EMBO reports - Peer Review Process File – EMBO-2016-42032
Manuscript EMBO-2016-42032
RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs Hiroaki Kajiho, Yuko Kajiho, Emanuela Frittoli, Stefano Confalonieri, Giovanni Bertalot, Giuseppe Viale, Pier Paolo Di Fiore, Amanda Oldani, Massimiliano Garre, Galina Beznusenko, Andrea Palamidessi, Manuela Vecchi, Philippe Chavrier, Franck Perez, Giorgio Scita Corresponding author: Giorgio Scita, IFOM, Fondazione Istituto FIRC di Oncologia Molecolare
Review timeline:
Submission date: Editorial Decision: Revision received: Accepted:
15 January 2016 17 February 2016 08 April 2016 28 April 2016
Editor: Barbara Pauly Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)
1st Editorial Decision
17 February 2016
Thank you very much for the submission of your research manuscript to our editorial office. We have now received the full set of reports from the referees that were asked to assess it. As they are pasted below I will only repeat the main points here. As you will see, the reviewers agree on the interesting nature of your data in principle, but point out a number of instances in which additional controls or data need to be added. Moreover, they all agree that the writing and data presentation needs to be more focused and streamlined to provide a coherent message. While referee 3 suggests splitting up the manuscript into two parts we do not feel that this would be a good option. Rather, re-writing and re-structuring the manuscript would be sufficient. Given the potential interest of your study and the constructive suggestions of the reviewers on how to improve it, I would like to give you the opportunity to revise your manuscript, with the understanding that the main concerns of the referees should be addressed. Acceptance of the manuscript will depend on a positive outcome of a second round of review and I should also remind you that it is EMBO reports policy to allow a single round of major revision only and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. I would also like to point out that we now strongly encourage the publication of original source data (whole western blots and raw microscopical images) with the aim of making primary data more
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accessible and transparent to the reader. The source data will be published in a separate source data file online along with the accepted manuscript and will be linked to the relevant figure. As one of the referees also raised this issue, I would strongly recommend that you send us these raw data when submitting your revised manuscript. I look forward to seeing a revised form of your manuscript when it is ready. REFEREE REPORTS Referee #1: The study by Kajiho et al. identifies RAB2A as a novel player in cancer cell behavior. The authors perform an extensive loss of function screen of RAB GTPases to identify RAB2A as a promoter of cell invasion and they provide a strong correlative analysis of RAB2A in aggressive breast cancer, suggestive of RAB2A levels as a predictor of prognosis. They then place RAB2A on a late endocytic compartment and show it interacts with the HOPS complex to control post-endocytic trafficking of MT1-MMP. Thus, this study not only identifies RAB2A as promoter of cell invasion, but shows that RAB2A likely promotes cell invasion by regulating a late-endocytic trafficking pathway. This is interesting because RAB2A is typically studied in an ER to Golgi pathway. The findings presented in this paper are novel and will be interesting for the readers of EMBO reports. Overall, the study is for the most part well conducted. I have outlined some major comments regarding controls and interpretation of the data that need to be addressed. In addition, this paper requires substantial editing as there are several problems with the writing. Major Comments 1. In Figure 2a, the authors show a Western blot of their inducible RAB2A KD in MDA-MB-231 cells. While this knockdown is certainly convincing, it is hard to appreciate how efficient the knockdown is when the loading control is also decreasing. The authors should quantify the knockdown and normalize to the loading control. There also appears to be a decrease in RAB2A in the absence of DOX. This quantification will also show whether this decrease occurs consistently 2. In Figures 2b and 2c, the authors perform 2D and 3D invasion assays using shRAB2A cells in the presence and absence of DOX. The authors need to show that DOX itself does not have any effect on the invasive capacity of these cells. 3. In Figures 2b and 2c, the authors perform 2D and 3D invasion assays. Since these assays are very different from the gelatin degradation assays, the experiments need either a second shRNA or a rescue to control for off target effects. 4. I'm not sure I agree with the interpretation of the pHluorin experiments. The authors state that the frequency of endocytic events is reduced. But the image in Figure 6d does not show that. It shows that MT1-MMP remains on the plasma membrane longer with RAB2A knockdown. There is not enough information in the paper to assess whether the exocytic events were quantified appropriately. The methods section refers to reference 20, but the references are not numbered. Furthermore, if RAB2A knockdown does in fact reduce exocytosis of MT1-MMP, why don't you see a decrease in surface levels in supplemental figure 5i-j. 5. In figure 5C, the authors say that expression of VPS39 is sufficient to induce the formation of enlarged Rab7-positive late endosomes. But there already appears to be enlarged endosomes in the absence of VPS39 expression. Perhaps they are made larger with VPS39? The authors should be more descriptive and quantify the size of these vesicles to show this is the case. This quantification should also be done with expression of RAB2A-Q65L in figure 4A. 6. In Figure 7A the authors show a loss of epithelial cell compaction with RAB2A expression. The authors need to show that the cells are not simply dying as expression of RABs can be toxic to the cells and the thin protrusions could be retraction of the cell body when a cell is dying. 7. It is not clear how RAB2A regulates MT1-MMP activity or E-cadherin trafficking. The authors need to provide a model of how RAB2A promotes MT1-MMP and E-cadherin trafficking. For
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instance, if the HOPs complex promotes late endosome to lysosome fusion, how does the RAB2Avps39 interaction promote recycling? Do they think that E-cadherin is regulated by retention at the Golgi while MT1-MMP trafficking on recycling endosomes? E-cadherin is also trafficking on the same early/recycling endosomes as integrin but in opposing conditions. Perhaps the same is true for MT1-MMP and E-cadherin? Minor Comments 8. The authors need to have figure numbers that correspond to the quantifications and then refer to those figures in the text. For instance, there are 4 graphs floating at the bottom of figure 1. 9. TCGA data is actually supplemental figure 2a, not 2b 10. In the text the authors state they use "a murine gene that is resistant to shRNA targeting the human sequence". This implies that the shRNA knockdown is DOX inducible. Where it seems according to the figure legends and labelling, they use DOX inducible RAB2-expression in cells transfected with a scramble or RAB2A siRNA. The authors need to better explain what they did in the text. 11. The survival plots in Figure 3c are very striking. However, using the KMplotter public database (http://kmplot.com/analysis/) there is no correlation between RAB2A levels and survival in any form of breast cancer. If RAB2A levels are as powerful for predicting outcome as you say, why is there such a difference between this analysis and your own? 12. In supplemental figure 5h, the authors states that MT1-MMP mRNA levels do not change, but the graph looks like mRNA levels are increasing with RAB2A knockdown. Perhaps the mRNA figure is unnecessary since they also show a western blot of protein levels. Then the authors should quantify protein levels normalized to a loading control. 13. The authors state that MTI-MMP/integrin delivery is mediated through recycling from early and late endosomal compartments. Then they show that RAB2A knockdown has no effect on MTIMMP/integrin surface levels. Then they claim this proves that RAB2A regulates matrix degradation independent from its role in Golgi-to-ER trafficking. While the fact that there is no change in total levels argues that RAB2A knockdown does not affect the synthesis. The surface levels are also unaffected arguing that RAB2A knockdown does not affect recycling from early or late endosomes. This section needs to be written to reflect this. 14. In Supplemental Figure 6B, the enlarged late endosomal phenotype is striking. However, the authors claim that RAB2A continues to localize to these structures but it is not apparent at the current magnification. The authors need to add a high magnification of the figure. 15. In Supplemental Figure 6C the authors presumably show that wild-type RAB2A does not colocalize with EEA1? This was already shown in Figure 4b with the mutants in a different cell line. The authors need to either refer to this figure in the text, or if the figure does not add to the story, remove the figure from the paper. 16. E-cadherin is not secreted, it is recycled back to the plasma membrane. The authors should adjust the text and title accordingly. Referee #2: This highly detailed study explores novel roles of the RAB GTPase, RAB2A in the regulation of aspects of endocytic trafficking of 2 key proteins, MT1-MMP and E-cadherin, implicated in tumor cell invasiveness. The data presented are an unusual combination of mechanistic cell biology studies and evidence that RAB2A is a predictor of metastatic recurrence in human breast cancer. Although the latter are interesting, the link with the cell biology of focal matrix degradation and the secretory pathway of E-cadherin is rather tenuous. Overall the work cannot really be considered as 'sharply focused' as per the journal remit, and contains a thorough and extremely detailed presentation of all experiments carried out, many of which are negative. As a consequence the paper is rather unwieldy
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and the broad biological significance of the study is somewhat lost. Much data has already been confined to the Supplement but it is possible that more of the earlier screening studies could be moved into an 'Expanded View' section? Overall the study is a moderately important contribution to the field. Further comments 1. siRNA and shRNA ablation of RABs in cancer cell lines is carried out in a number of experiments using fluorescent gelatin film degradation assays as a readout. Degradation is exhibited as tiny black foci that are often not visible without extensive magnification of the images. These images need to be improved throughout the manuscript. A number of these images could be confined to the expanded view section. 2. P.10, Suppl.Fig.5 h-k. Silencing of RAB2a in MCF10.DCIS cells did not alter the levels of surface MT1-MMP or alpha5 beta1 integrin. Why was this study only carried out using unstimulated cells? Cell degradation of the gelatin films was observed after HGF stimulation in many of the previous experiments and it would thus seem important to evaluate the location of MT1-MMP at the cell surface, and the effect of RAB2A ablation, under equivalent stimulatory conditions. Referee #3: This is a very nice and scientifically sound piece of work. However, the MS is nearly impossible to follow in its current, overly long and unrefined format. Unless the author re-write and re-arrange the MS, their findings would be lost because of the poor presentation of the very interesting results. My suggestions are as follows: 1) To split the existing massive material and to present two papers, one on MT1-MMP and another on E-cadherin. 2) To arrange the customary, individual Results and Discussion sections that would allow to reducing the enormous length of the current paper. 3) To present only the relevant experiments as now the MS is more similar with the progess report rather than a focused scholarly publication I would gladly review each individual paper or both papers after these or similar modifications are made. Without these modifications and changes, the MS is nearly impossible to follow and understand. Naturally, this is multi-year work by many professional co-workers. Accordingly, the exciting results the authors managed to acquire deserve to be presented in a better fashion rather than in a laundry list of experiments.
1st Revision - authors' response
08 April 2016
Thanks a lot for the letter of February 17, 2016 regarding our manuscript entitled “RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs”. (We change slightly the title as per reviewers’ suggestions). We greatly appreciated yours and reviewers’ comments and addressed each of them with new experiments and by rewriting the manuscript to streamline its message as per reviewers’ suggestions. To this end, we moved some of the control experiments and “negative data” onto Appendix Figures and shorten the related text. We also limited Expanded View files to 5 figures as per your request. We also shorten the main text, and in the discussion, we highlighted the dual role played by RAB2A in the regulation of MT1-MMP late endosomal trafficking and of E-cadherin Golgi-to-PM transport. The main changes in revised text are in blue.
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Referee #1: The study by Kajiho et al. identifies RAB2A as a novel player in cancer cell behavior. The authors perform an extensive loss of function screen of RAB GTPases to identify RAB2A as a promoter of cell invasion and they provide a strong correlative analysis of RAB2A in aggressive breast cancer, suggestive of RAB2A levels as a predictor of prognosis. They then place RAB2A on a late endocytic compartment and show it interacts with the HOPS complex to control post-endocytic trafficking of MT1-MMP. Thus, this study not only identifies RAB2A as promoter of cell invasion, but shows that RAB2A likely promotes cell invasion by regulating a late-endocytic trafficking pathway. This is interesting because RAB2A is typically studied in an ER to Golgi pathway. The findings presented in this paper are novel and will be interesting for the readers of EMBO reports. Overall, the study is for the most part well conducted. I have outlined some major comments regarding controls and interpretation of the data that need to be addressed. In addition, this paper requires substantial editing as there are several problems with the writing. R. We thank the reviewer for the insightful comments and suggestions. We addressed below each of the issue raised and edited the paper following the suggestions of the other reviewers to streamline the manuscript. Major Comments 1. In Figure 2a, the authors show a Western blot of their inducible RAB2A KD in MDA-MB231 cells. While this knockdown is certainly convincing, it is hard to appreciate how efficient the knockdown is when the loading control is also decreasing. The authors should quantify the knockdown and normalize to the loading control. There also appears to be a decrease in RAB2A in the absence of DOX. This quantification will also show whether this decrease occurs consistently R. We have run additional immunoblotting in order to have similar level of total proteins and further quantified the efficacy of silencing. The data are presented in revised Figure 2a and show that RAB2A levels were reduced by more than 95% after siRNA silencing. 2. In Figures 2b and 2c, the authors perform 2D and 3D invasion assays using shRAB2A cells in the presence and absence of DOX. The authors need to show that DOX itself does not have any effect on the invasive capacity of these cells. R. We originally performed these assays in the presence or absence of doxycycline and did not observed any effects of drug treatment on cell invasion. We have included quantification of this control in the revised Figure 2d. 3. In Figures 2b and 2c, the authors perform 2D and 3D invasion assays. Since these assays are very different from the gelatin degradation assays, the experiments need either a second shRNA or a rescue to control for off target effects. R. We would like to point out that this set assays was carried out with an shRNA targeting a sequence different from those targeted by the siRNA oligos used in inversed invasion assays shown in Figure 1f-h. This notwithstanding, we also performed an additional set of Transwell invasion assays, as requested, with two different targeting siRNAs that consistently show that RAB2A removal hampers BC cell invasion (revised Figure EV1a and 1b).
4. I'm not sure I agree with the interpretation of the pHluorin experiments. The authors state that the frequency of endocytic events is reduced. But the image in Figure 6d does not show that. It shows that MT1-MMP remains on the plasma membrane longer with RAB2A knockdown. There is not enough information in the paper to assess whether the exocytic events were quantified appropriately. The methods section refers to reference 20, but the references are not numbered. Furthermore, if RAB2A knockdown does in fact reduce exocytosis of MT1-MMP, why don't you see a decrease in surface levels in supplemental figure 5i-j.
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R. We apologize for the insufficient experimental information provided. Indeed, what we measured is the frequency of the appearance of EGFP-positive dots in various cells over time exactly as described by Monteiro et al. JCB 2013 (corresponding to ref. 20), which is now properly numbered in the revised methods. We also would like to point out that a significant amount of MT1-MMP is localized at the plasma membrane in strict association with Tissue inhibitor of metalloproteases II (TIMP II). TIMP II binds to surface MT1-MMP rapidly inhibiting its activity [1, 2]. Hence, the need to have a constant flow of MT1-MMP delivered from endosomal vesicles to the PM, where its transient and localized activity is exerted before being inhibited by TIMP II association. Elegant experimental and mathematical modeling supports this mode of action [3, 4]. The important implications/predictions of this mechanism of operation are: i) the absolute need of membrane recycling for MT1-MMP proteolytic activity to be effective; ii) impairing late endosomal membrane recycling is expected to diminish peri-cellular proteolysis without affecting significantly total cell surface levels of MT1-MMP. We verified these predictions experimentally in our cell system. We also discussed this point in the discussion of the revised manuscript (pg. 15)
5. In figure 5C, the authors say that expression of VPS39 is sufficient to induce the formation of enlarged Rab7-positive late endosomes. But there already appears to be enlarged endosomes in the absence of VPS39 expression. Perhaps they are made larger with VPS39? The authors should be more descriptive and quantify the size of these vesicles to show this is the case. This quantification should also be done with expression of RAB2A-Q65L in figure 4A. R. We quantify the size of endosomes following VPS39 expression and show that they are significantly enlarged with respect to control. We included this quantification in revised Figure 5c. Enlargement of Rab7-positive vesicles by RAB2A-Q65L are also quantified and shown in revised Figure 4a. 6. In Figure 7A the authors show a loss of epithelial cell compaction with RAB2A expression. The authors need to show that the cells are not simply dying as expression of RABs can be toxic to the cells and the thin protrusions could be retraction of the cell body when a cell is dying. R. We performed a vital dye staining under exactly the same conditions used to monitor cell shape described in Figure 7a (see Appendix Figure S5 of the revised manuscript). We observed no cell toxicity or death under these conditions. Similarly, a time lapse recording reveals that RAB2A expression causes increase cell elongation as compared to control, but no sign of cell toxicity. 7. It is not clear how RAB2A regulates MT1-MMP activity or E-cadherin trafficking. The authors need to provide a model of how RAB2A promotes MT1-MMP and E-cadherin trafficking. For instance, if the HOPs complex promotes late endosome to lysosome fusion, how does the RAB2Avps39 interaction promote recycling? Do they think that E-cadherin is regulated by retention at the Golgi while MT1-MMP trafficking on recycling endosomes? Ecadherin is also trafficking on the same early/recycling endosomes as integrin but in opposing conditions. Perhaps the same is true for MT1-MMP and E-cadherin? R. The data are consistent with a dual role of RAB2A: a “canonical” one where RAB2A regulates ER-Golgi biosynthetic trafficking controlling Golgi stacking, and thus presumably affecting the maturation of proteins, including E-cadherin. The latter protein must be specifically and efficiently processed in the Golgi before reaching the PM. Within this context, ectopic expression of RAB2A, mimicking the alterations found in breast cancer, causes enlarged Golgi stacking (Figure 7i) that leads to E-cadherin mis-processing (Figure 7e), reduced furin cleavage (Figure 7g), and altered biosynthetic polarized trafficking (accumulation of E-cadherin into Golgi stacks -Figure 7j).
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RAB2A, in its active GTP-bound state, acts also to regulate late endosomal trafficking of cargo internalized from the PM, such as MT1-MMP. In this latter process, we have now collected a new set of evidence showing that RAB2A is essential for the motility of late endosomes. In particular, silencing of RAB2A as well as VPS39 impairs MT1-MMPvesicle motility and causes the accumulation of these vesicles in peri-nuclear regions, preventing MT1-MMP-endosomes to reach their delivery site at the PM. However, we are currently investigating in more details the underlying molecular mechanisms. We feel that this latter set of data represents the beginning of entirely new study requiring a number of additional experiments to be completed. Nevertheless, we discussed this possible mode of action of RAB2A in the revised discussion (pg. 15). The two functional roles of RAB2A are distinct. Indeed, RAB2A endosomal function requires binding to the HOPS components VPS39, which is instead dispensable for Golgi trafficking and processing of E-cadherin. We summarized this mode of action in a graphic summary included in the revised manuscript and commented it in the revised discussion (see pg. 15 of the revised manuscript). Minor Comments 8. The authors need to have figure numbers that correspond to the quantifications and then refer to those figures in the text. For instance, there are 4 graphs floating at the bottom of figure 1. R. We agree. We relabeled Figure 1 accordingly. 9. TCGA data is actually supplemental figure 2a, not 2b R. Both Supplemental Fig. 2a and 2b report TCGA data. We clarify this point in the revised legend to this figure, which we included as Appendix Figure S2 of the revised manuscript. 10. In the text the authors state they use "a murine gene that is resistant to shRNA targeting the human sequence". This implies that the shRNA knockdown is DOX inducible. Where it seems according to the figure legends and labelling, they use DOX inducible RAB2expression in cells transfected with a scramble or RAB2A siRNA. The authors need to better explain what they did in the text. R. The reviewer is correct and we apologize for this oversight. Indeed, we used siRNA in this experiment as indicated in Figure 2f-h and the related figure legend. We corrected the main text (pg. 6 of the revised text). 11. The survival plots in Figure 3c are very striking. However, using the KMplotter public database (http://kmplot.com/analysis/) there is no correlation between RAB2A levels and survival in any form of breast cancer. If RAB2A levels are as powerful for predicting outcome as you say, why is there such a difference between this analysis and your own? R. We also performed a set of analysis using data set from KM plotter. Regardless of the probe set analyzed, we found that elevated expression of RAB2A is significantly associated with Distant Metastasis Free Survival (DFMS) in BC, similarly to what we obtained with the analysis of the protein levels we carried out on our TMA cohort, and consistent with what was recently reported [5]. An example of one of the probe set analyzed is shown below.
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12. In supplemental figure 5h, the authors states that MT1-MMP mRNA levels do not change, but the graph looks like mRNA levels are increasing with RAB2A knockdown. Perhaps the mRNA figure is unnecessary since they also show a western blot of protein levels. Then the authors should quantify protein levels normalized to a loading control. R. We agree. We removed the mRNA quantification. 13. The authors state that MTI-MMP/integrin delivery is mediated through recycling from early and late endosomal compartments. Then they show that RAB2A knockdown has no effect on MTIMMP/integrin surface levels. Then they claim this proves that RAB2A regulates matrix degradation independent from its role in Golgi-to-ER trafficking. While the fact that there is no change in total levels argues that RAB2A knockdown does not affect the synthesis. The surface levels are also unaffected arguing that RAB2A knockdown does not affect recycling from early or late endosomes. This section needs to be written to reflect this. R. We have modified the text to clarify these issues. In particular, as pointed out by the reviewers, we showed that RAB2A impact on matrix degradation without affecting the total and surface levels of Integrin β1 or the secretion of various MMPs. RAB2A also has marginal effects on total and surface levels of MT1-MMP. This latter finding is likely accounted by the mechanisms regulating the activity of this transmembrane MMP. MT1MMP, as detailed in reply to point# 4, is thought to be rapidly inactivated by binding to TIMP II at the cell surface, and hence it requires to be constantly mobilized from endosomes to the PM to act as effective pericellular proteolytic enzyme. We discussed this point in the discussion of the revised manuscript (pg. 15) 14. In Supplemental Figure 6B, the enlarged late endosomal phenotype is striking. However, the authors claim that RAB2A continues to localize to these structures but it is not apparent at the current magnification. The authors need to add a high magnification of the figure. R. We added a magnification to highlight the enlarged endosomes caused by RAB2A elevation. Despite this obvious phenotype, wild type RAB2A only marginally localizes to these structures. This is likely due the fact that RAB2A can be seen in enlarged endosome (as shown in Figure 4a) only when it is in its active states (in its GTP-bound form). It is therefore likely that the intrinsic hydrolysis of GTP-RAB2A leads not only to the inactivation of the protein, but it is also responsible for its transient endosomal localization. We corrected the statement (pg. 8 of the revised manuscript) and showed magnified representative examples of enlarged endosome caused be elevation of RAB2A levels in the revised Figure EV2c.
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15. In Supplemental Figure 6C the authors presumably show that wild-type RAB2A does not colocalize with EEA1? This was already shown in Figure 4b with the mutants in a different cell line. The authors need to either refer to this figure in the text, or if the figure does not add to the story, remove the figure from the paper. R. Agree. We removed, as suggested, Suppl. Fig. 6C. 16. E-cadherin is not secreted, it is recycled back to the plasma membrane. The authors should adjust the text and title accordingly. R. Agree. We adjust the text accordingly throughout the manuscript (including the title) to point out that RAB2A affects E-cadherin transport and biosynthetic processing on its path from Golgi-to-PM.
Referee #2: This highly detailed study explores novel roles of the RAB GTPase, RAB2A in the regulation of aspects of endocytic trafficking of 2 key proteins, MT1-MMP and E-cadherin, implicated in tumor cell invasiveness. The data presented are an unusual combination of mechanistic cell biology studies and evidence that RAB2A is a predictor of metastatic recurrence in human breast cancer. Although the latter are interesting, the link with the cell biology of focal matrix degradation and the secretory pathway of E-cadherin is rather tenuous. Overall the work cannot really be considered as 'sharply focused' as per the journal remit, and contains a thorough and extremely detailed presentation of all experiments carried out, many of which are negative. As a consequence the paper is rather unwieldy and the broad biological significance of the study is somewhat lost. Much data has already been confined to the Supplement but it is possible that more of the earlier screening studies could be moved into an 'Expanded View' section? Overall the study is a moderately important contribution to the field. R. To stream line the manuscript we moved some of the “negative results” to the Expanded View section and Appendix Figures, and significantly shorten the description of this set of data, focusing on the new findings. We also reduced the text related to the description of the screening. We shorten the discussion and included comment as to the modes of action of RAB2A. Further comments 1. siRNA and shRNA ablation of RABs in cancer cell lines is carried out in a number of experiments using fluorescent gelatin film degradation assays as a readout. Degradation is exhibited as tiny black foci that are often not visible without extensive magnification of the images. These images need to be improved throughout the manuscript. A number of these images could be confined to the expanded view section. R. We improve some of the images of the focal matrix degradation by presenting magnified images where need (revised Figure 1c). 2. P.10, Suppl.Fig.5 h-k. Silencing of RAB2a in MCF10.DCIS cells did not alter the levels of surface MT1-MMP or alpha5 beta1 integrin. Why was this study only carried out using unstimulated cells? Cell degradation of the gelatin films was observed after HGF stimulation in many of the previous experiments and it would thus seem important to evaluate the location of MT1-MMP at the cell surface, and the effect of RAB2A ablation, under equivalent stimulatory conditions. R. All the experiments using MCF10.DCIS.com were carried out in the presence of HGF, which is necessary to induce matrix degradation. We include explicitly this information in the revised figure legend.
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Referee #3: This is a very nice and scientifically sound piece of work. However, the MS is nearly impossible to follow in its current, overly long and unrefined format. Unless the author rewrite and re-arrange the MS, their findings would be lost because of the poor presentation of the very interesting results. R. We thanks the reviewer for appreciating the effort in putting together a nice and sound set of experiments My suggestions are as follows: 1) To split the existing massive material and to present two papers, one on MT1-MMP and another on E-cadherin. 2) To arrange the customary, individual Results and Discussion sections that would allow to reducing the enormous length of the current paper. 3) To present only the relevant experiments as now the MS is more similar with the progess report rather than a focused scholarly publication I would gladly review each individual paper or both papers after these or similar modifications are made. Without these modifications and changes, the MS is nearly impossible to follow and understand. Naturally, this is multi-year work by many professional co-workers. Accordingly, the exciting results the authors managed to acquire deserve to be presented in a better fashion rather than in a laundry list of experiments. R. As suggested by the reviewer, we attempted to streamline the manuscript by presenting primarily the novel set of data and confining “negative results’ to the Expanded view section and Appendix Figures. We would also like to point that most of the negative data were included since the canonical accepted function of RAB2A is in the control of ERGIC function and Golgi trafficking, which instead appear dispensable roles, at least for the regulation of MT1-MMP. Hence, the description of properly controlled, negative data was felt as necessary to rule out obvious functional roles of RAB2A. Nevertheless, we decided to limit the description of this set of finding according to reviewers’ suggestions. We also re-wrote the manuscript in various parts as suggested by this and other reviewers. However, following editorial indications, we did not split the manuscript in two parts.
Reference 1. 2. 3. 4. 5. 6. 7.
Imai K, Ohuchi E, Aoki T, Nomura H, Fujii Y, Sato H, Seiki M, Okada Y (1996) Membranetype matrix metalloproteinase 1 is a gelatinolytic enzyme and is secreted in a complex with tissue inhibitor of metalloproteinases 2. Cancer Res 56: 2707-10 Clark IM, Swingler TE, Sampieri CL, Edwards DR (2008) The regulation of matrix metalloproteinases and their inhibitors. Int J Biochem Cell Biol 40: 1362-78 Watanabe A, Hoshino D, Koshikawa N, Seiki M, Suzuki T, Ichikawa K (2013) Critical role of transient activity of MT1-MMP for ECM degradation in invadopodia. PLoS computational biology 9: e1003086 Saitou T, Itano K, Hoshino D, Koshikawa N, Seiki M, Ichikawa K, Suzuki T (2012) Control and inhibition analysis of complex formation processes. Theoretical biology & medical modelling 9: 33 Luo ML, Gong C, Chen CH, Hu H, Huang P, Zheng M, Yao Y, Wei S, Wulf G, Lieberman J, et al. (2015) The Rab2A GTPase promotes breast cancer stem cells and tumorigenesis via Erk signaling activation. Cell reports 11: 111-24 Marchesin V, Castro-Castro A, Lodillinsky C, Castagnino A, Cyrta J, Bonsang-Kitzis H, Fuhrmann L, Irondelle M, Infante E, Montagnac G, et al. (2015) ARF6-JIP3/4 regulate endosomal tubules for MT1-MMP exocytosis in cancer invasion. J Cell Biol 211: 339-58 Quintero OA, DiVito MM, Adikes RC, Kortan MB, Case LB, Lier AJ, Panaretos NS, Slater SQ, Rengarajan M, Feliu M, et al. (2009) Human Myo19 is a novel myosin that associates with mitochondria. Curr Biol 19: 2008-13 Addendum Figure for reviewer [Data removed per author’s request.]
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EMBO reports - Peer Review Process File – EMBO-2016-42032
Reference 1. ImaiK,OhuchiE,AokiT,NomuraH,FujiiY,SatoH,SeikiM,OkadaY(1996)Membranetypematrixmetalloproteinase1isagelatinolyticenzymeandissecretedinacomplexwithtissueinhibitorofm etalloproteinases2.Cancer Res 56:2707-10 1 ClarkIM,SwinglerTE,SampieriCL,EdwardsDR(2008)Theregulationofmatrixmetalloprotein asesandtheirinhibitors.Int J Biochem Cell Biol 40:1362 2 WatanabeA,HoshinoD,KoshikawaN,SeikiM,SuzukiT,IchikawaK(2013)Criticalroleoftransie ntactivityofMT1-MMPforECMdegradationininvadopodia.PLoS computational biology 9:e1003086 3 SaitouT,ItanoK,HoshinoD,KoshikawaN,SeikiM,IchikawaK,SuzukiT(2012)Controlandinhib itionanalysisofcomplexformationprocesses. Theoretical biology & medical modelling 9:33 4 LuoML,GongC,ChenCH,HuH,HuangP,ZhengM,YaoY,WeiS,WulfG,LiebermanJ, et al. (2015)TheRab2AGTPasepromotesbreastcancerstemcellsandtumorigenesisviaErksignalingactivation. Cell reports 11:111-24 5 MarchesinV,Castro-CastroA,LodillinskyC,CastagninoA,CyrtaJ,BonsangKitzisH,FuhrmannL,IrondelleM,InfanteE,MontagnacG, et al. (2015)ARF6JIP3/4regulateendosomaltubulesforMT1-MMPexocytosisincancerinvasion.J Cell Biol 211:339-58 6 QuinteroOA,DiVitoMM,AdikesRC,KortanMB,CaseLB,LierAJ,PanaretosNS,SlaterSQ,Ren garajanM,FeliuM, et al. (2009)HumanMyo19isa novelmyosinthatassociateswithmitochondria.Curr Biol 19:2008-13
[Data removed per author’s request]
Reference 1. 2.
Marchesin V, Castro-Castro A, Lodillinsky C, Castagnino A, Cyrta J, Bonsang-Kitzis H, Fuhrmann L, Irondelle M, Infante E, Montagnac G, et al. (2015) ARF6-JIP3/4 regulate endosomal tubules for MT1-MMP exocytosis in cancer invasion. J Cell Biol 211: 339-58 Quintero OA, DiVito MM, Adikes RC, Kortan MB, Case LB, Lier AJ, Panaretos NS, Slater SQ, Rengarajan M, Feliu M, et al. (2009) Human Myo19 is a novel myosin that associates with mitochondria. Curr Biol 19: 2008-13
2nd Editorial Decision
28 April 2016
I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to our journal. REFEREE REPORTS Referee #1 The original version of the manuscript was interesting and novel. However, the construction of the manuscript was flawed (writing, figure labelling, etc). Moreover, there were several specific issues with experimentation and interpretation that needed to be addressed. The revised version addresses all of the concerns that were raised. Referee #3 As a reviewer, I am impressed with the effort the authors have made to improve their MS. This appears to be a monumental study, with a plethora of the well-performed and adequatly controlled experiments. In my humble opinion, the MS in its correct, revised form reads better and is convincing and sound.
© European Molecular Biology Organization
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Corresponding Author Name: GIORGIO SCITA Journal Submitted to: EMBO REPORT Manuscript Number: EMBOR-‐2016-‐42032V2
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In the pink boxes below, provide the page number(s) of the manuscript draft or figure legend(s) where the information can be located. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable).
B-‐ Statistics and general methods
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return)
1.a. How was the sample size chosen to ensure adequate power to detect a pre-‐specified effect size?
The sample size was selected based on preliminary experiments and previous analysis. No power calculation was perfomed.
1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
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2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria pre-‐ established?
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3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe.
Analysis of invadopodia formation, cell invasion, endosome size and number were done blindly to the experimenters.
For animal studies, include a statement about randomization even if no randomization was used.
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Each of the cell line used has been identified through fingerprinting and is tested regularly for mycoplasma contamination by the IFOM cell culture facility.
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10. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, 2010) to ensure N/A that other relevant aspects of animal studies are adequately reported. See author guidelines, under ‘Reporting Guidelines’. See also: NIH (see link list at top right) and MRC (see link list at top right) recommendations. Please confirm compliance.
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The analysis of archival matierial from human breast cancer patients has been approved by the European Institute of Oncology ethical committee
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All samples were collected after obtaining inform consents and were anonimyzed to the experimenters and conformed the priniciple of WMA Declaration of Helsinki and the Department of Human Health Service Belmont Report.
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Data deposition in a public repository is mandatory for: a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data for small molecules d. Functional genomics data e. Proteomics and molecular interactions 19. Deposition is strongly recommended for any datasets that are central and integral to the study; please consider the N/A journal’s data policy. If no structured public repository exists for a given data type, we encourage the provision of datasets in the manuscript as a Supplementary Document (see author guidelines under ‘Expanded View’ or in unstructured repositories such as Dryad (see link list at top right) or Figshare (see link list at top right). 20. Access to human clinical and genomic datasets should be provided with as few restrictions as possible while N/A respecting ethical obligations to the patients and relevant medical and legal issues. If practically possible and compatible with the individual consent agreement used in the study, such data should be deposited in one of the major public access-‐ controlled repositories such as dbGAP (see link list at top right) or EGA (see link list at top right). 21. As far as possible, primary and referenced data should be formally cited in a Data Availability section. Please state N/A whether you have included this section. Examples: Primary Data Wetmore KM, Deutschbauer AM, Price MN, Arkin AP (2012). Comparison of gene expression and mutant fitness in Shewanella oneidensis MR-‐1. Gene Expression Omnibus GSE39462 Referenced Data Huang J, Brown AF, Lei M (2012). Crystal structure of the TRBD domain of TERT and the CR4/5 of TR. Protein Data Bank 4O26 AP-‐MS analysis of human histone deacetylase interactions in CEM-‐T cells (2013). PRIDE PXD000208 22. Computational models that are central and integral to a study should be shared without restrictions and provided in a N/A machine-‐readable form. The relevant accession numbers or links should be provided. When possible, standardized format (SBML, CellML) should be used instead of scripts (e.g. MATLAB). Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right). If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.
G-‐ Dual use research of concern 23. Could your study fall under dual use research restrictions? Please check biosecurity documents (see link list at top right) and list of select agents and toxins (APHIS/CDC) (see link list at top right). According to our biosecurity guidelines, provide a statement only if it could.
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