Dear Sandy,

Thank you for submitting your manuscript entitled "FCHSD2 Controls Oncogenic ERK1/2 Signaling Outcome by Regulating Endocytic Trafficking" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Feb 14 2020 11:59PM.

Login to Editorial Manager here: https://www.editorialmanager.com/pbiology

During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

+44 1223–442810

Dear Sandy,

Thank you very much for submitting your manuscript "FCHSD2 Controls Oncogenic ERK1/2 Signaling Outcome by Regulating Endocytic Trafficking" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by three independent reviewers.

As you will see, all the reviewers are very positive and find the conclusions interesting and significant for the field, however they also raise a few points that would need to be addressed with additional experiments to strengthen the results. The reviewers have also requested several clarifications.

In light of the reviews (attached below), we will not be able to accept the current version of the manuscript, but we would welcome re-submission of a revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.

We expect to receive your revised manuscript within 2 months.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Re-submission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this re-submission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

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. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

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: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Best wishes,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

+44 1223–442810

---------------------------------------------------------------

Reviewers’ comments

Rev. 1:

In the present manuscript, Xiao G. et al., characterized the role of FCHSD2 in endocytosis and trafficking of RTKs. Authors reported that FCHSD2 has a role in ligand-induced trafficking of EGFR and Met receptors, and, in particular, they proposed that it acts as a positive regulator of receptor recycling. They also characterized the role of FCHSD2 in unstimulated basal conditions, where depletion of FCHSD2 causes the increased transcription of EGFR and MET mRNA, possibly through the activation and nuclear translocation of ERK1/2/c-Jun. They also showed that Rab7 is required for the decreased recycling of TfR and EGFR upon siFCHSD2, suggesting that FCHSD2 function could be that of counteracting Rab7. In addition, siRab7 also reverted the increase in EGFR and MET levels induced upon FCHSD2 depletion. FCHSD2 loss correlates with high grade NSCLC; patients with high FCHSD2 have an elevated survival at variance with patients with high Rab7 that display low survival rates.

The manuscript provides novel insight into the connection between endocytosis and signaling in cancer. Therefore, is of high interest for the cell biology and cancer community. However, there are a number of issues that need to be solved prior publication.

1) The assay described in Fig. 1B, C does not measure properly recycling, but it just detects disappearance of internalized Tf/EGF signal. For TfR, which is almost exclusively recycled back to the PM, it could be used as a proxy for recycling. However, this is not the case for EGFR, which is both recycled and degraded after internalization. Thus, the experiment in Fig. 1C suggests that disappearance of internalized EGF signal is delayed, which is compatible with either defective recycling or defective degradation. This methodological issue need to be better discussed. In addition, the nomenclature on the Y axis in Fig. 1B, C need to be changed accordingly. As it is represented now, it is misleading as it seems that recycling decreases with time.

2) Fig. 1F and Fig. 2B. It is not clear how the assay is performed. Are all samples (from 0.5h to 6h of EGF stimulation) treated with cycloheximide for the same period of time? As cycloheximide could really affect the biosynthesis of many factors involved in endocytosis, trafficking and degradation (also considering the effects of FCHSD2 on transcription/biosynthesis), I would recommend to repeat EGFR degradation curve (-/+ siFCHSD2) without this treatment.

3) Fig. 3B. The treatment with Afatinib is not convincing, as it is not substantially reducing EGFR activation (measure as p-EGFR). More consistent inhibition is required to exclude a role for EGFR kinase activity in the process. Also, the WB shown on the left does not reproduce the increase in EGFR and MET upon siFCHSD2 observed in previous experiments. As Erk1/2 is involved in the process, another standard loading control should be used.

4) Fig. 3D. Authors concluded from this experiment that STAT3 is not involved in the process. However, its phosphorylation is affected upon siFCHSD2. Authors should better discuss their conclusion.

5) Fig. S1. The inhibition of Erk1/2 with SCH compound has the same effect of the Rab7 inhibition on TfR recycling (in Fig. 5A). It seems that the decrease in TfR recycling upon siFCHSD2 requires the action of Erk1/2, as it is reverted by SCH treatment. I did not understand how the authors interpret this results in Fig. S1 at variance with results shown in Fig 5 A.

In addition, in Fig. S1, the effects of SCH treatment are mild - if any - as well as the effects of siFCHSD2 in ARPE-19 cells. The magnitude is minimal and, although some time points are statistically significant, it is hard to draw conclusion on the biological significance of these effects. In my opinion, these data on SCH treatment should be more critically discussed.

6) Fig. 4F. The WB provided is not completely convincing. The levels are variable and there are some inconsistencies. For instance, MET levels seem not to be rescued by both treatments in H1975, just all levels are decreased. Similarly, in HCC4079, MET levels are variable, sometimes increasing (SCH) sometimes decreasing (GSK) after treatments. It is not clear what these treatments are really doing. In addition, the blots are not representative of the quantification on the right. In the quantitation, samples should be all normalized to the same reference, i.e. the not treated Ctrl sample.

An important experiment that would corroborate this finding is the analysis of EGFR and MET mRNA levels upon siFCHSD2, to investigate if they are rescued by these treatments.

7) The effect of siRab7 are strong and convincing. However, it is not clear how Rab7 function is linked to Erk1/2 nuclear translocation and activation, and to the induced EGFR/MET transcription. The model shown in Fig. 6A is too complicated and does not convey the message.

Which is the mechanism though which Rab7 KD revert the increased EGFR and MET levels? Authors should show if siRab7 is able to revert the MET and EGFR mRNA levels.

Does siRab7 revert the increased Erk1/2, c-Jun, Ets-1 levels in the nucleus?

These connections need to be investigated and further discussed in the text.

Rev. 2:

This is an excellent study interrogating how membrane trafficking aletrations downstream of FCHSD2 depletion impact ERK1/2 signaling. The primary conclusions are that FCHSD2, by negative regulation of Rab7, diverts tyrosine kinase receptors (TKRs) from late endosomes, limiting access of TKRs to ERK1/2 activation at the late endosome. Loss of FCHSD2 increases traffic of TKRs to LE and subsequent activation of ERK1/2. The data, for the most part, support the stated conclusions. My main comments are as follows.

1. The authors should be more attentive to alerting the reader what was done in the KRAS/p53 line and what was done in EGFR mutant. For the non-cancer specialist, the potential importance of the distinction might be lost. The authors might also consider adding a discussion paragraph on the topic of the oncogenic driver mutation and the impact of FCHSD2 (loss thereof).

2. The authors might consider extending the main conclusion, that KD of FCHSD2 increases expression of EGFR and MET, in analyses of other human NSCLC cell lines, both EGFR mutant and KRAS.

3. The authors might provide some speculation (or perhaps data) on how FCHDS2 acts as a negative regulator of Rab7. Does FCHSD2 activate a Ran7 GAP or inhibit a Rab7 GEF?

4. Fig 6 is not particularly strong, and I do not feel it strengthens the paper but rather detracts from the otherwise strong cell biology. To appreciate the data in panel B one would want to see expression of FCHSD2 in adjacent normal lung tissue. Pushing a link between the expression of a single protein (FCHSD2) and grade is not particularly compelling. I assume the analyses in Fig6C are based on RNAseq. Have they correlated IHC with RNAseq in their own cohort (panel B) to establish the RNAseq data agrees with the IHC? The author's need to present data on the expression distribution of FCHSD2 and Rab7 so the reader can assess how meaningful the distinction is. Did the author's use above and below the median or upper and lower quartiles? They should also look at the TCGA database to determine how specific this is for lung.

5. Throughout the manuscript the authors should reanalyze the recycling curves. In many instances it looks like the extent of recycling rather than the rate of recycling is changed. For example, in Fig 1B it is the extent of transferrin receptor recycling that is changed by FCHSD2 depletion not the rate, suggesting a fraction of transferrin receptor is trapped and no longer recycled rather than a slowing of recycling. Although I do not think it will change the conclusions it might change how one thinks about the molecular mechanism(s) underlying the effects.

Rev. 3: Peter S McPherson – Please note that this reviewer has waived anonymity

This is a clear, concise, well-executed study, exactly what we have come to expect from Dr. Schmid and her colleagues. I was left a little wanting, how exactly does the FCHSD2-depeletion induced upregulation of the RTKs occur, what specifically does Rab7 do in this pathway, why the seeming conundrum between increased trafficking to late endosome/lysosome and yet upregulation of expression? But there is valuable information in this study and no one study ever has all the answers.

Abstract. "…can reciprocally regulate endocytic trafficking by creating feedback loops in non-small-cell lung cancer (NSCLC) cells…". The sentence is awkward. Presumably these feedback loops are present in other cancer cells, perhaps in non-cancer cells.

Line 53, can you please be more specific as to what you mean by "…cancer-specific activation…", in particular, what is meant by activation?

Figure 1 and throughout, use 2 siRNAs or better describe the specificity of the single one used or perhaps validate 1-2 key phenotypes in a knockout line.

Figure 1D, please, a word or two about the specificity of the PO4-EGFR antibody in the results section.

The upregulation of mRNA in Figure 3 is relatively minor, and in light of the enhanced degradation its seems the balance should not be towards such a strong upregulation of the protein. Please discuss.

Figure 5, one Rab7 siRNA?

Figure 5, higher levels of active Rab7 in FCHSD2 knockdown cells. It would be nice if there was some additional data here. What explains the activation, regulation of a GEF or a GAP?

Figure 5. There is some uncertainty regarding the specificity of the antibodies recognizing the active Rabs. The results state that an antibody specific to active Rab7 was used without reference or validation. The authors need to address this issue.

Dear Sandy,

Thank you for submitting your revised Research Article entitled "FCHSD2 Controls Oncogenic ERK1/2 Signaling Outcome by Regulating Endocytic Trafficking" for publication in PLOS Biology. I have now obtained advice from the original Academic Editor, who has assessed the revision along with the team of editors.

We're delighted to let you know that we're now editorially satisfied with your manuscript. However before we can formally accept your paper and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication. Please also make sure to address the data and other policy-related requests noted at the end of this email.

*Copyediting*

Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that 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. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

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: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

*Submitting Your Revision*

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include a cover letter, a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable), and a track-changes file indicating any changes that you have made to the manuscript.

Please do not hesitate to contact me should you have any questions.

Best wishes,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

+44 1223–442810

------------------------------------------------------------------------

DATA POLICY:

Thank you for submitting all the data underlying the graphs shown in the figures. Please indicate in each figure legend (including the supplementary figures) where the underlying data can be found.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

Dear Dr Schmid,

On behalf of my colleagues and the Academic Editor, Frederick M. Hughson, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology.

The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. This will ensure that any requested corrections reach the production department in time for publication.

Early Version

The version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.

Kind regards,

Vita Usova

Publication Assistant,

PLOS Biology

on behalf of

Ines Alvarez-Garcia,

Senior Editor

PLOS Biology