α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling

Murat Oz; Justin R. King; Keun-Hang Susan Yang; Sarah Khushaish; Yulia Tchugunova; Maitham A. Khajah; Yunus A. Luqmani; Nadine Kabbani

doi:10.1371/journal.pone.0289098

Peer Review History

Original SubmissionMay 17, 2023
7 Jun 2023 Decision Letter - Israel Silman, Editor PONE-D-23-15038α7 nicotinic acetylcholine receptor interaction with G proteins mediates breast cancer cell proliferation, motility, and calcium signalingPLOS ONE Dear Dr. Kabbani, 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. Please submit your revised manuscript by Jul 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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. 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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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: 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 Reviewer #2: No ****** 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 Reviewer #2: 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 experiment in figure 7B should be repeated with a control group for α7345-348a that was not exposed to choline to show that transfection with α7345-348a does not alter proliferation by itself. The results in Figure 4 are not mentioned in the abstract or the discussion. It would be good if the authors discuss how the increase in α7 nAChR expression might affect the proliferation, motility, and/or calcium signaling of cancer cells. Additionally, the authors did not show if these effects could be blocked by MLA or another antagonist, or whether this upregulation is related to g-protein mediated signaling via α7 nAChRs. Further experiments to answer these questions would help to tie these results into the rest of the paper. YM 254890 is mentioned in the abstract as a G protein blocker, but in the results section of the manuscript, the authors never define what specifically YM 254890 is or why it is being used. The proliferation assay in Figure 7 plots Cell count x 105, whereas the other proliferation assays measure Net OD. Could the authors please explain why this assay was quantified differently? There are two “B” panels in figure 5 The deltaF/F scales are not consistent across figures. For example, in Figure 5, A and C plot deltaF/F (x100), whereas panel B1 writes out the 100s, and the second panel B just has deltaF/F without the x100 clarification. Similarly, in Figure 7, panel A, the first graph is x100, while the second graph is not. Scale bars should be added to Figure 3A and Figure 4A Scale bar should be defined in Figure 7B. In the figure legend for Figure 6 it is noted that the “Asterix denotes significant difference from control,” whereas the figure seems to indicate that the Asterix represents significant difference from the choline treated group. Could the authors clarify what comparisons are being measured in this figure, and what is significantly different from what? The figure legend for Figure 2 states “Asterix denoted significant difference from control” but the figure seems to indicate that significance is measured as difference from the choline group. Figure 7 has only 2 panels, A and B, but the figure legend has A, B, and C. Figure 7 states “statistical significance relative to the control or α7 transfected group.” This is confusing. Could the authors clarify what comparisons are being made and what significance is being denoted. Does this mean that the α7345-348a group is significantly different from both the control and α7 group? If so, this should be denoted by two different symbols. In the text, the mutant α7 is called “α7345-348a” whereas in the figure legend and figure it is “α7345-8a”, this nomenclature should be consistent throughout the paper In the figure legend for Figure 5, the definition of a double Asterix is given, but there is no double Asterix in Figure 5 Reviewer #2: The paper by Oz et al, shows that in MCF-7 cells activation by choline of α7 receptor increases proliferation, motility, and calcium signaling and this is partially blocked by the application of G protein blocker, Gαi/o inhibitor and by the presence of a G protein binding dominant negative α7 subunit (α7345-348A). The work is interesting and well done, and it demonstrated that the α7 subtype is involved in mediating choline induced proliferation, motility, and calcium signalling. However the effect of choline was blocked using antagonists that act on both α7 and α9 subtypes. Why the authors did not used the α7 selective antagonist AR (Whitaker et al. 2007) and the α9 selective antagonist RGIA4 (Romero et al 2017) ? This certainly will help in clarifying the subtypes involved and their contribution to the effects reported Minor points: In the abstract it is written "Nicotine activates various nicotinic acetylcholine receptors (nAChRs)" and in the introduction it is written"Mammalian nAChRs assemble from α (α1–α6) and β (β2–β4) subunit combinations forming homo- or hetero-pentameric channels [9, 10]. Nicotine does not activates a1 containing receptors and nicotine activates various nAChRs that assemble from α (α2–α7, α9, α10) and β (β2–β4) subunit combinations. Fig 7: Please add C to the figure ****** 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 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.] 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. 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Revision 1
7 Jul 2023 Author Response Please find the revision of our manuscript entitled “α7 nicotinic acetylcholine receptor interaction with G proteins mediates breast cancer cell proliferation, motility, and calcium signaling” for consideration as a research article in PLOS One. We have taken into consideration the comments of both reviewers and have addressed them throughout the revised manuscript. This is enumerated in a point-by-point response letter to the reviewers showing specific changes in the revised submission: Reviewer #1: • The experiment in figure 7B should be repeated with a control group for α7345-348a that was not exposed to choline to show that transfection with α7345-348a does not alter proliferation by itself. RESPONSE: We thank the reviewer for this comment. In fact, the experiment was conducted as a control however it was not included in the original submission. Therefore, the revised manuscript shows data on mutant receptor subunit transfection within Figure 7 and corresponding text in Results page 8 (last sentences of the section) . • The results in Figure 4 are not mentioned in the abstract or the discussion. It would be good if the authors discuss how the increase in α7 nAChR expression might affect the proliferation, motility, and/or calcium signaling of cancer cells. Additionally, the authors did not show if these effects could be blocked by MLA or another antagonist, or whether this upregulation is related to g-protein mediated signaling via α7 nAChRs. Further experiments to answer these questions would help to tie these results into the rest of the paper. RESPONSE: We thank the reviewer for pointing this out and we have now incorporated a discussion of this on Page 9 (end of the second paragraph). As stated, alteration in nicotinic receptor expression at the cell surface is likely an important driver of calcium and other signaling responses that can support cancer cell growth, and the experiments suggested by the reviewer are clearly important. However, due to limitation in experimental resources, we are not able to perform these experiments currently. Furthermore, we would like to suggest that studies on the mechanisms that participate in the regulation of nAChR at the cell surface, fall outside of the scope of the current manuscript. We now mention this as an important direction in future study in the revised manuscript in the Discussion on Page 9 (end of the second paragraph). • YM 254890 is mentioned in the abstract as a G protein blocker, but in the results section of the manuscript, the authors never define what specifically YM 254890 is or why it is being used. RESPONSE: The correction is now included on Page 7, last sentences of the 3rd paragraph within the Results section. The clarification is made that YM254890 is a Gq inhibitor and used to confirm Gq involvement. • The proliferation assay in Figure 7 plots Cell count x 105, whereas the other proliferation assays measure Net OD. Could the authors please explain why this assay was quantified differently? RESPONSE: Proliferation assays in the manuscript were performed in the laboratory of Dr. Oz however those presented in Figure 7 were performed in the lab of Dr. Kabbani. The two assays are virtually identical (i.e., using the same cell line and plating and MTT counting protocol). The results are also highly robust and show a comparable effect of choline on MCF-7 cells proliferation when comparing % change in cell count relative to the control group. To clarify this in the revised manuscript a newly added Figure 7 C shows cell count results as “% control (vehicle)” as done in earlier figures. • There are two “B” panels in figure 5 Response: This is now corrected. • The deltaF/F scales are not consistent across figures. For example, in Figure 5, A and C plot deltaF/F (x100), whereas panel B1 writes out the 100s, and the second panel B just has deltaF/F without the x100 clarification. Similarly, in Figure 7, panel A, the first graph is x100, while the second graph is not. Scale bar should be defined in Figure 7B. Response: These figures are significantly revised to correct for these errors. • Scale bars should be added to Figure 3A and Figure 4A Response: The scale bars are now added into Figs. 3 and 4. • In the figure legend for Figure 6 it is noted that the “Asterix denotes significant difference from control,” whereas the figure seems to indicate that the Asterix represents significant difference from the choline treated group. Could the authors clarify what comparisons are being measured in this figure, and what is significantly different from what? Response: Thank you for pointing out this error. We have revised the legends for figures 2, 6, and 7 with the new legends clearly stating which groups are used for statistical comparison. • The figure legend for Figure 2 states “Asterix denoted significant difference from control” but the figure seems to indicate that significance is measured as difference from the choline group. Response: This is now corrected. • Figure 7 has only 2 panels, A and B, but the figure legend has A, B, and C. Response: This is now corrected. • Figure 7 states “statistical significance relative to the control or α7 transfected group.” This is confusing. Could the authors clarify what comparisons are being made and what significance is being denoted. Does this mean that the α7345-348a group is significantly different from both the control and α7 group? If so, this should be denoted by two different symbols. In the text, the mutant α7 is called “α7345-348a” whereas in the figure legend and figure it is “α7345-8a”, this nomenclature should be consistent throughout the paper Response: This is now corrected. • In the figure legend for Figure 5, the definition of a double Asterix is given, but there is no double Asterix in Figure 5 Response: This is now corrected. Reviewer #2: The paper by Oz et al, shows that in MCF-7 cells activation by choline of α7 receptor increases proliferation, motility, and calcium signaling and this is partially blocked by the application of G protein blocker, Gαi/o inhibitor and by the presence of a G protein binding dominant negative α7 subunit (α7345-348A). The work is interesting and well done, and it demonstrated that the α7 subtype is involved in mediating choline induced proliferation, motility, and calcium signalling. • However the effect of choline was blocked using antagonists that act on both α7 and α9 subtypes. Why the authors did not used the α7 selective antagonist AR (Whitaker et al. 2007) and the α9 selective antagonist RGIA4 (Romero et al 2017) ? This certainly will help in clarifying the subtypes involved and their contribution to the effects report. Response: α-Conotoxin RgIA has higher potency for α9/α10 than α7 nAChRs (Ellison et al., 2008), and was thus chosen in complementary experiments to α bungarotoxin. The use of additional pharmacological ligands with greater selectivity, as suggested by the reviewer, is definitely important and to that end this point is now stated in the Discussion on Page 10, last two sentences. Minor points: In the abstract it is written "Nicotine activates various nicotinic acetylcholine receptors (nAChRs)" and in the introduction it is written"Mammalian nAChRs assemble from α (α1–α6) and β (β2–β4) subunit combinations forming homo- or hetero-pentameric channels [9, 10]. • Nicotine does not activates a1 containing receptors and nicotine activates various nAChRs that assemble from α (α2–α7, α9, α10) and β (β2–β4) subunit combinations. • Fig 7: Please add C to the figure Response: We thank the reviewer for pointing these mistakes and have corrected them within the revised manuscript. Attachments Attachment Submitted filename: Reviewer_Comments.docx https://doi.org/10.1371/journal.pone.0289098.r002
12 Jul 2023 Decision Letter - Israel Silman, Editor α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling PONE-D-23-15038R1 Dear Dr. Kabbani, 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, Israel Silman Academic Editor PLOS ONE Additional Editor Comments (optional): 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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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) ****** 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 ******** https://doi.org/10.1371/journal.pone.0289098.r003
Formally Accepted
17 Jul 2023 Acceptance Letter - Israel Silman, Editor PONE-D-23-15038R1 α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling Dear Dr. Kabbani: 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 Prof. Israel Silman Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289098.r004

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