Expression profile of sonic hedgehog signaling-related molecules in basal cell carcinoma

Hye Sung Kim; Young Sil Kim; Chul Lee; Myung Soo Shin; Jae Wang Kim; Bo Gun Jang

doi:10.1371/journal.pone.0225511

Peer Review History

Original SubmissionAugust 26, 2019
9 Oct 2019 Decision Letter - Amanda Ewart Toland, Editor PONE-D-19-24006 Expression profile of Sonic hedgehog signaling-related molecules in basal cell carcinoma PLOS ONE Dear Dr Jang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Many of the points to be addressed are in the attached comments. 1. The authors should include in their interpretation of results and in the discussion consideration that GLI1 and GLI2 can be full-length or cleaved which impacts their function. Details on where in the protein antibodies bind (and implications for protein function) should be included. (A reminder that full length westerns need to be included in the supplemental materials). 2. More discussion on how their results fit into previous findings of increased SMO levels in BCC, differences in approaches or why there may be differences should be considered. (See reviewer's comments). 3. Address reviewer 1's queries about correlations between stem cell markers and HH-related molecules. 4. Fix the graphs and figures per reviewer 1's suggestions. 5. In the methods and results clarify the details on number of samples that were used for each analysis. 6. Respond to the additional points raised by the reviewer. 7. Proofread the manuscript carefully and fix the typographical errors that were identified by the reviewer. We would appreciate receiving your revised manuscript by Nov 23 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Amanda Ewart Toland, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Additional Editor Comments (if provided): [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript reveals compelling evidence about mRNA expression levels of Hedgehog pathway components (GLI1, GLI2, PTCH1, PTCH2, SHH and SMO) in different skin tumors and the expression profile observed was compared to stem cells markers (LGR4, LGR5, LGR6, and LRIG1) content. Also, protein expression and subcellular localization was studied by IHC technique. In this setting, Gli1 was found overexpressed in BCC at mRNA and protein levels. The manuscript reveal evidence that support the conclusions but some points need to be solved to be published. Please, answer the questions (provided in the attach file) in a "point by point" manner. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: PONE-D-19-24006 revision comments.docx https://doi.org/10.1371/journal.pone.0225511.r001
Revision 1
18 Oct 2019 Author Response We highly appreciate the reviewer’s careful evaluation of our manuscript and constructive comments. We agree with the reviewer and tried our best to revise our manuscript in order to address all the concerns raised. The manuscript reveals compelling evidence about mRNA expression levels of Hedgehog pathway components (GLI1, GLI2, PTCH1, PTCH2, SHH and SMO) in different skin tumors and the expression profile observed was compared to stem cells markers (LGR4, LGR5, LGR6, and LRIG1) content. Also, protein expression and subcellular localization was studied by IHC technique. In this setting, Gli1 was found overexpressed in BCC at mRNA and protein levels. - In fact, some discrepancy was shown between both RNA and protein expression pattern in the other HH components. This is particularly important in GLI1 and GLI2, since both proteins can be found as activator (full-length) as well as repressor (proteolytically cleaved) forms. The authors have to take this consideration when interpret and discuss your IHC results. : The dual role of GLI2 and GLI3 as activator and repressor is included in the Introduction (In page3, line 20-23), and its significance in IHC result has been discussed in Discussion (In page9, line 7-9). In page3, line 20-23: “GLI1 appears to exclusively act as a transcriptional activator, whereas GLI2 and GLI3 can display both activator and repressor functions (9). In the absence of upstream signal, GLI3, and to a lesser degree GLI2, are proteolytically cleaved and play a role of transcriptional repressors (10).” In page9, line 7-9: “For example, the lower GLI2 protein expression may be in part associated with the high turnover rate considering the fact that GLI2 can be proteolytically processed into the truncated-repressor form in the absence of HH ligands.” � Additionally, the authors not found increased SMO mRNA level in BCC, when have been recently reported increases in this messenger in BCCs from Gorlin syndrome, even after adjustment by germinal and somatic mutation carrier status (Martinez MF; Cells, 2019). This is also interesting to include when this particular issue is discussed. : As suggested, the discrepancy in SMO expression in BCC between studies has been discussed and included in the revised manuscript (In page 8, line 20-25). “Interestingly, SMO transcription was not altered in BCCs, whereas Martinez et al. recently showed an increased mRNA expression of SMO in BCCs from 20 nevoid basal cell carcinoma syndrome (NBCCS) patients mainly caused by PTCH1 gene mutations (24). This discrepancy is probably due to the different study groups because our study only includes sporadic BCCs that have different genetic profiles from those of NBCCS patients even though both BCCs share a common signaling abnormality.” � The correlation analysis between HH-related molecules and stem cells markers results challenging to this reviewer: � . In result text (page 8, lines 2-5), the correlation scores and P values for GLI1 probably correspond to the GLI2 (see Figure 6A and 7). Please, check the data and correct both values and its interpretation. � : The error in the result text has been fixed as pointed out. � � . Why the correlation of SMO and SHH mRNA levels with LGR4, LGR5, LGR6, and LRIG1 are missing? This issue need to be solved to understand overall relationship between HH pathway and stem cells markers. � : The correlation of SHH mRNA with stem cell markers has been included in the Figure 7 and result and figure 7 legend has been fixed. However, the SMO mRNA levels are too low to analyze the correlation. � Some bars in the graphs, such as the EP in Figure 2E and the GLI IHC of Figure 5B, have no dispersion. Check carefully the data presentation throughout the manuscript. : There is no error bar in the IHC scores of EP because all values are same as 3. � The figure 6B are confusing about the methodology used: • . If the authors try to show the in situ hybridization of GLI1 and LRG5, no mention concerning GLI1 in methods were found. • . If the authors try to show IHC of GLI1 and LRG5 proteins, the entire legend of figure 6 need to be rewritten to clearly state which variable is represented in each case. : LGR5 staining was performed by RNA in situ hybridization because there is no reliable antibody to LGR5 for immunohistochemical anaylsis with FFPE specimens, whereas GLI1 expression was detected by IHC. As suggested, the legend of figure 6 was rewritten to clarify the methodology. “Fig. 6 Correlations of GLI1 expression with other stem cell markers in basal cell carcinomas. (A) Scatter plots with regression lines showing the correlations between GLI1 and stem cell marker mRNA expression. (B) Representative case of basal cell carcinoma expressing both LGR5 mRNA and GLI1 protein (RNA in situ hybridization for LGR5 and immunohistochemistry for GLI1).” � Would important to add a comment in Introduction about the reason/s to study the stem cell markers and its relationship with HH pathway. : As suggested, the reason to study the relationship between HH pathway and stem cell markers has been included in the Introduction. (Page4, line 2-5) Page4, line 2-6: “Although the precise cellular origin of BCC has been controversial, recent studies have demonstrated that BCC-like tumors can arise from multiple hair follicle (HF) stem cell populations (15-17). Since several distinct stem cell markers of HF have been identified by lineage-tracing experiments, we also assessed the correlation of HH molecules with the established markers including LGR4, LGR5, LGR6, and LRIG1.” � The amount of samples studied are confusing. In the first paragraph of subjects section of Materials and methods stated “…152 formalin-fixed, paraffin-embedded (FFPE) human skin tissues (normal skin, n = 5; skin tumors, n = 147)…” but in the next sentence and in Tissue microarray construction declared 168 skin tumors. Is important to correct and uniform the number of samples studied. In addition, since the clinicopathological features of patients are not analyzed, its mention in the text is unnecessary. : The tumor number has been changed from 147 to 168 and the sentence of clinicopathological features has been removed. � The number of samples of each subtype of BCC have to be included in figure 5B. : As suggested, the number of samples have been included. � The final sentence of Discussion point out the relationship between HH and Wnt signaling in BCCs, but this affirmation are not supported by the results. Then, would more appropriate a suggestion of link between both pathways. : As suggested, the sentence of “indicating a functional relationship between~” has been replaced by “suggesting a link between~” Minor changes: - Second paragraph of Introduction, line 2: “…12transmembrane receptor for SHH that has a regulatory effect on the pathway (8). in the absence of…” replace by “…12 transmembrane receptor for SHH that has a regulatory effect on the pathway (8). In the absence of…” - In page 5, line 4 replace Anti-PTCH2 by anti-PCTH2. - In page 6, last paragraph, include HDA samples in “SHH expression was slightly higher in several tumors including BCC, TE, EP, and SPA (Fig. 1E)” - In page 7, close de parenthesis in “…PMC (n = 13)…” - In page 8, line 5, replace PTC2 by PTCH2. : All minor errors have been fixed in the revised manuscript. https://doi.org/10.1371/journal.pone.0225511.r002
5 Nov 2019 Decision Letter - Amanda Ewart Toland, Editor PONE-D-19-24006R1 Expression profile of Sonic hedgehog signaling-related molecules in basal cell carcinoma PLOS ONE Dear Dr Jang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. 1. Address the writing for page 8, lines 7-8 as specified in reviewer 1's attachment. 2. Ensure that when a gene/RNA is being referred to that the name is in italics (e.g. for in RNA in situs). Protein names are not italicized. We would appreciate receiving your revised manuscript by Dec 20 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Amanda Ewart Toland, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The revised version of the manuscript has been carefully checked and the author’s responses are satisfactory and greatly improve the overall quality of the paper. In this version, only one issue has to be corrected (please, see the attached file). ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Reviewer response to PONE-D-19-24006R1.docx https://doi.org/10.1371/journal.pone.0225511.r003
Revision 2
5 Nov 2019 Author Response We appreciate the careful evaluation of our manuscript and revised our manuscript to address the issue raised by reviewer and editor. 1. The revised version of the manuscript has been carefully checked and the author’s responses are satisfactory and greatly improve the overall quality of the paper. In the view of this version, only one issue has to be corrected: In page 8, lanes 7-8, “… GLI1 expression showed a strong positive correlation with LGR4 (r2=0.62, P < 0.001), LGR5 (r2=0.60, P < 0.001), and LGR6 7 (r2=0.21, P = 0.05), but not with LRIG1…” needs to be rewrite as “… GLI1 expression showed a strong positive correlation with LGR4 (r2=0.62, P < 0.001), LGR5 (r2=0.60, P < 0.001), but not with LGR6 (r2=0.21, P = 0.05) and LRIG1…” . As is stated in statistical analysis section, “A P-value < 0.05 was considered statistically significant”, both LGR6 and LRIG1 have to be considered not significant. : As suggested, the sentence has been rewritten in the revised manuscript. Attachments Attachment Submitted filename: Rebuttal letter.doc.docx https://doi.org/10.1371/journal.pone.0225511.r004
7 Nov 2019 Decision Letter - Amanda Ewart Toland, Editor Expression profile of Sonic hedgehog signaling-related molecules in basal cell carcinoma PONE-D-19-24006R2 Dear Dr. Jang, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Amanda Ewart Toland, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0225511.r005
Formally Accepted
12 Nov 2019 Acceptance Letter - Amanda Ewart Toland, Editor PONE-D-19-24006R2 Expression profile of Sonic hedgehog signaling-related molecules in basal cell carcinoma Dear Dr. Jang: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Amanda Ewart Toland Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0225511.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .