Inhibition of GLI Transcriptional Activity and Prostate Cancer Cell Growth and Proliferation by DAX1
Round 1
Reviewer 1 Report (Previous Reviewer 4)
Comments and Suggestions for AuthorsThe authors have satisfactorily addressed the concerns raised in the original version. The revised version is significantly improved. No further concerns.
Comments on the Quality of English Language
Minor editing of English language required.
Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsThis re-submitted manuscript has been greatly improved that results can support the conclusions, and the reviewer has no additional comments.
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 Authors1. Grammar and spell check throughput the manuscript
2. Write names in full, then abbrv or acronyms after on first mention
3. Line 25 and 26, please rectify as PCa mortality rates are higher in LMICs such as African Countries, even though HICs have high PCa incidence rates
4. Line 45-46, did the authors mean PCa development and progression?
5. Line 89- pen/strep %?
6. Line 159, standard error of the mean can be abbreviated as SEM
Methods are briefly described, can be improved
7. A comprehensive research approach has been used. However, this does not really identify novel targets in DAX1- GLI1/2 interaction. A brief overview of an in silico analysis may assist with this and also enhance the conclusions.
Author Response
We thank the Reviewer #1 for insightful and positive comments.
Detailed point-by-point answers to comments are provided below.
Point 1. Grammar and spell check throughput the manuscript
Response 1: According to the reviewer’s suggestion, we again received the English editing service
Point 2: Write names in full, then abbrv or acronyms after on first mention
Response 2: According to the reviewer’s suggestion, we corrected the manuscript
Point 3: Line 25 and 26, please rectify as PCa mortality rates are higher in LMICs such as African Countries, even though HICs have high PCa incidence rates
Response 3: According to the reviewer’s suggestion, we corrected the manuscript (Line 31-32).
Point 4: Line 45-46, did the authors mean PCa development and progression?
Response 4: GLI1 and GLI2 are important in both the development and progression of cancer. So, we revised the sentenc (Line 70).
Point 5: Line 89- pen/strep %?
Response 5: According to the reviewer’s suggestion, we corrected the manuscript(Line 144). We used 1% penicillin–streptomycin
Point 6: Line 159, standard error of the mean can be abbreviated as SEM
Response 6: According to the reviewer’s suggestion, we corrected the manuscript (Line 260).
Point 7: A comprehensive research approach has been used. However, this does not really identify novel targets in DAX1- GLI1/2 interaction. A brief overview of an in silico analysis may assist with this and also enhance the conclusions.
Response 7: Response 6: According to the reviewer’s suggestion, we corrected the manuscript (Line 455-461). As suggested by the reviewer, we expected to find new targets through DAX1- GLI1/2 interaction by in silico analysis, but we were very disappointed that we did not get satisfactory results.
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsSpecific comments to the authors
The authors Sung Pyo Hong et al. of the submitted manuscript “Inhibition of GLI transcriptional activity and prostate cancer cell growth and proliferation by DAX1” studied the possible regulative mechanisms of DAX1 on the activity of GLI transcription. In summary, based on their different molecular techniques investigations authors could show that (i) DAX1 inhibits the Hh pathway, (ii) DAX1 interacts with GLI1 and GLI2, (iii) DAX1 induces G1/S cell cycle arrest, (iv) DAX1 suppresses the proliferation and growth of prostate cancer cells. Therefore, the authors postulated a novel regulatory mechanism of Hh signaling in prostate cancer cells, which could be relevant for new therapeutical strategies.
Overall, the manuscript give some interesting information and insights on the regulative influence of DAX1 on GLI expression in prostatic cancer cell lines. The manuscript (including the presentation) is mostly comprehensible and convincing. The methods are mostly well described. Although the results are mostly presented in the tables and figures, the authors must do some minor additional explanations/experiments to improve the impact of the presented study. In conclusion, the data presented are interesting.
The main concerns relate to the fact that no definitive relationship between DAX1 and the Hh pathway has been published to date. In silico analysis using the STRING online database (see https://string-db.org/), no association was found, too.
After incorporating the mentioned specific comments (see below), the manuscript has the potential to be accepted.
Specific comments
Abstract: The end of conclusion (“…with potentially relevant implications in cancer therapy.” is largely speculative.
Material&Methods: Please describe basic molecular and pathological aspects of the two chosen cell lines HEK293 and DU145, since HEK293 is not a prostatic cancer cell line (HEK293 = Human embryonic kidney 293 cells).
Results:
# Figure 1: The effect of DAX1 on the expression of Smoothened should be investigated by the authors, too. Furthermore, western blots should be performed to support the findings of the mRNA expression.
# Figure 2: The co-localization/merged images should be presented in higher magnification.
# Figure 3: Resolution of the graphics in Fig 3A is poor.
# Figure 4: Resolution of the graphics in Fig 4B is poor. The cell density should be quantified for further statistical analysis. The proliferation should be studied by cell blocks and by consecutive immunohistochemistry with Ki-67 antibody.
Discussion: The authors could demonstrate the DAX-GLI-axis in the chosen experimental in-vitro setting. Nevertheless, the protein expression of DAX1 and GLI in different prostatic cancer specimen should be investigated. Overall, more question than answers are evident by the submitted manuscript. Therefore, the authors should mentioned the limitations of the investigations (no in-vivo or in-situ experiments). The authors should discuss how the findings could be definitely transferred from a theoretical to a practical view (especially in the clinical setting (drug-development, drug-combination)) in the future.
Author Response
We thank the Reviewer #4 for insightful and positive comments.
Detailed point-by-point answers to comments are provided below.
Point 1. Abstract: The end of conclusion (“…with potentially relevant implications in cancer therapy.” is largely speculative.
Response 1: According to the reviewer’s suggestion, we corrected the manuscript. (Line 25-28).
Point 2: Material&Methods: Please describe basic molecular and pathological aspects of the two chosen cell lines HEK293 and DU145, since HEK293 is not a prostatic cancer cell line (HEK293 = Human embryonic kidney 293 cells).
Response 2: HEK293 is not a prostatic cancer cell line. Due to the high transfection efficiency of HEK293 cells, we overexpressed GLI1/2 and DAX1 to confirm protein binding. In addition, we utilized a luciferase reporter vector containing a GLI-binding site to investigate the potential regulation of GLI1 and GLI2 activity by DAX1 in HEK293 cells. However, it is worth noting that we used HEK293 cells in our study, which may be considered a limitation as they are not prostate cancer cell. Nonetheless, when we overexpressed and knocked down DAX1 in DU145 cells, we obtained clear evidence that DAX1 suppresses the expression of the GLI1/2 target gene.
Point 3: Figure 1: The effect of DAX1 on the expression of Smoothened should be investigated by the authors, too. Furthermore, western blots should be performed to support the findings of the mRNA expression.
Response 3: The reviewer made a valid point that we should have investigated whether DAX1 has an effect on SMOs, which are upstream of the Hedgehog signal. However, we couldn't evaluate the effect of DAX1 on the expression of Smoothened.
Since GLI1/2 is a transcription factor, we thought it would be sufficient to show that DAX1 represses the target gene of GLI1/2 at the transcriptional level. In order to ensure the accuracy of our results, it would have been necessary to confirm any changes in protein expression levels. Unfortunately, due to the limited remaining revision period, it was difficult to carry out the necessary verification.
Point 4: Figure 2: The co-localization/merged images should be presented in higher magnification.
Response 4: According to the reviewer’s suggestion, we corrected the Figure2C.
Point 5: Figure 3: Resolution of the graphics in Fig 3A is poor.
Response 5: According to the reviewer’s suggestion, we corrected the Figure3A.
Point 6: Figure 4: Resolution of the graphics in Fig 4B is poor. The cell density should be quantified for further statistical analysis. The proliferation should be studied by cell blocks and by consecutive immunohistochemistry with Ki-67 antibody.
Response 6: According to the reviewer’s suggestion, we corrected the Figure4B,4C.
However, The suggested experiment by the reviewer to study proliferation using cell blocks and consecutive immunohistochemistry with Ki-67 antibody could not be performed due to the short revision period.
Point 7: Discussion, The authors could demonstrate the DAX-GLI-axis in the chosen experimental in-vitro setting. Nevertheless, the protein expression of DAX1 and GLI in different prostatic cancer specimen should be investigated. Overall, more question than answers are evident by the submitted manuscript. Therefore, the authors should mentioned the limitations of the investigations (no in-vivo or in-situ experiments). The authors should discuss how the findings could be definitely transferred from a theoretical to a practical view (especially in the clinical setting (drug-development, drug-combination)) in the future.
Response 7: According to the reviewer’s suggestion, we corrected the manuscript (Line 466-476).
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIn this study, the authors tried to identify proteins that directly regulate the activity of GLI proteins in prostate cancer cells. The authors concluded that DAX1 strongly repressed the transcriptional activity of GLI1 and GLI2, thereby causing an inhibition of the Hh signaling pathway in prostate cancer cells.
Comments:
The reviewer has some concerns as follows:
1. In this study, the authors drew an exaggerated conclusion. There are no evidence to support that “DAX1 strongly repressed the transcriptional activity of GLI1 and GLI2, thereby causing an inhibition of the Hh signaling pathway in prostate cancer cells”. The authors found that DAX1 interacted with GLI1 and GLI2 in HEK293 cells (an embryonic kidney cell line), but not DU145 prostate cancer cells.
2. Only one prostate cancer cell line was used in this study, which made the results unreliable.
3. The method for immunoprecipitation can be described in the Methods section.
4. Figure 1A and 1B showed the DAX1 and Hh target gene expression levels in DU145 cells. How about the protein expression for DAX1 and Hh signaling molecules?
5. Figure 3 showed the DAX1 overexpression induces G1/S cell cycle arrest in prostate cancer cells. How about the effect of endogenous DAX1 on cell cycle? Can DAX1 SiRNA affect the effect of Smoothened agonist (SAG) on cell cycle?
6. In Figure 4B, the images for colony formation are really unclear. It can be revised.
7. How about the effect of endogenous DAX1 on the proliferation and growth of prostate cancer cells? Can DAX1 SiRNA affect the proliferation and growth of prostate cancer cells?
8. In general, the presented results cannot support the conclusions.
Author Response
We thank the Reviewer #2 for insightful and positive comments.
Detailed point-by-point answers to comments are provided below.
Point 1. In this study, the authors drew an exaggerated conclusion. There are no evidence to support that “DAX1 strongly repressed the transcriptional activity of GLI1 and GLI2, thereby causing an inhibition of the Hh signaling pathway in prostate cancer cells”. The authors found that DAX1 interacted with GLI1 and GLI2 in HEK293 cells (an embryonic kidney cell line), but not DU145 prostate cancer cells.
Response 1: Reviewer 2 points out a valid point. Due to the high transfection efficiency of HEK293 cells, we overexpressed GLI1/2 and DAX1 to confirm protein binding. In addition, we utilized a luciferase reporter vector containing a GLI-binding site to investigate the potential regulation of GLI1 and GLI2 activity by DAX1 in HEK293 cells. However, it is worth noting that we used HEK293 cells in our study, which may be considered a limitation as they are not prostate cancer cell. Nonetheless, when we overexpressed and knocked down DAX1 in DU145 cells, we obtained clear evidence that DAX1 suppresses the expression of the GLI1/2 target gene.
Point 2: Only one prostate cancer cell line was used in this study, which made the results unreliable.
Response 2: It appears that Reviewer 2 has raised a valid point. The limitation of this study is that only one prostate cancer cell line was utilized, which may compromise the reliability and generalizability of the results.
Point 3: The method for immunoprecipitation can be described in the Methods section.
Response 3: According to the reviewer’s suggestion, we corrected the manuscript (Line 242-257).
Point 4: Figure 1A and 1B showed the DAX1 and Hh target gene expression levels in DU145 cells. How about the protein expression for DAX1 and Hh signaling molecules?
Response 4: Unfortunately, we couldn't evaluate the protein levels of DAX1 and Hh signaling molecules.
Point 5: Figure 3 showed the DAX1 overexpression induces G1/S cell cycle arrest in prostate cancer cells. How about the effect of endogenous DAX1 on cell cycle? Can DAX1 SiRNA affect the effect of Smoothened agonist (SAG) on cell cycle?
Response 5: Unfortunately, the impact of endogenous DAX1 on the cell cycle was not assessed in this study. Additionally, the effect of DAX1 siRNA on the impact of Smoothened agonist (SAG) on the cell cycle was not investigated.
Point 6: In Figure 4B, the images for colony formation are really unclear. It can be revised.
Response 6: According to the reviewer’s suggestion, we corrected the manuscript (Figure 4B)
Point 7: How about the effect of endogenous DAX1 on the proliferation and growth of prostate cancer cells? Can DAX1 SiRNA affect the proliferation and growth of prostate cancer cells?
Response 7: Unfortunately, we could not measure "the effect of endogenous DAX1 on the proliferation and growth of prostate cancer cells". Moreover, the impact of DAX1 siRNA on the proliferation and growth of prostate cancer cells was not measured.
Point 8: In general, the presented results cannot support the conclusions.
Response 8: There may be limitations to the findings presented in this study
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for Authors Title: “Inhibition of GLI transcriptional activity and prostate cancer cell growthand proliferation by DAX1”
Authors: Sung Pyo Hong, Kil Won Kim, Soon Kil Ahn
Summary:Here, the authors present work focused on identifying proteins that can directly regulate GLI protein activity. To this end, they have studied different prostate cancer cell lines using gene expression and reporter assays. This will allow them to reveal a new molecular mechanism by which Hh signaling is regulated in prostate cancer cells, opening up potential therapeutic opportunities. This manuscript is an interesting piece of work, however, the missing information must be filled in, and if it is not available, the limitations of the available results must be discussed in detail.
Several major points are listed below:
1: The abstract is too short and lacks content and should be rewritten as it seems unstructured.
2: The novelty of the article should be clearly emphasized throughout.
3: The introduction is too short and uniform. Much more should be written about GLs and DAX1.
4: Material and methods are very short and incomplete.
5: The results can be displayed and described even more clearly.
6: The search strategy used for the literature review should be stated.
7: The limitations associated with the use of DAX1 should be discussed.
8: I miss images, enlargements, bars, letters in Figure 2. The quality of the images is not sufficient.
9: All abbreviations should be defined at their first mention and used thereafter.
10: Please use consistent abbreviations throughout the manuscript.
11: The paper contains many complicated abbreviations. A list of the most important abbreviations would be helpful to the reader.
12: The English language in general needs improvement. The entire text needs to be revised by a native English speaker.
Author Response
We thank the Reviewer #3 for insightful and positive comments.
Detailed point-by-point answers to comments are provided below.
Point 1. The abstract is too short and lacks content and should be rewritten as it seems unstructured.
Response 1: According to the reviewer’s suggestion, we corrected the abstract in the manuscript.
Point 2: The novelty of the article should be clearly emphasized throughout.
Response 2: According to the reviewer’s suggestion, we corrected the manuscript. (Line 114-125).
Point 3: The introduction is too short and uniform. Much more should be written about GLs and DAX1.
Response 3: According to the reviewer’s suggestion, we corrected the manuscript. (Line 49-57) (Line 76-83) (Line 87-103).
Point 4: Material and methods are very short and incomplete.
Response 4: According to the reviewer’s suggestion, we corrected the “Material and methods” in the manuscript. (Line 171-179) (Line 185-191) (Line 242-258).
Point 5: The results can be displayed and described even more clearly.
Response 5: According to the reviewer’s suggestion, we corrected the manuscript.
Point 6: The search strategy used for the literature review should be stated.
Response 6: According to the reviewer’s suggestion, we corrected the manuscript.
(Line 76-83) (Line 92-103)
The search strategy used for our literature review was as follows.
Most small molecule inhibitors targeting the Hedgehog pathway are designed to inhibit SMO, making them ineffective against cancers that arise from mutations downstream of SMO or GLI1/GLI2 overexpression. Researchers are now exploring inhibitors that target GLI1/GLI2 directly to overcome this limitation.
DAX1 can affect the activity of several nuclear receptors. By influencing the activity of these receptors, DAX1 can impact their ability to bind to DNA and regulate target gene expression, emphasizing its crucial role as a co-regulator protein in gene expression regulation.
Point 7: The limitations associated with the use of DAX1 should be discussed.
Response 7: According to the reviewer’s suggestion, we corrected the manuscript (Line 454-465).
Point 8: I miss images, enlargements, bars, letters in Figure 2. The quality of the images is not sufficient.
Response 8: According to the reviewer’s suggestion, we corrected the Figure2C.
Point 9: All abbreviations should be defined at their first mention and used thereafter.
Response 9: According to the reviewer’s suggestion, we corrected the manuscript.
Point 10: Please use consistent abbreviations throughout the manuscript.
Response 10: According to the reviewer’s suggestion, we corrected the manuscript.
Point 11: The paper contains many complicated abbreviations. A list of the most important abbreviations would be helpful to the reader.
Response 11: According to the reviewer’s suggestion, we corrected the manuscript. (Line 485-489).
Point 12: The English language in general needs improvement. The entire text needs to be revised by a native English speaker.
Response 12: According to the reviewer’s suggestion, we again received the English editing service
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsSpecific comments to the authors
In the revised version of the manuscript, the authors have attempted to address the previously mentioned concerns, but the responses to points 2, 3 and 6 are not adequate and convincing. Therefore, I suggest that the revised manuscript "Inhibition of GLI transcriptional activity and prostate cancer cell growth and proliferation by DAX1" should not be accepted.
Reviewer 3 Report
Comments and Suggestions for AuthorsNo further comments. This revised manuscript can be accepted.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe authors have satisfactorily addressed the concerns raised in the original version. The revised version is significantly improved. No further concerns.
