Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells

Julie Pratibha Singh; Manisha Dagar; Gunjan Dagar; Sudhir Kumar; Sudhir Rawal; Ravi Datta Sharma; Rakesh Kumar Tyagi; Gargi Bagchi

doi:10.1371/journal.pone.0226056

Peer Review History

Original SubmissionNovember 18, 2019
14 Jan 2020 Decision Letter - MOHAMMAD Saleem, Editor PONE-D-19-31997 Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells PLOS ONE Dear Dr. Gargi Bagchi: 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. Reviewer #1: Singh and coworkers conducted a study entitled “Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells” in which the authors indicated that androgens activate Rho-signaling pathway through the activation of GPR56. GPR56 knockdown blocks the nuclear translocation of AR and its downstream transcription factors. To validate the in vitro studies, GPR56 was upregulated in prostate cancer (PCa) tumor specimens when compared with normal tissues. General comments: The manuscript was well-designed, technical aspects used in this study are appropriate, results are supported with enough data and the size of the text is reasonable. However, I have some few comments need be addressed in the current study. - Using LNCaP as a model is appropriate here but PC-3 representing CRPC cells should be also included as a control. - Examining the expression of GPR56 transcript in prostate cancer tissue is not enough because posttranslational modification can change the whole story. It is better to stain the tissue with GPR56 specific antibody. In addition, the authors showed that GPR56 triggers cytosolic translocation of AR using immunofluorescence, and it would be more believable if the authors use cell fractionation to validate the results. Specific comments: - In the abstract: Line 4, the phrase of “a role for these in CRPC” should be fixed. Please include bioinformatics results of AR-ligand similarity and the number of examined PCa tissues (25 tissue samples) in the abstract. - In Methods section, please replace (u: micron) with Latin symbol (µ). Clinical characteristics of human prostate cancer samples are needed. - In results section, all figures need an improvement because of quality was very poor. Figure legends must be included together after the MS text not included in the results. In results, page 14, please replace S 1 Fig. with Supl. Fig. 1. Statistical analysis should be included in Figure 7A&B. In Fig. 7C, quantification of band densities will improve the results of GRP56 in tissues samples. - In discussion section, page 21, “Cancerous prostate” should be replaced. I suggest to move Figure 8 A&C and also Tables 3&4 to the results section (The Authors should discuss the results but not to include new results). At the end of discussion, summary or conclusion should be include in which you summarize your findings. Discussion should be more concise, focus and comprehensive. - The manuscript should be edited again because it has several typos and incorrect structures. Reviewer #2: In the current manuscript titled “Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells” demonstrates the role of GPR56 in regulating AR downstream signaling. The authors have demonstrated how binding of the testosterone to the GPR56 can activate PKA and Rho signaling promoting AR nuclear translocation demonstrating crosstalk between two signaling pathways. However, following concerns need to be addressed prior to publication. 1- Since CRPC occurs in patients having very low levels of testosterone it does not explain how GPR56 can lead to CRPC which the authors are claiming (the docking score for AR is much higher for testosterone than GPR56, table 2) this needs to be clarified in the discussion. 2- AR receptor signaling studies require the testosterone induction to be performed in phenol red free charcoal stripped serum media, authors have used serum starvation instead that is not ideal for these studies. 3- The authors need to demonstrate if GPR56 siRNA can block AR nuclear localization using cell fractionation studies. 4- Fig3H-The PSA levels go down after testosterone treatment whereas GPR56 expression go up. Testosterone upregulates AR signaling and PSA expression, authors need to explain this abnormal result. Also they need to show effect on another AR regulated gene. Minor concerns: 1- Authors have included the figure legends in between the manuscript text without the figure that is not reader friendly. 2- Constructs- is it at the HindIII site or between HindIII and another restriction enzyme? 3- The GTP Rho pull down experiment not described properly in methods section. 4- Was the cyclic AMP assay performed 2-4 hrs after serum starvation or after 24 hrs of starvation? 5- Page 15, correct that GPR56 protein is not expressed in HEK cells 6- Fig 3F-Is the control scrambled siRNA? 7- Rephrase “androgen transcription” to AR downstream signaling, authors are not looking into AR mRNA levels. 8- Fig4A-PKAsiRNA set is still showing response to ISO. 9- Fig 7- provide a western blotting with N and C terminal GPR56 antibodies. We would appreciate receiving your revised manuscript by Feb 28 2020 11:59PM. 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Revision 1
4 Mar 2020 Author Response Dear Editor, Thank you for considering our article. Also, thanks to the reviewers who have read the manuscript meticulously and provided constructive comments. According to the reviewers’ suggestions, we have now performed more experiments and have included them in the manuscript text and figures. The pointwise response is also included below. Reviewer #1: Singh and coworkers conducted a study entitled “Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells” in which the authors indicated that androgens activate Rho-signaling pathway through the activation of GPR56. GPR56 knockdown blocks the nuclear translocation of AR and its downstream transcription factors. To validate the in vitro studies, GPR56 was upregulated in prostate cancer (PCa) tumor specimens when compared with normal tissues. General comments: The manuscript was well-designed, technical aspects used in this study are appropriate, results are supported with enough data and the size of the text is reasonable. However, I have some few comments need be addressed in the current study. 1. - Using LNCaP as a model is appropriate here but PC-3 representing CRPC cells should be also included as a control. Response: The PC3 cell line has now been used along with LNCaP cells for performing Luciferase assay and MTT assay and the results have now been included in the text and figures ( 3H and 8C). It is important to note that the response in PC3 cells is similar to that of LNCaP. 2. Examining the expression of GPR56 transcript in prostate cancer tissue is not enough because posttranslational modification can change the whole story. It is better to stain the tissue with GPR56 specific antibody. Response: To demonstrate the changes in GPR56 expression at the protein level in normal versus tumour tissues, Western Blot analysis has been performed using normal and tumour tissues using GPR56 antibody. The result is now included as Figure 8A and the intensity quantitation is depicted as Fig. 8B. The expression of GPR56 is significantly higher at the protein level in all tumour tissues analysed. 3.In addition, the authors showed that GPR56 triggers cytosolic translocation of AR using immunofluorescence, and it would be more believable if the authors use cell fractionation to validate the results. Response: As per the reviewer’s suggestion, we have now performed Western Blot experiment using nuclear and cytoplasmic fractions of LNCaP cells transfected using scrambled siRNA or GPR56 siRNA, and probed using anti-AR antibody and the results have been included as Fig. 6C. The results clearly demonstrate that in LNCaP cells expressing GPR56, the AR is in the cytoplasm in untreated cells, but moves to the nucleus on testosterone treatment, whereas in the cells transfected using GPR56 siRNA, upon testosterone stimulation, a significant proportion of AR continues to reside in the cytoplasm and does not migrate into the nucleus. Specific comments: - In the abstract: Line 4, the phrase of “a role for these in CRPC” should be fixed. Response: This sentence has now been corrected. Please include bioinformatics results of AR-ligand similarity and the number of examined PCa tissues (25 tissue samples) in the abstract. Response: The details have now been included in the Abstract. - In Methods section, please replace (u: micron) with Latin symbol (µ). Response: Corrected Clinical characteristics of human prostate cancer samples are needed. Response: Clinical Characteristics of patients have now been included in the M and M section as Supplementary Table 1. - In results section, all figures need an improvement because of quality was very poor. Response: High resolution images for all figures have now been included in Tiff format 300 Dpi. Figure legends must be included together after the MS text not included in the results. Response: Figure legends have now been included after the MS text. In results, page 14, please replace S 1 Fig. with Supl. Fig. 1. Response: Corrected Statistical analysis should be included in Figure 7A&B. In Fig. 7C, quantification of band densities will improve the results of GRP56 in tissues samples. Response: Statistical Analysis has now been included for Fig. 7A, B and C and quantification of band densities has also been included for Fig. 7D. - In discussion section, page 21, “Cancerous prostate” should be replaced. Response: Has been replaced. I suggest to move Figure 8 A&C and also Tables 3&4 to the results section (The Authors should discuss the results but not to include new results). Response: Figures 8A and C have now been moved to Results section as Fig. 3A and Fig. 8C. Tables 3 and 4: Have now been moved to Results Section At the end of discussion, summary or conclusion should be include in which you summarize your findings. Response: A Summary section has now been included. Discussion should be more concise, focus and comprehensive. - The manuscript should be edited again because it has several typos and incorrect structures. Response: We have edited the MS again to correct typos. Reviewer #2: In the current manuscript titled “Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells” demonstrates the role of GPR56 in regulating AR downstream signaling. The authors have demonstrated how binding of the testosterone to the GPR56 can activate PKA and Rho signaling promoting AR nuclear translocation demonstrating crosstalk between two signaling pathways. However, following concerns need to be addressed prior to publication. 1- Since CRPC occurs in patients having very low levels of testosterone it does not explain how GPR56 can lead to CRPC which the authors are claiming (the docking score for AR is much higher for testosterone than GPR56, table 2) this needs to be clarified in the discussion. Response: While in CRPC testosterone levels are reduced, yet the expression of most GPCRs and their ligands are significantly enhanced (Daaka, 2005). Increase in expression of GPR56 in tumour samples over their normal counterparts has been demonstrated in this study (Fig.7 and 8) and through TCGA analysis of previous studies (Fig. 8C). Increased expression of GPR56 via any means is likely to result in increased activation of PKA, which would result in increased nuclear migration of AR due to phosphorylation of HSP90 (Dagar et al, 2019). This would result in enhanced cell proliferation in low/no testosterone conditions as observed in CRPC. This has now been explained more clearly in Discussion section in _______ 2- AR receptor signaling studies require the testosterone induction to be performed in phenol red free charcoal stripped serum media, authors have used serum starvation instead that is not ideal for these studies. Response: In literature, some studies have used serum starvation while others have used charcoal stripped serum. We repeated few of the experiments in Charcoal stripped serum to verify that the results were similar to those obtained with serum starvation. These results are now included as Supplementary Figure 2. 3- The authors need to demonstrate if GPR56 siRNA can block AR nuclear localization using cell fractionation studies. Response: This has now been included in the text and Results section as Figure 6C. 4- Fig3H-The PSA levels go down after testosterone treatment whereas GPR56 expression go up. Testosterone upregulates AR signaling and PSA expression, authors need to explain this abnormal result. Also they need to show effect on another AR regulated gene. Response: To verify the effect of GPR56 knockdown, LNCaP cells were transfected with GPR56 siRNA or scrambled and expression levels of genes such as PSA and GPR56 was analysed. This experiment has now been repeated to verify the results and expression of both PSA and GPR56 are upregulated upon Testosterone treatment. Expression of another androgen target gene, namely TMPRSS2 was also analysed and found to increase upon Testosterone treatment. Expression of all the three genes were negligible in LNCaP cells transfected using GPR56 siRNA. This has now been included in text, page number 16. Minor concerns: 1- Authors have included the figure legends in between the manuscript text without the figure that is not reader friendly. Response: Figure legends have now been included at the end of the text. 2- Constructs- is it at the HindIII site or between HindIII and another restriction enzyme? Response: It is at the Hind III site and has been corrected in the M and M section. 3- The GTP Rho pull down experiment not described properly in methods section. Response: Has now been explained in detail. 4- Was the cyclic AMP assay performed 2-4 hrs after serum starvation or after 24 hrs of starvation? Response: This has now been corrected in the M and M section regarding cAMP assay. 5- Page 15, correct that GPR56 protein is not expressed in HEK cells Response: This has now been corrected and included on page 15. 6- Fig 3F-Is the control scrambled siRNA? Response: The results were similar in both LNCaP cells wildtype and that transfected with scrambled GPR56 siRNA. In the figure results for LNCaP cells transfected using scrambled GPR56 has been included. 7- Rephrase “androgen transcription” to AR downstream signaling, authors are not looking into AR mRNA levels. Response: Has now been rephrased. 8- Fig4A-PKAsiRNA set is still showing response to ISO. Response: The experiment with Isoproterenol has been repeated and the results of PKA activity have been revised. 9- Fig 7- provide a western blotting with N and C terminal GPR56 antibodies. Response: The Western Blot experiment has now been performed using GPR56 N-terminal antibody (C-terminal antibody was not available at short notice) and the results have been modified accordingly in text and included as Fig. 8A. Attachments Attachment Submitted filename: Plos one - Response to reviewers comments (1).docx https://doi.org/10.1371/journal.pone.0226056.r002
2 Apr 2020 Decision Letter - MOHAMMAD Saleem, Editor PONE-D-19-31997R1 Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells PLOS ONE Dear Dr Bagchi, 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. We would appreciate receiving your revised manuscript by May 17 2020 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, M. Saleem 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 Reviewer #2: (No Response) ******** 2. 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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 Reviewer #2: 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 Reviewer #2: 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: (No Response) Reviewer #2: Authors have addressed most of the comments; I recommend a minor revision for figure 6C. The authors have used GAPDH for loading control instead; they need to use lamin or PCNA as nuclear marker and GAPDH/tubulin as cytoplasmic marker. The presence of GAPDH in nuclear fraction indicates that the fractions are not properly separated. ****** 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 Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0226056.r003
Revision 2
4 Jul 2020 Author Response Dear Editor, We thank you for the review of the article. We are happy to see that the reviewers were satisfied on almost all accounts. As per the request of Reviewer 2, point 6, a fresh Western blot experiment has been performed, where presence of androgen receptor (AR) in the cytoplasmic and nuclear fraction was probed. As control we have probed the blot using both GAPDH (cytoplasmic marker) and Histone H3B (nuclear marker). While GAPDH expression is observed in cytoplasmic fractions only, Histone H3B expression was restricted to the nucleus. Accordingly, changes have been made in the text and the figure has been replaced. In addition, the name of Mr. Sudhir Kumar has been added to the list of authors as he helped us in performing the last experiment. Also, the Histone H3B antibody was a kind gift from Dr. Rakesh Saxena whose name has now been added in acknowledgement and Materials and Methods. Hope the manuscript is acceptable to you in its current form. Thanks, and regards, Gargi Bagchi Attachments Attachment Submitted filename: Rebuttal Letter 2nd July.docx https://doi.org/10.1371/journal.pone.0226056.r004
10 Jul 2020 Decision Letter - MOHAMMAD Saleem, Editor Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells PONE-D-19-31997R2 Dear Dr. Gargi Bagchi We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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. Kind regards, Mohammad Saleem Academic Editor-PLOS ONE, Masonic Cancer Center, University of Minnesota-Minneapolis https://doi.org/10.1371/journal.pone.0226056.r005
Formally Accepted
6 Aug 2020 Acceptance Letter - MOHAMMAD Saleem, Editor PONE-D-19-31997R2 Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells Dear Dr. Bagchi: I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. MOHAMMAD Saleem Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226056.r006

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