HDAC9 and miR-512 Regulate CAGE-Promoted Anti-Cancer Drug Resistance and Cellular Proliferation

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

 The auhtors' findings highlight the regulatory axis of CAGE-HDAC9-miR-512 in modulating anticancer drug resistance, cellular proliferation, and autophagic flux in gastric cancer cells. These results provide insights into the molecular mechanisms underlying HDAC9-mediated drug resistance and its crosstalk with autophagy, contributing to our understanding of therapeutic strategies targeting these pathways in cancer treatment. This is an interesting story, but I have several following concerns:

1. Abbreviations should be defined when they first appear in the text. Such as "CAGE" in Line 8, "cDNA" in Line 31, "S.E.M" in Line 187...

2. Please analyze the results of RT-qPCR used 2 ^-△△ct2 to compare the differences in gene expression between the experimental group and the control group more intuitively.

3. In Line 141, "50 mm" should be "50 mM".

4. Please quantify the results of the ChIP assay in Figure 2D. 

5. Please analyse the correlation among HDAC9, miR-512 and CAGE in clinical gastric cancer samples.

6. There are three "Data Availability" section, please combine them togather or rephrase them.

7. The nucleic acid sequences (including gene names, regulatory sequences, and primer names) should be in italics.

8. Please unify the format of references in the article, including the author's name, the case of words in the title of the article, the writing of the name of the journal, and the page number.

Comments on the Quality of English Language

Moderate editing of English language required.

Author Response

Dear Sir

 

Thanks for excellent suggestions. I made changes according to your suggestions. I hope that changes I made are suitable. In this revision, I let professionals handle English problems. I also upload English certificate.

 

 

 

Comments and Suggestions for Authors

 The auhtors' findings highlight the regulatory axis of CAGE-HDAC9-miR-512 in modulating anticancer drug resistance, cellular proliferation, and autophagic flux in gastric cancer cells. These results provide insights into the molecular mechanisms underlying HDAC9-mediated drug resistance and its crosstalk with autophagy, contributing to our understanding of therapeutic strategies targeting these pathways in cancer treatment. This is an interesting story, but I have several following concerns:

Q1. Abbreviations should be defined when they first appear in the text. Such as "CAGE" in Line 8, "cDNA" in Line 31, "S.E.M" in Line 187...

Ans. Thanks. I defined it as you suggested. Please take look at lines 8 and 195.

Q2. Please analyze the results of RT-qPCR used 2 ^-△△ct2 to compare the differences in gene expression between the experimental group and the control group more intuitively.

Ans. Thanks. I analyze the results of qRT-PCR as you suggested. Please take look at new figures (1C, 5A, 6A, 7B, 7D). They are repented as relative levels.       

Q3. In Line 141, "50 mm" should be "50 mM".

Ans. Thanks. I corrected it. Please take look at line 148.

Q4. Please quantify the results of the ChIP assay in Figure 2D. 

Ans. I quantify it. Please take look at new figure 2D. 

Q5. Please analyse the correlation among HDAC9, miR-512 and CAGE in clinical gastric cancer samples.

Ans. TCGA-STAD dataset was obtained from the GDC data portal, and subsequently filtered to include all miR-512-3p, CAGE, and HDAC9 sequencing results. Kaplan-Meier survival curves were then generated using GraphPad Prism 7.

 

Figure A: We analyzed the relationship between miR-512 and HDAC9. Based on this analysis, there was not close relationship between miR-512 and HDAC9.

Figure B: Gastric cancer patients with low level of miR-512 show shorter survival compared to cancer patients with high level of miR-512.

Figure C: We examined gastric cancer patients (82) with low level of miR-512 and high level of CAGE. For analysis, we divided these patients into two groups based on the level of HDAC9.    Those cancer patients with low level of HDAC9 show longer survival compared to cancer patients with high level of HDAC9.

We need more clinical samples to analyze the relationship between CAGE, HDAC9, and miR-512. I am afraid that I may not give enough information at this point. 

Q6. There are three "Data Availability" section, please combine them togather or rephrase them.

Ans. Thanks. I merge them. Please take look at lines 480-483. 

Q7. The nucleic acid sequences (including gene names, regulatory sequences, and primer names) should be in italics.

Ans. Thanks. In this revision, I try to use italics with nucleic acid sequences. Please take look at new manuscript.

Q8. Please unify the format of references in the article, including the author's name, the case of words in the title of the article, the writing of the name of the journal, and the page number.

Ans. Thanks. I found mistakes. I corrected them as you suggested. Please take look at new references.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript titled "HDAC9 and miR-512 regulate CAGE-promoted anti-cancer drug resistance and cellular proliferation" details the influence of the expression of HDAC9 and CAGE in gastric carcinoma making the cells drug resistant, and also shown the regulatory effect miR-512 on HDAC and CAGE thus containing the disease. The study is important especially when there is the huge occurrence of drug resistant cancers and also their relapse. The manuscript is interesting and the authors have performed wide range of studies. It can be accepted after authors had addressed my minor concerns:

1) The abstract is bit too long. I ask the authors to make to more concise and precise.

2) Secton 2.3: Mention the procedure for cell counting here.

3) Section 3.2 what is -2: Line 224. Also the authors need to specify the need to use 2 promoter sites in this study.

4) Section 3.3 So authors have to give a brief overview of the other proteins assess in this study. This will help the readers to understand the readers the involvement of. these proteins in autophagic flux.

5) Line 398:399: Authors should elaborate more on this.

6) Line 401 can be removed

7) Line 433 and 434 can be merged together.

8) Curious whether targetScan predicted any other miRNA as possible interactors of HDAC/CAGE?  I think the predictions should be submitted as supplementary data.

Comments on the Quality of English Language

Minor English editing required

Author Response

Dear Sir

 

Thanks for excellent suggestions. I made changes according to your suggestions. I hope that changes I made are suitable. In this revision, I let professionals handle English problems. I also upload English certificate.

 

 

Comments and Suggestions for Authors

The manuscript titled "HDAC9 and miR-512 regulate CAGE-promoted anti-cancer drug resistance and cellular proliferation" details the influence of the expression of HDAC9 and CAGE in gastric carcinoma making the cells drug resistant, and also shown the regulatory effect miR-512 on HDAC and CAGE thus containing the disease. The study is important especially when there is the huge occurrence of drug resistant cancers and also their relapse. The manuscript is interesting and the authors have performed wide range of studies. It can be accepted after authors had addressed my minor concerns:

Q1.The abstract is bit too long. I ask the authors to make to more concise and precise.

Ans. Thanks. In this revision, I try to make abstract more clear by removing unnecessary sentences. Please take look at new abstract. 

Q2. Secton 2.3: Mention the procedure for cell counting here.

Ans. I mention the procure. Please take look at new section 2.3. Please take look at lines 83-85.   

Q3. Section 3.2 what is -2: Line 224. Also the authors need to specify the need to use 2 promoter sites in this study.

Ans. - 2 is promoter site 2 of HDAC9. I change it. Please take look at lines 228-230.  

I add this sentence: Promoter luciferase activity assays showed that promoter sites 1 and 2 (P1 and P2) of HDAC9 were both necessary for increased expression of HDAC9 in AGS^R cells (Figure 2C).  

Q4. Section 3.3 So authors have to give a brief overview of the other proteins assess in this study. This will help the readers to understand the readers the involvement of. these proteins in autophagic flux.

Ans. Thanks. In this revision, I add these sentences: CAGE induced anti-cancer drug resistance by decreasing the expression of p53 in melanoma cells [7]. Anti-cancer drug resistance induced by CAGE was accompanied by increased autophaguc flux [11]. Overexpression of HDAC9 promotes cancer cell proliferation by suppressing expression of p53 [14]. Increased expression of pBeclin1^Ser15 and LC3II is known to be associated with anti-cancer drug resistance [11]. Please take look at lines 248-251 and 256-257. I hope that these sentences make things clear. 

Q5. Line 398:399: Authors should elaborate more on this.

Ans. Thanks. I add this sentence: Downregulation of PAICS can enhance the sensitivity of gastric cancer cells to cisplatin and inhibit gastric cancer cell growth [30]. I hope that this is helpful. Please take look at lines 384-386.

Q6. Line 401 can be removed

Ans. Thanks. I remove it.

Q7. Line 433 and 434 can be merged together.

Ans. Thanks. I delete this sentence: The expression of miR-512 is decreased in cisplatin-resistant ovarian cancer cells [37]. Please take look at new manuscript.

8) Curious whether targetScan predicted any other miRNA as possible interactors of HDAC/CAGE?  I think the predictions should be submitted as supplementary data.

Ans. I submit it as supplementary data. Please take look at figure S1.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Congratulations on your manuscript. Overall, the study is very interesting and well-designed. However, I have found a few minor editorial errors and some lack of methodology and results.

1.            The methodology should be more detailed. Some descriptions are too similar to https://doi.org/10.3389/fcell.2021.666387 (problem of self-plagiarism and magazine copyrights?)

2.3-        How many cells were seeded per surface to the test?

2.4-        Why did the medium in this test change for RPMI 1640 (in 2.2 I found information that DMEM was used)?

2.8-        The lack of transfection conditions (siRNA, microRNA, pFALAG-CAGE1, pGL constructs- concentrations, sequences);

2.9-        qRT-PCR was used to determine the expression of CAGE and HDAC as well (normalization to actin). Please supplement the protocol and add information about primer sequences and thermal conditions.

Fig. 1D presents results for modified cell lines AGS^RΔCAGE#5 and ^#7, while the methodology of creation of these cells is not included in the manuscript

2.            Some conclusions are not supported by results i.e.:

Line 65-66: HDAC9 was shown to function as a negative regulator of miR-512. – It was shown that miR-512-3p is a negative regulator of the HDAC9 (its target gene)

Line227-228: Thus CAGE-HDAC9 complex binds to the promoter sequence of HDAC…- presented data suggests only interactions of CAGE1 with protein and fragment DNA (P1, P2) of HDAC9 promoter, there are no evidences for CAGE1-HDAC9 complexes binding to HDAC9 promoter

Line251: HDAC9 downregulation increases cleaved-PARP level only in celastrol and taxol-treated AGSR, not as you mention AGS^R cells

Line 298: Please verify your statistical analysis in Fig. 5B  (NS looks significant- the data from this analysis is not included in supplemental data)

3.            Editorial mistakes i.e:

-              CAGE1 (according to gene cards is a cancer antigen 1 or cancer/testis antigen 3- CTAG3); I recommend changing the title for: HDAC9 and mir-512-3p regulate CAGE1-promoted anti-cancer drug resistance and cellular proliferation

-              Line 73: lowercase after dot

-              Line 95: double dot

-              Line 97, 99: RPMI 1640 (is 1,640)

-              Line 101: a lack of sentence end

-              Line 125: the algorithm's notation is misleading, I propose 2^-Δct

-              Line 229: Fig.2C -the title of the horizontal axis is not visible

-              Line 297: lack of dot

-              Line 344: SAGSR (AGS^R?)

-              Please revise of language style, especially in the introduction

4.            Some aspects are not clear in the manuscript. Could you explain them by additional experimental data or discussion :

-              How CAGE1 level impacts on HDAC9 promoter activity (luciferase assays in AGS^RΔCAGE#5 or  #7  or siCAGE1 AGS^R ) and  whereas HDAC9 protein is crucial for transcription activation of HDAC9 as you suggested?

-              Medium from AGSR culture stimulated expression of CAGE1 and HDAC9 in AGS cells. Can this effect be reversed by treating AGS cells with siCAGE1 truncated AGS^R?

 

-              What is the role of MDR1 in aspects of regulation CAGE1-HDAC9-mir-512-3p? You presented MDR1 results in samples obtained from in vivo experiments. What was the expression of MDR1 in cells with silencing CAGE1 and HDAC9?

Author Response

Dear Sir

 

Thanks for excellent suggestions. I made changes according to your suggestions. I hope that changes I made are suitable. In this revision, I let professionals handle English problems. I also upload English certificate.

Comments and Suggestions for Authors

Congratulations on your manuscript. Overall, the study is very interesting and well-designed. However, I have found a few minor editorial errors and some lack of methodology and results.

 

Q1.            The methodology should be more detailed. Some descriptions are too similar to https://doi.org/10.3389/fcell.2021.666387 (problem of self-plagiarism and magazine copyrights?)

Ans. In this revision, I make changes to give more detailed information on method. Please take look at new materials and methods. Throughout this revision, I try to reduce similarity as much as possible.

I use copy killer program run by our university to reduce similarity. I am sorry to cause concern. Similarity was down to 15 % throughout this manuscript.

 

 

Q2. 2.3- How many cells were seeded per surface to the test?

Ans. I add these sentences. Please take look at lines 83-85.

Cells were mixed with 0.4 % Trypan Blue staining solution in a 1:1 ratio and counted using a hemocytmeter. Two-hundred cells were seeded onto 6-well plates and maintained at 37°C in 5% CO₂  for 7 days. Colonies were stained with 0.01% crystal violet and counted.

 

Q3. 2.4- Why did the medium in this test change for RPMI 1640 (in 2.2 I found information that DMEM was used)?

Ans. Thanks. I change it to DMEM. I am sorry to cause confusion. Please atke look at new section 2.5.

 

Q4. 2.8-        The lack of transfection conditions (siRNA, microRNA, pFALAG-CAGE1, pGL constructs- concentrations, sequences);

 

Ans. I provide information on the sequences of primers, mimics, siRNAs. Please take look at supplementary tables for these sequences.

miRNAs and mimics (10 nM), constructs (each at 1 μg): please take look at figure legends.  

Please take look at new section 2.8 (transfections).

 

2. 2.9- qRT-PCR was used to determine the expression of CAGE and HDAC as well (normalization to actin). Please supplement the protocol and add information about primer sequences and thermal conditions.

 

Ans. In this revision, I give detailed information on PCR. Please take look at lines 121-135.

Please take look at the primer sequences (supplementary table 3).  

 

 

1. Fig. 1D presents results for modified cell lines AGS^RΔCAGE#5and ^#7, while the methodology of creation of these cells is not included in the manuscript

Ans. In this revision, I describe method for creation of AGS^RΔCAGE#5 and ^#7 . Please take look at lines 77-80. 

 

2. Some conclusions are not supported by results i.e.:
3. Line 65-66: HDAC9 was shown to function as a negative regulator of miR-512. – It was shown that miR-512-3p is a negative regulator of the HDAC9(its target gene)

Ans. Thanks. I agree. I add this sentence: HDAC9 was shown to function as a target of miR-512. Please take look at line 62-63.

 

1. Line227-228: Thus CAGE-HDAC9 complex binds to the promoter sequence of HDAC…- presented data suggests only interactions of CAGE1 with protein and fragment DNA (P1, P2) of HDAC9promoter, there are no evidences for CAGE1-HDAC9 complexes binding to HDAC9promoter.

 

Ans. Thanks. I agree. I change the sentence into: Thus, CAGE can bind to promoter sequences of HDAC9 to exert direct regulation of HDAC9 expression. Please take look at lines 234-235.

 

Line251: HDAC9 downregulation increases cleaved-PARP level only in celastrol and taxol-treated AGSR, not as you mention AGS^R cells

 

Ans. Thanks. I agree. I change sentence into: However, downregulation of HDAC9 increased the expression of cleaved PARP in AGS^R cells in response to celastrol and taxol (Figure 3D). Please take look at lines 259-260.

 

Line 298: Please verify your statistical analysis in Fig. 5B  (NS looks significant- the data from this analysis is not included in supplemental data)

Ans. I agree. I made a mistake. I add new figure 5B and legend. 

 

Q3. Editorial mistakes i.e:

 

1. CAGE1 (according to gene cards is a cancer antigen 1 or cancer/testis antigen 3- CTAG3); I recommend changing the title for: HDAC9 and mir-512-3p regulate CAGE1-promoted anti-cancer drug resistance and cellular proliferation

 

Ans. I think there is a misunderstanding. CAGE1 is a different gene from CAGE. We previously published paper concerning CAGE1. I am sorry that I do not have to change the title. CAGE is alternatively known as CT26 or DDX53.    

 

1. Line 73: lowercase after dot

Ans. I delete the sentence: anti-mouse and anti-rabbit IgG-horseradish peroxidase conjugate antibodies were purchased from Pierce Company (Rockford, IL, United States). Please take look at new section 2.1.  

 

1. Line 95: double dot

Ans. I corrected it. Please take look at new section 2.4.

 

1. Line 97, 99: RPMI 1640 (is 1,640)

Ans. It is DMEM, nor RPMI.

 

1. Line 101: a lack of sentence end

 

Ans. I change the sentence into: The invaded cells were stained and counted as described [11]. Please take look at lines 98-99.

 

1. Line 125: the algorithm's notation is misleading, I propose 2^-Δct

 

Ans. I agree. I change in into 2^{− (Ct of miR 512)−(Ct of U6 )}. Please take look line 125.

 

1. Line 229: Fig.2C -the title of the horizontal axis is not visible

Ans. I change it. Please take look at new figure 2C.

1. Line 297: lack of dot
   Ans. Thanks. I change it.

 

1. Line 344: SAGSR (AGS^R?)

 

Ans. Thanks. I change it.

 

1. Please revise of language style, especially in the introduction

 

Ans. Thanks. I change the introduction in a way to make this manuscript more readable. I let professionals handle English problems.    

,

Q4.  Some aspects are not clear in the manuscript. Could you explain them by additional experimental data or discussion :

 

1. How CAGE1 level impacts on HDAC9 promoter activity (luciferase assays in AGS^RΔCAGE#5or  #7  or siCAGE1 AGS^R) and  whereas HDAC9 protein is crucial for transcription activation of HDAC9 as you suggested?

 

Ans. Our results show that CAGE binds to the promoter sequences of HDAC9. However, this does not suggest that CAGE directly affects HDAC9 promoter activity.

I add this sentence: Transfection HDAC9 promoter luciferase construct into AGS ^R△CAGE#5 or AGS^{R △CAGE#7} cells might provide clue to the possibility of direct regulation of HDAC9 expression by CAGE.. Please take look at lines 236-237. In this study, we show that overexpression or downregulation of CAGE regulates the expression of HDAC9 (Figure 2A). Human recombinant CAGE protein increases the expression of HDAC9 in AGS cells (Figure 7E).

 

1. Medium from AGSRculture stimulated expression of CAGE1 and HDAC9 in AGS cells. Can this effect be reversed by treating AGS cells with siCAGE1 truncated AGS^R?

Ans. Our previous reports show that the culture medium of anti-cancer drug resistant cancer cells (AGS^R) increased the expression of CAGE and autophagic flux in anti-cancer drug sensitive AGS cells (Yeon M et al., 2021, Frontiers in Cell and Developmental Biology). Previously, we also showed that the culture medium of AGS^R increased the expression of CAGE and autophagic flux in AGS^RΔCAGE#5 and #7 cells (Yeon M et al., 2021, Frontiers in Cell and Developmental Biology). It is probable that the culture medium of AGS^R cells transfected with siCAGE does not increase the expression of CAGE or autophagic flux in AGS cells. It is also probable that the culture medium of AGS^R cells transfected with siCAGE may decrease the expression of CAGE or autophagic flux in AGS^R cells.   

The culture medium of AGS^R cells transfected with siHDAC9 decrease the expression of CAGE and autopahgic flux in AGS^R cells (Figure below). 

 

1. What is the role of MDR1 in aspects of regulation CAGE1-HDAC9-mir-512-3p? You presented MDR1 results in samples obtained from in vivo experiments. What was the expression of MDR1 in cells with silencing CAGE1 and HDAC9?

 

Ans. For this study, we did not focus on the role of MDR1. I believe that MDR1 is necessary for anticancer drug resistance displayed by AGS^R cells. It is known that taxol-resistant HeLa cells show the increased expression of CAGE and MDR1 (Park S et al., 2018, BBRC). The downregulation of CAGE decreases the expression of MDR1 and enhances the sensitivity to taxol and celastrol (Park S et al., 2018, BBRC). It is probable that MDR1 is necessary for CAGE-promoted anti-cancer drug resistance. I would like to work on the role of MDR1 in anti-cancer drug resistance in relation with autophagy in the future.     

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have addressed all my concerns. I recommend accepting this manuscript in current form.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper “HDAC9 mediates CAGE-promoted anti-cancer drug resistance and cellular proliferation” presents an interesting subject and actuality in medical field about using the miR-512 as a regulator of HDAC9 in gastric cancer cell lines by passive transfection, that is the reason, which I suggest some recommendations to improve the quality of your manuscript.

1.- Celastrol is a triterpenoid drug with anti-inflammatory, antioxidant, and anti-cancer effects. The major cell signaling pathways modulated by Celastrol include the NF-kB pathway, MAPK pathway, JAK/STAT pathway, PI3K/Akt/mTOR pathway, and antioxidant defense mechanisms. Please specify the gastric cell lines used an their specific and also the cytotoxic concentration of Celastrol (details of treatments) to create these resistant-drug cell line cultures (fractions). Do you think that cellular metabolism or phenotype status of cells may be modify in function of metabolic substrate which you are used for cell cultures?

2.-Please added method and equipment that you used to measure the cell viability. In figure 1, you mention IC50, but in methods you didn’t specify. Please correlate the methods with results.

3.-In results section you mentioned to create the drug resistance cell cultures Celastrol and Taxol? In methods you specify only Celastrol. Please, make the correlation between methods and results.

4.-in line 181-182-RNA sequencing analysis showed that HDAC9 was the most upregulated among HDACs in AGS cells (data not shown). If you didn’t showed data is better to remove this paragraph.

5.-In lines 185-186-In this study, we also found that 185 AGS^R cells showed increased tumorigenic and metastatic potential (data not shown). If you didn’t showed data is better to remove this paragraph.

6.-In 3.1. Anti-Cancer Drug Resistant Gastric Cancer Cells Show Increased Expressions of CAGE and HDAC9 section of results, cell viability (% of control) was calculated for experimentals and controls that means you may write only cell viability (%).

7. -The cancer/testis antigen CAGE with oncogenic potential stimulates cell proliferation by up-regulating cyclins D1 and E implied in cell apoptosis linked with autophagy mechanisms. Target of rapamycin is mTOR signaling pathway that is a regulator of cell growth and metabolism. Deregulation of the mTOR pathway is already known to be implied in cancer and genetic disorders. Rapamycin is a specific inhibitor of mTOR pathway. Also, changes in DNA methylation status are implicated in cancer-associated miR deregulation. miR-512-3p exerts pro-tumoral properties by increasing cell proliferation, migration, invasion, or decreasing apoptosis. Also, in figure 3, results section, DAPI is a fluorescent nucleic acid stain that binds to A-T rich regions of double-stranded DNA, which is exclude from viable cells, but penetrate cell membranes of dead or dying cells. Please added in discussion section more references about apoptosis –autophagy-DNA damage linked mechanisms to highlight better your obtained results.

8. In this study, line 415 we found the increased expression of PD-L1 in AGSR cells (data not shown)-Please added the obtained results in manuscript or remove this paragraph.

9.- In this study, we found that recombinant CAGE protein increased 420 HDAC9 expression in AGS cells (data not shown). Please added the obtained results in manuscript or remove this paragraph.

10.- AGSR cells showed the increased expression of PD-L1 compared with 426 AGS cells (data not shown). In this study, we found that AGSR cells showed increased 427 expression of BCL6 compared with AGS cells (data not shown). Please added the obtained results in manuscript or remove this paragraph.

11.-Please add the future directions of your research. Also provide limitations of your study.

 

Author Response

Dear Sir

I thank for excellent suggestions made by reviewers. I make changes to accommodate suggestions made by reviewers. I make revisions throughout the manuscript. I provide extra data. I hope that changes I made are suitable. In this revision, I let professionals manage English problems. I provide English certificate.

 

Sincerely yours

Jeoung Dooil, Ph.D.

Professor of Biochemistry

Kangwon National University

Chuncheon 24341, Kore a

 

Reviewer 1

 

The paper “HDAC9 mediates CAGE-promoted anti-cancer drug resistance and cellular proliferation” presents an interesting subject and actuality in medical field about using the miR-512 as a regulator of HDAC9 in gastric cancer cell lines by passive transfection, that is the reason, which I suggest some recommendations to improve the quality of your manuscript.

Q1.- Celastrol is a triterpenoid drug with anti-inflammatory, antioxidant, and anti-cancer effects. The major cell signaling pathways modulated by Celastrol include the NF-kB pathway, MAPK pathway, JAK/STAT pathway, PI3K/Akt/mTOR pathway, and antioxidant defense mechanisms. Please specify the gastric cell lines used an their specific and also the cytotoxic concentration of Celastrol (details of treatments) to create these resistant-drug cell line cultures (fractions). Do you think that cellular metabolism or phenotype status of cells may be modify in function of metabolic substrate which you are used for cell cultures?

Ans. Thanks. In our experiment, we used the AGS human gastric adenocarcinoma cell line. This cell line harbors the following genetic mutations: CTNNB1 G34E, KRAS G12D, and PIK3CA E453K and E545A. The parental AGS cell line showed an IC50 of 1.48 μM in response to celastrol. Compared to other cell lines, the AGS cell line showed higher sensitivity to celastrol and taxol (Fig.1A). To establish celastrol-resistant AGS (AGS^R) cell line, AGS cells were initially treated with 0.5 μM celastrol for 3 days. The medium was replaced with fresh medium to allow the cells to recover. The recovered cells were replated at 50% confluency and treated again with 0.5 μM celastrol for 3 days. This process was repeated with gradually increasing the celastrol concentration up to 1.5 μM for 6 months.

AGS^R cells show low expression levels of pAkt^Ser473, pERK^Tyr204, pJAK2^Tyr1007/1008, pSTAT3^Tyr705, pCyclinD1^Thr286, pSTAT3^{Tyr 705}, pGSKβ^Thr126, p53, p62, and EGFR compared to AGS cells (Figure below). AGS^R cells show high expression levels of cyclin D1, SOX2, HER2, MDR1, ATG5-12, S1PR1, pIkB ^Ser32, pAMPKα^Thr172, and pBeclin1^Ser15 compared to AGS cells (unpublished observations). It is probable that celastrol treatment leads to celastrol resistance by modulating cellular signaling (metabolic) pathways. Figure (right) shows nuclear translocation of NF-kB in AGS^R cells. This indicates that anticancer drug resistance involves the activation of NF-kB. It is therefore probable that the resistance to celastrol involves changes in signaling pathway.

 

Q2.-Please added method and equipment that you used to measure the cell viability. In figure 1, you mention IC50, but in methods you didn’t specify. Please correlate the methods with results.

Ans. The relative cell viability (the response of each cancer cell line to each anticancer drug) was measured by MTT assays. I mentioned software for measuring IC₅₀. Please take look at figure 1 legend. Please take look at new materials and methods. I add new section 2.4. as is seen in below. 

[Cell viability assay (section 2.4)]

MTT assays were employed to determine the response to anticancer drugs. The indicated cancer cells were plated in 48-well plates and incubated with culture medium for 24 hours. The culture medium was then replaced with medium containing the indicated anticancer drug for 48 hours. To determine the response to each anticamcer drug, the medium was replaced with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and incubated for 1 hour. After removing solution, MTT formazans were dissolved with DMSO, and the plates were read at 570 nm. Cell viability was calculated using Graphpad Prism 7.  

 

Q3.-In results section you mentioned to create the drug resistance cell cultures Celastrol and Taxol? In methods you specify only Celastrol. Please, make the correlation between methods and results.

Ans. AGS^R cells were established by stepwise addition of celastrol to anticancer drug sensitive AGS cells (Materials and methods section). I described procedures to establish AGS^R cells in response to Q1. AGS^R cells seem to display multi drug resistance phenotype (Fig. 1). Fig.2A shows that AGS^R cells show resistance to both celastrol and taxol. It is probable that AGSR cells may display resistance to various chemotherapeutic anticancer drugs.

Q4.-in line 181-182-RNA sequencing analysis showed that HDAC9 was the most upregulated among HDACs in AGS cells (data not shown). If you didn’t showed data is better to remove this paragraph.

Ans. Thanks. I remove it.

Q5.-In lines 185-186-In this study, we also found that 185 AGS^R cells showed increased tumorigenic and metastatic potential (data not shown). If you didn’t showed data is better to remove this paragraph.

Ans. Thanks. I remove it.

Q6.-In 3.1. Anti-Cancer Drug Resistant Gastric Cancer Cells Show Increased Expressions of CAGE and HDAC9 section of results, cell viability (% of control) was calculated for experimentals and controls that means you may write only cell viability (%).

Ans. In this study, we perform MTT assays. Viability is expressed as % of controls. MTT assays do not count cell number.

Q7. -The cancer/testis antigen CAGE with oncogenic potential stimulates cell proliferation by up-regulating cyclins D1 and E implied in cell apoptosis linked with autophagy mechanisms. Target of rapamycin is mTOR signaling pathway that is a regulator of cell growth and metabolism. Deregulation of the mTOR pathway is already known to be implied in cancer and genetic disorders. Rapamycin is a specific inhibitor of mTOR pathway. Also, changes in DNA methylation status are implicated in cancer-associated miR deregulation. miR-512-3p exerts pro-tumoral properties by increasing cell proliferation, migration, invasion, or decreasing apoptosis. Also, in figure 3, results section, DAPI is a fluorescent nucleic acid stain that binds to A-T rich regions of double-stranded DNA, which is exclude from viable cells, but penetrate cell membranes of dead or dying cells. Please added in discussion section more references about apoptosis –autophagy-DNA damage linked mechanisms to highlight better your obtained results.

Ans. Thanks. In the discussion, I mention the effect of miR-512 on apoptosis, metastasis, and anticancer drug resistance. In the discussion, I mention miR-512 as positive regulator of apoptosis (refs. 41-43). We previously reported that anticancer drug resistance was positively associated with the enhanced autophagic flux [refs. 12,14,15]. It is well known that autophagy is a double-sword. In early phase of cancer, autophagy inhibits spread of cancer. However, in later phase of cancer, autophagy promotes spread of cancer. In the discussion, I also mention that [The downregulation of HDAC9 has been shown to induce apoptosis and growth arrest in gastric cancer cells (lines 400-401, ref. 20)]. These reports indicate close relationship between apoptosis and autophagy. I also mention these sentences: In the present study, we found the increased expression of HDAC6 in AGS^R cells compared to AGS cells (data not shown). HDAC6 inhibition leads to apoptosis in gastric cancer cells [37]. HDAC6 is known to play an essential role in autophagy [38]. Please take look at lines 381-384.

Q8. In this study, line 415 we found the increased expression of PD-L1 in AGSR cells (data not shown)-Please added the obtained results in manuscript or remove this paragraph.

Ans. Thanks. I remove it.

Q9.- In this study, we found that recombinant CAGE protein increased 420 HDAC9 expression in AGS cells (data not shown). Please added the obtained results in manuscript or remove this paragraph.

Ans. Thanks. I remove it.

Q10.- AGSR cells showed the increased expression of PD-L1 compared with 426 AGS cells (data not shown). In this study, we found that AGSR cells showed increased 427 expression of BCL6 compared with AGS cells (data not shown). Please added the obtained results in manuscript or remove this paragraph.

Ans. Thanks. I remove it.

Q11.-Please add the future directions of your research. Also provide limitations of your study.

Ans. In this revision, I amend discussion. I remove unnecessary and repetitive sentence. I change order of some sentences. I add two references. I suggest several experiments: in vivo anticancer drug resistance of AGS^R cells, determination of the resistance of AGS^R cells to immune therapeutics such as anti-PD-L1 antibody, identification of molecular network involving CAGE and HDAC9, the binding of CAGE to the promoter sequences of miR-512. I also mention the possibility of CAGE and HDAC9 as prognostic markers of cancers. Please take look at new discussion and conclusion (lines 360-485).

Reviewer 2 Report

Comments and Suggestions for Authors

Review comment

 

The manuscript titled "HDAC9 mediates CAGE-promoted anti-cancer drug resistance and cellular proliferation" presents a study on the role of Histone deacetylase 9 (HDAC9) in mediating cancer drug resistance and cell proliferation through interaction with CAGE, a cancer/testis antigen. The research delves into the molecular interactions between CAGE and HDAC9, highlighting how CAGE upregulation promotes anti-cancer drug resistance and autophagic flux through HDAC9 expression regulation. It also tried to investigate the role of miR-512 in modulating these processes and the effects of HDAC9 on autophagy, invasion, migration, and tumor spheroid formation. However, apparent flaws can be observed in the manuscript. Thus, Major revision is recommended. Main issues have been listed as following:

 

1.Since the authors mentioned that HDAC9 is overexpressed in gastric cancer patients, the effects of HDAC9 on the prognosis of gastric cancer patients must be presented.

 

2.Authors claimed they established Anticancer drug-resistant cancer cell lines for all the experiments. What kind of drugs can those cell lines resist? Chemo-drug? Targeted-agent？ Or immunotherapy agent? Those key and indispensable information is missing.

 

3.There is no direct evidence in this manuscript that can prove the direct regulatory effects among CAGE, HDAC9 and miR-512 .

 

4.Each experimental group should be represented by a set of three images to adequately illustrate the findings (such as fig4A 4c and 4E)

 

5.The conclusion should be tested in vivo.

 

6.A more detailed description of the control experiments and the statistical methods used for data analysis is required for the manuscript's clarity and reliability.

 

7.The title of this manuscript should include “miR-512” since authors reported the regulatory role of miR-512 and the effect of HDAC9 on cancer cell behavior.

 

8.Authors should provide a more critical examination of potential limitations and the implications of the study for future therapeutic strategies. Additionally, discussing alternative mechanisms that might contribute to CAGE-promoted drug resistance could provide a more comprehensive overview.

 

9.Editorial revisions for grammar are required.

Comments on the Quality of English Language

Language editing is recommended.

Author Response

Reviewer 2

The manuscript titled "HDAC9 mediates CAGE-promoted anti-cancer drug resistance and cellular proliferation" presents a study on the role of Histone deacetylase 9 (HDAC9) in mediating cancer drug resistance and cell proliferation through interaction with CAGE, a cancer/testis antigen. The research delves into the molecular interactions between CAGE and HDAC9, highlighting how CAGE upregulation promotes anti-cancer drug resistance and autophagic flux through HDAC9 expression regulation. It also tried to investigate the role of miR-512 in modulating these processes and the effects of HDAC9 on autophagy, invasion, migration, and tumor spheroid formation. However, apparent flaws can be observed in the manuscript. Thus, Major revision is recommended. Main issues have been listed as following:

 

Q1. Since the authors mentioned that HDAC9 is overexpressed in gastric cancer patients, the effects of HDAC9 on the prognosis of gastric cancer patients must be presented.

 

Ans. Zhang T, Wang B, Gu B, Su F, Xiang L, Liu L, Li X, Wang X, Gao L, Chen H. Genetic and Molecular Characterization Revealed the Prognosis Efficiency of Histone Acetylation in Pan-Digestive Cancers.J Oncol. 2022 Apr 5;2022:3938652. doi: 10.1155/2022/3938652. eCollection 2022.

* The above article shows that HDAC9 displayed widespread copy number amplification across five pan-digestive cancers. This implies that HDAC9 can function as diagnostic and/or prognostic marker of digestive cancers. So far, there have not been extensive studies on potential of HDAC9 as a prognostic marker of gastric cancer.

 

Fu Y, Sun S, Bi J, Kong C, Shi D. An HDAC9-associated immune-related signature predicts bladder cancer prognosis. PLoS One. 2022 Mar 3;17(3):e0264527. doi: 10.1371/journal.pone.0264527. eCollection 2022.

* The above article indicates that HDAC9 expression level can predict prognosis of bladder cancer patients.

 

The figure below shows that the expression of HDAC9 is higher in gastric cancer patients with high grade. High expression of HDAC9 is correlated with stomach adenocarcinoma (STAD) grades and stages based on TCGA database. Below data was modified from UALCAN cancer database at Dana-Farber Cancer Institute.
   

* Xiong et al shows that HDAC9 can be target for developing anticancer drugs in gastric cancer (2019, EMM). They show clinical implication of HDAC9: Expression of HDAC9 in gastric cancer patients, role of HDAC9 in cell proliferation and cell cycle.

 

Since CAGE functions as a prognostic marker of pulmonary adenocarcinoma (Yeon et al., 2023, Scientific reports, ref 52), HDAC9 can function as a prognostic marker of cancers.

It will be necessary to examine clinical validation of HDAC9 by conducting clinical studies employing tissues, sera, and various specimen of cancer patients.

 

 

Q2. Authors claimed they established Anticancer drug-resistant cancer cell lines for all the experiments. What kind of drugs can those cell lines resist? Chemo-drug? Targeted-agent？ Or immunotherapy agent? Those key and indispensable information is missing.

 

Ans. AGS^R cells show resistance to cilantro and taxol (Fig. 1). We have not studied resistance of AGS^R cells to immunotherapy agents. It is necessary to examine the resistance of AGS^R to immunotherapeutic reagents such as Anti-PD-L1 antibody. Please take look at new discussion. In this study, we found that AGSR cells showed higher expression of PD-L1 than AGS cells (unpublished observations). Thus, it is probable that AGS^R cells may display resistance to anti-PD-L1 antibody. We will conduct this experiment in the near future.            

 

Q3.There is no direct evidence in this manuscript that can prove the direct regulatory effects among CAGE, HDAC9 and miR-512 .

 

Ans. Thanks. I agree. HDAC9 was identified as one of the most upregulated genes in AGS cells with CAGE overexpression based on RNA sequencing profiling (personal observations). First, I would like to summarize findings in this manuscript. 

• CAGE binds to HDAC9.CAGE binds to HDAC9.
• CAGE binds to the promoter sequences of HDAC9.
• Since CAGE binds to HDAC9, CAGE may exert direct effect on HDAC9.
• miR-512 acts as a negative regulator of HDAC9.
• Based on luciferase activity assays, it seems that miR-512 directly regulates the expression of HDAC9.

I add this sentences: Since miR-512 directly regulates the expression of HDAC9 (Figure 5), it is necessary to examine whether CAGE and HDAC9 can directly regulate the expression of miR-512. It is necessary to examine the possibility of binding of CAGE to the promoter sequence of miR-512.   Please take look at lines (424-427).

 

 

Q4.Each experimental group should be represented by a set of three images to adequately illustrate the findings (such as fig4A 4c and 4E)

 

Ans. Thanks. I agree. I provide raw images (figure 4A, C, and E).

Figure below (figure 4A) shows the effect of the downregulation of HDAC9 on the migration potential of AGS^R cells.

 

 

 

Figure below (figure 4A) shows the effect of the downregulation of HDAC9 on the invasion of AGS^R cells. 

.

 

 

Figure below (figure 4C) shows the effect of the downregulation of HDAC9 on the tumor spheroid forming potential of AGS^R cells.

 

 

 

Figure below (figure 4E) shows the effect of the downregulation of HDAC9 on the colony forming potential of AGS^R cells.

 

 

 

 

Q5.The conclusion should be tested in vivo.

 

Ans. Thanks. I agree. We measured oncogenic potential of AGS and AGS^R cells. AGS^R cells, but not AGS cells, developed tumor as is seen in figure below. Immunoblot of tissue lysates show that tumor derived from AGS^R cells express CAGE, MDR1, pBeclin1^Ser15, LC3, and S1PR1 (sphingosine-1-phosphate receptor 1). Anticancer drug sensitive AGS cells did not develop tumors (figure below). We would like to examine  in vivo anticancer drug resistance of AGS^R cells to various anticancer drugs in near future. I suggest that CAGE and HDAC9 can function as prognostic markers of gastric cancer. Please take look at new conclusions.

 

 

 

The figure below shows that high HDAC9 epxression is correlated with high grade gastric carcinoma. It is probable that HDAC9 can be employed as a prognostic marker of gastric cancer. It is known that CAGE is present in the sera of patients with various cancers [refs 1-3]. It is probable that CAGE can function as a prognostic marker of cancers. We reported that CAGE can finction as a prognostic marker of pulmonary adenocarcinoma [Yeon M et al., 2023, Scientific reports, ref 52].           

 

 

I also mention that [It is necessary to examine the role of CAGE, HDAC9 and miR-512 in in vivo anticancer drug resistance of AGS^R cells]. Please take look at lines 482-483.   

 

Q6. A more detailed description of the control experiments and the statistical methods used for data analysis is required for the manuscript's clarity and reliability.

 

Ans. I mentioned that student’s t test was employed throughout this study. Please take look at legends.

I also mentioned that [Representative images of three independent experiments were shown. The uncropped blots are shown in supplementary materials]. Please take look at figure legends. In this revision, I try to give a detailed description of the experiments. Please take look at new figure legends. In this revision, I changed figure legends in a way to give a detailed description of the experiments. In this revision, I try to give a detailed description of cell viability determination. Please take look at new materials and methods (Section 2.4).

   

Figure below (Figure 6C) shows the effect of miR-512 mimic on the migration potential of AGS^R cells.

 

 

 

Figure below (Figure 6C) shows the effect of miR-512 mimic on the invasion potential of AGS^R cells. 

 

 

 

Figure below (Figure 6D) shows the effect of miR-512 mimic on the colony forming potential of AGS^R cells. 

 

 

 

 

Q7.The title of this manuscript should include “miR-512” since authors reported the regulatory role of miR-512 and the effect of HDAC9 on cancer cell behavior.

 

Ans. Thanks. I change the title as you suggested. New title is: HDAC9 and miR-512 regulate CAGE-promoted anti-cancer drug resistance and cellular proliferation.

   

Q8. Authors should provide a more critical examination of potential limitations and the implications of the study for future therapeutic strategies. Additionally, discussing alternative mechanisms that might contribute to CAGE-promoted drug resistance could provide a more comprehensive overview.

 

Ans. Thanks. I agree. This manuscript does not fully cover the mechanism of CAGE-promoted anticancer drug resistance. It is likely that CAGE-promoted anticancer drug resistance is closely related with anti-apoptotic effects and autophagy. In this revision, I suggest experiments: a) in vivo anticancer drug resistance; b) identification of molecular network involving CAGE and HDAC9; c) the resistance of AGS^R cells to immunotherapeutic agents such as anti-PD-L1 antibody; d) effect of CAGE expression on cell signaling pathways; e) the binding of CAGE to the promoter sequences of miR-512. These experiments may reveal novel mechanisms of CAGE-promoted anticancer drug resistance. Please take look at new discussion and conclusion. In this revision, I mention these sentences: We previously reported that CAGE forms a negative feedback loop with miR-181b and promotes anticancer drug resistance in gastric cancer cells [15]. CAGE was shown to directly regulate the expression of sphingosine-1-phosphate receptor 1 (S1PR1) by binding to the promoter sequences of S1PR1 [15]. miR-181b was shown to act as a direct regulator of S1PR1. Thus, CAGE-S1PR1-miR-181b loop may provide clues to understand the mechanism of CAGE-promoted anticancer drug resistance. It is necessary to examine the role of S1PR1 for better understanding of CAGE-promoted anticancer drug resistance. Please take look at new discussion (lines 438-444).

 

Q9. Editorial revisions for grammar are required.

Ans. Thanks. In this revision, I let professionals manage English problems. I provide English certificate.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This is an experiment conducted solely using gastric cancer cells, but the article title does not specify the type of cancer. The study focuses on chemotherapy-resistant gastric cancer cells, and in today's era where gastric cancer treatment has advanced to targeted and immunotherapy（such as PD-1/PI-L1i, CTLA-4i,etc）, this research apparently lacks novelty and practicality. Additionally, authors failed to address several important issues mentioned in the previous review, inclduing: Q4.Each experimental group should be represented by a set of three images to adequately illustrate the findings (such as fig4A 4c and 4E); Q5.The conclusion should be tested in vivo. Therefore, evidence of this study is too weak to be published on CIMB.