2019 Jul 1;9(7):1354-1366. PMID: 31392074.
9. Yan X et al., Comprehensive Genomic Characterization of Long Non-coding RNAs across Human Cancers. Cancer Cell. 2015 Oct 12;28(4):529-540. PMID: 26461095. (The authors should briefly describe the lncRNAs related to OC from this paper in the text and in the table if not already included.)
10. Akrami R et al., Comprehensive analysis of long non-coding RNAs in ovarian cancer reveals global patterns and targeted DNA amplification. PLoS One. 2013 Nov 12;8(11):e80306. PMID: 24265805.

Author Response

We would like to thank both editor and reviewers for putting an effort to read our manuscript very thoroughly and for providing valuable comments and suggestions. We sincerely believe that we have significantly improved this review paper. We have re-written, expanded, and added new parts of the manuscript, because of which reading it with turned on track changes would be impossible. Therefore, we have highlighted the new or changed text in yellow.

Here we are providing our answers and responses to the reviewers’ comments and suggestions:

 

Reviewer 1

 

The review manuscript titled “(In)distinctive Role of Long Non-Coding RNAs in Common and Rare Ovarian Cancers” by Dr. Sabol and colleagues provides a detailed overview on the role/currently known molecular mechanisms of long non-coding RNAs in the pathogenesis of both common and rare Ovarian Cancer subtypes. The current state of the art about the use of these RNA molecules as predictive biomarkers alongside a detailed description of the current strategies for targeting lncRNAs as a treatment for OCs is also provided.

 

The review is interesting, well written and easy to follow. Overall, this topic is quite important and interesting. This review can help to better understand the role of ncRNAs dysregulation in Ovarian Cancer. While I recommend the article for publication, I have some minor observations to be reviewed by authors before such publication:

Introduction

1. “Ovarian malignancies mostly share the same problem, which is late diagnosis.” ---> this sentence, which underlies an important point for the purpose of the review, is lacking in supporting reference, for instance PMID: 20230172

We added the reference you suggested.

2. “diagnostic, prognostic and predictive biomarkers” diagnostic and predictive are quite redundant, as expressing the same notion

We changed the phrase to “diagnostic and prognostic”, omitting predictive.

LncRNA Classification and Mechanisms of Action

1. A figure describing the lncRNA biogenesis would be helpful for the reader. A very detailed description/representation of lncRNA biogenesis can be found here PMID: 26332907

New Figure 1 has been made which better describe the lncRNA biogenesis. However, we did not go to much into the details but put reference you have mentioned.

2. “Even though lncRNAs were originally defined as RNA which do not encode proteins, recent reports suggest that some lncRNAs contain open reading frames (ORF) and may encode peptides.” It has also been reported that lncRNAs are capable to encode microRNAs. For instance, lncRNA H19 generates miR-675 (PMID: 26417995). Please include this information/ref

Thank you for the suggestion, this was added to the text.

 

The Role of lncRNAs in Various Biological Processes Related to Ovarian Carcino-genesis

1. LncRNAs also play a pivotal role in osteogenic differentiation (PMID: 33898434). Please include this information/ref

This information was elaborated in the new paragraph added to section 5 (former section 4).

2. “LncRNAs can change this process either directly by attacking the binding sites [36] or interacting with associated enzymes [37].” Please describe which lncRNAs present these functions

This sentence was re-written to be more accurate and to provide the examples of these lncRNA. It now states: LncRNAs can change this process either by interrupting the histone acetylation process, such as lncPRESS1 binding to SIRT6 and preventing its binding to histones [38], or by acting as scaffolds regulating histone acetylation and methylation, such as GClnc1 which acts as a scaffold for WDR5 and KAT2A proteins [39].

3. “Dysregulated phosphorylation and proteins activated through..”

Dysregulated histone phosphorylation? Or proteins/enzymes in general?

This refers to protein phosphorylation in general. The sentence was re-written to clarify this. It is well-known that phosphorylation of proteins is one of the most common mechanisms that regulates protein activity, and phosphorylation is often dysregulated in cancer.

 

H19

1. “This lncRNA acts as a molecular scaffold, and binds the Poly-comb Repressive Complex 2 (PRC2) and the lysine-specific histone demethylase 1A (LSD1), bringing them in close proximity and enabling trimethylation of the H3K27 his-tone and consequent gene silencing” – The role of LSD1 in this process is unclear as this enzyme removes mono-/di-methylation marks on lysine 9 in histone H3.

We have changed the sentence for a more detailed description and added an additional reference for this process (Tsai et al 2010, Science). This lncRNA acts as a molecular scaffold, and binds the Polycomb Repressive Complex 2 (PRC2) on its 5’ domain and the lysine-specific histone demethylase 1A (LSD1)/CoREST/REST complex on its 3’ domain, bringing them in close proximity and methylating lysine 27 and demethylating lysine 4 of histone H3, leading to consequent gene silencing.

 

HOTAIR

1. “HOTAIR expression is also associated with chemoresistance, and its knockdown can lead to increased sensitivity to cisplatin and carboplatin [63,72–74].” In vitro? In vivo? Both?

The references provided here cover in vitro experiments on cell lines, tumor xenograft models in nude mice and clinical samples from carboplatin-treated patients compared to non-treated patients. It now reads: HOTAIR expression is also associated with chemoresistance in clinical samples and in vitro models, and its knockdown can lead to increased sensitivity to cisplatin and car-boplatin both in vitro and on a mouse xenograft model.

 

2. “The potential use of this lncRNA as a biomarker has led to development of various methods for detection of this lncRNA from samples derived from patients with OC, primarily plasma [75,76].” I would move this sentence at the end of the paragraph as the last sentence is describing an additional functional role for H19 as the previous ones in the paragraph

Thank you for the suggestion, it makes more sense putting it at the end.

 

MALAT1

1. “significantly overexpressed in various cancers,” Please include supporting references. For instance PMID: 31186750, PMID: 25481511

We have added the suggested references.

 

MEG3

It is unclear why MEG3 is considered a tumor suppressor, as (i) it is downregulated in EOC and (ii) its overexpression is related with a better progression-free survival and overall survival in HGSOC, while, as described, it also regulates tumor suppressor genes such as TP86 and RB1. Is this regulation positive or negative?

Thank you for noticing this discrepancy, it is now corrected. The regulation of TP53 is positive, resulting in TP53 upregulation and increased stability. The paper describing better survival of MEG3-high expressing patients also demonstrated on cell lines and xenograft models that MEG3 upregulation leads to inhibition of tumor growth. This may be due to the fact that the HGSOC patients already underwent platinum therapy before sampling, but as the authors do not elaborate on this fact we decided only to present the facts as they were shown in the paper.

 

NEAT1

“Based on all this, NEAT1 has been proposed as a considerable biomarker in…” better prognostic biomarker. In addition, studies underling its role as diagnostic biomarker should be, at least briefly, mentioned. As an example, PMID: 33917553 for prostate cancer

Thank you for the suggestion. We added your suggested reference, along with two more, one referring to breast cancer and the other to EOC serum biomarker panel for early detection of EOC.

 

UCA1

“This is an important factor in OC metastasis [121].” Who? UCA1, miR-485-5p or MMP14?

The statement refers to MMP14, and to strengthen the point we have added an additional reference showing the role of MMP14 in metastasis of OC.

 

Reviewer 2

This manuscript by Sabol et al. reviews the current state of the role of long non-coding RNAs (lncRNAs) in ovarian cancer (OC) with a special focus on the rare ovarian cancer (ROC) types. They describe the regulatory mechanisms related to lncRNAs function and general roles of lncRNAs in cancer. Then, an extensive survey of the specific roles of lncRNAs in OC and ROC is provided. Finally, they provide a general account of targeting lncRNAs using different strategies. The topic of the review is important as it tries to look into the similarities and differences in lncRNA expression and function between common OCs and ROCs. This review will encourage researchers in the field to look for specific lncRNA biomarkers for ROCs. That said, the review misses a mark in being more ROC-centric and also more OC-centric. There is a lot of general information about lncRNA mechanisms, role of lncRNAs in cancer, and lncRNA targeting that have been extensively reviewed elsewhere. The review could describe these topics more in relation to OC and ROC. Especially the differences and similarities between OC and ROC in relation to lncRNA function need to be more clearly described. Also, the review is missing important references related to the topics presented. The suggestions to improve the review are provided below.

Major changes:

1. A separate section describing the general differences between ROCs and common OCs would be great to have in the beginning and will set the stage for later sections. The authors can describe the differences in relation to gene expression patterns, pathways, mutations, structural variations, and epigenetics. The authors can add a table describing different types of ROCs and OCs (especially the subtypes and cell lines described in the review).and features relevant to these subtypes. That way it will be easier for the readers to find this information. Also, the authors can describe the problems related to studying ROCs like availability of pre-clinical models.

As suggested, we added a new section (now section 2) describing the genetic and clinical features of ROCs and common OCs. A summary table (now Table 1) was also added as suggested. We also added new subsection 2.1 in which we described problems with using available cell lines as models for different OCs. However, we did not go too much into details because we plan to conduct a comprehensive literature and database survey, as well as bioinformatic analyses on ovarian cancer cell lines and publish results in a separate paper.

1. In Section 4, the authors can describe the various functions of lncRNAs more in relation to OC, ROC, and other gynecological cancers.

As suggested, section 5 (former section 4) has been expanded with more cellular processes related to OCs in which lncRNAs have known roles. However, in general we did not want to involve other gynecological cancers besides OCs in this review.

1. In Section 5, the authors can clearly point out the similarities and differences between common OC and ROC in relation to all the lncRNAs described in this section. There can be more emphasis on that as that is the main topic of the review. It is not always immediately clear which studies are for common OCs and which ones are for ROCs. The authors can try to add all the original references instead of citing reviews in Section 5.

We believe that now, after adding new section 2 and new Table 1, as well as after providing more detailed explanation what Tables 2 and 3 present, readers, when they come to section 6 (former section 5), would comprehend which part of text (lncRNAs) are related to which type of OC.

1. In Section 5.1, the authors can check the sentence related to ectopic expression and silencing of H19 that comes from reference 47 to see if that is correct or not.

Thank you for noticing this, it was indeed an error. We corrected the sentence according to the data presented in the paper.

1. In Table 1, the authors can check if all the OC and ROC types described in Section 5 are included or not. It can be made clearer which OCs are common and which ones are rare types in Table 1.

Not all references are included in the tables, but only those with the described miRNA interactors and mechanisms of action. Many studies only reported the effect of overexpression or silencing of a certain lncRNA without going into mechanisms. If the table was expanded to include those papers, then all the data in the text and tables would be overlapping and redundant. By limiting our tables to only those papers with known miRNA targets we focus the attention only on the work that demonstrates actual lncRNA-miRNA interactions and their consequences in OCs. Here is an example how the table rows would look like if all the references were added, and you can see many of the fields would be “undefined”.

	undefined	HGSOC	undefined	Increases cisplatin resistance	Oncogene	[53]
	undefined	EC	undefined	Increases cisplatin resistance through downregulation of EZH2	Oncogene	[54]
	undefined	EC	undefined	Increases cisplatin resistance through regulation of glutathione metabolism	Oncogene	[55]

We added the abbreviations of rare and common OC types in the headings of Tables 1 and 2 (now Table 2 and 3) so it can be easily distinguishable which are common, and which are rare subtypes.

1. Section 7 is quite general and the authors should add more examples of lncRNA targeting in OC or other gynecological cancers.

Thank you for your observation and suggestion, we expanded section 7 (now section 8) with examples of targeting lncRNAs related to OC. However, in general we did not want to involve other gynecological cancers besides OCs in this review.

1. The authors can cite all the articles mentioned in the reference list below and look at instructions provided for some of the references.

Minor changes:

1. In the Introduction section, information about 5-year survival rates or other clinical outcome metrics can be added.

Thank you for suggestion, this information about 5-year overall survival rates has been provided in new section 2.

1. In the second paragraph of the Introduction section, the authors cite only one review (reference 7) in relation to lncRNAs and OC. There are other recent reviews published on this topic and the authors should cite them as well.

As suggested, much more recent references on lncRNAs in OCs have been cited.

1. For Section 2, the authors should cite more recent reviews on lncRNAs in addition to references 10 and 11. Also, the authors should check if all lncRNAs are polyadenylated or not and modify the sentence accordingly. They can check this recent review on lncRNA and cite this and other recent reviews and update the section 2: Statello L, Guo CJ, Chen LL, Huarte M. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol. 2021.

We have added the suggested information about the percentage of polyadenylated lncRNA and cited the suggested reference, as well as added more recent references.

1. The authors should try to cite the original references wherever possible and not just cite the review article.

We have implemented this suggestion.

References that need to be included:

1. Tripathi MK et al., Role of lncRNAs in ovarian cancer: defining new biomarkers for therapeutic purposes. Drug Discov Today. 2018 Sep;23(9):1635-1643. PMID: 29698834.

Reference has been included in section 10.

1. Arun G, Diermeier SD, Spector DL. Therapeutic Targeting of Long Non-Coding RNAs in Cancer. Trends Mol Med. 2018 Mar;24(3):257-277. PMID: 29449148.

Reference and information from it have been added to section 8.

1. Schmitt AM, Chang HY. Long Noncoding RNAs in Cancer Pathways. Cancer Cell. 2016 Apr 11;29(4):452-463. PMID: 27070700.

Reference and information from it have been added to section 5.

1. Liu SJ et al., Long noncoding RNAs in cancer metastasis. Nat Rev Cancer. 2021 Jul;21(7):446-460. PMID: 33953369.

Reference and information from it have been added to section 5.

1. Carlevaro-Fita J et al., Cancer LncRNA Census reveals evidence for deep functional conservation of long noncoding RNAs in tumorigenesis. Commun Biol. 2020 Feb 5;3(1):56. PMID: 32024996. (This paper needs to be discussed in relation to Ovarian cancer.)

Reference and information from it have been added to section 5.

1. Papp E et al. Integrated Genomic, Epigenomic, and Expression Analyses of Ovarian Cancer Cell Lines. Cell Rep. 2018 Nov 27;25(9):2617-2633. PMID: 30485824. (The authors can check what information is available for common and rare OC cell lines in this paper and summarize that in the review.)

Reference and information from it have been included in the new section 2.

1. Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature. 2011 Jun 29;474(7353):609-15. PMID: 21720365

This reference has been added to the new section 2. However, this paper contains only the data on HGSOC available from the TCGA, and no data on ROC.

1. Jiang MC et al., Emerging roles of lncRNA in cancer and therapeutic opportunities. Am J Cancer Res. 2019 Jul 1;9(7):1354-1366. PMID: 31392074.

Reference and information from it have been added to section 8.

1. Yan X et al., Comprehensive Genomic Characterization of Long Non-coding RNAs across Human Cancers. Cancer Cell. 2015 Oct 12;28(4):529-540. PMID: 26461095. (The authors should briefly describe the lncRNAs related to OC from this paper in the text and in the table if not already included.)

This reference has been added to the new section 2. However, this paper contains only the data on HGSOC available from the TCGA, and no data on ROC.

1. Akrami R et al., Comprehensive analysis of long non-coding RNAs in ovarian cancer reveals global patterns and targeted DNA amplification. PLoS One. 2013 Nov 12;8(11):e80306. PMID: 24265805.

This reference has been added to section 6 but we had to remove one of the cited references describing the role of MALAT1 in epithelial OC as the paper has been retracted.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Thanks to the authors for making all the changes. The manuscript is much improved now. It will be a good resource for the researchers interested in lncRNAs related to ovarian cancer.