GENETIC ANALYSIS OF RAS SIGNALING PATHWAYS IN CELL PROLIFERATION, MIGRATION AND SURVIVAL

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1 Manuscript EMBO GENETIC ANALYSIS OF RAS SIGNALING PATHWAYS IN CELL PROLIFERATION, MIGRATION AND SURVIVAL Matthias Drosten, Alma Dhawahir, Eleanor Sum, Jelena Urosevic, Carmen Lechuga, Luis Esteban, Esther Castellano, Carmen Guerra, Eugenio Santos and Mariano Barbacid Corresponding author: Mariano Barbacid, Centro Nacional de Investigaciones OncolÛgicas - Member Review timeline: Submission date: 09 September 2009 Editorial Decision: 13 October 2009 Revision received: 11 January 2010 Accepted: 18 January 2010 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 13 October 2009 Thank you for submitting your manuscript to the EMBO journal. Your study has now been seen by three referees and their comments to authors are provided below. As you can see, referee #2 is not persuaded that we gain enough new insight into how Ras proteins function in order to consider publication here. However, both referees #1 and 3 are more supportive and find the analysis interesting, insightful and suitable for publication in the EMBO Journal. Given the strong support provided by both those referees, I will go with their overall recommendation and ask you to submit a suitably revised manuscript for our consideration. In the revised version please extend the analysis along the lines as indicated by referees #1 and #3 and also keep in mind the specific points raised by referee #2. When you send us your revision, please include a cover letter with an itemised list of all changes made, or your rebuttal, in response to comments from review. Thank you for the opportunity to consider your work for publication. I look forward to reading the revised manuscript. Yours sincerely, Editor The EMBO Journal REFEREE REPORTS Referee #1 (Remarks to the Author): European Molecular Biology Organization 1

2 This is an interesting and novel paper. In it the authors take work on the function of Ras proteins to its logical genetic endpoint by studying mouse embryo fibroblasts in which all three Ras genes have been removed. The results reported provide a number of unexpected observations, for example the apparent equivalence of Ras with B-Raf in this system. This paper is well executed and adds significantly to our understanding of the function of Ras proteins in cellular growth and transformation. A few minor issues could be addressed. The experiments in figures 5-7 address the ability of Ras and its effectors to support normal proliferation in serum, but the ability to induced transformed growth is not assessed. It would be interesting to see an analysis of how well B-Raf CAAX can induce transformed growth of Rasless MEFs (e.g. growth in soft agar) and whether or not this approaches what activated Ras can do. It is quite likely that the requirement for other effectors is more important in transformed than normal growth. In figure 7A, an indication of the number of colonies formed by MEK1 Q56P alone would be very useful in determining the synergistic impact of RlfCAAX or PTEN shrna. Referee #2 (Remarks to the Author): This manuscript characterizes the consequence of knocking out individual Ras genes and in various combinations in mouse embryo fibroblasts on cell proliferation, survival and migration. It also reveals which known Ras effectors can substitute for the loss of all Ras proteins in promoting cell proliferation. Overall, the main findings of the paper are that any of the three main Ras proteins can support fibroblast proliferation, with K-Ras appearing to be the most efficient. This does not however, help explain why multiple Ras forms exist nor advance understanding of why others have found differences between them in different types of assays. The paper also clearly shows that other closely related GTPases, like TC21, do not mimic this function of Ras in fibroblasts, even though this is the implication one gets from previous reports. Finally, the paper shows that the Raf/Mek/Erk pathway from Ras is most efficient at supporting cell proliferation in these cells. Overall, this is a very creative and well carried out study on the role of individual Ras protein family and Ras effector contribution to fibroblast proliferation. However, I do not feel enough novel information about how Ras promotes cell proliferation in fibroblasts warrants publication in EMBO J. A more speciality journal seems more appropriate. In addition, some weaknesses are listed below. 1) The approach used to test whether a specific Ras effector can compensate for the lack of all Ras function has some serious weaknesses. Differing versions of activated signaling molecules were used without really knowing how active they were in these cells and whether secondary effects could mislead results. For example, it is known that too much activity of some signaling molecules actually shuts down its effectors so that a negative finding in these studies could be hard to interpret. In fact, the authors document such a phenomenon when they show that activated C-Raf fails to rescue cell proliferation in this context. The authors knew this did not truly represent C-Rafs role in Ras mediated cell proliferation. That is because previous experiments showed that too much C-Raf causes a stress response that can be overcome by knocking down p16. The authors do not know if analogous phenomenon occur when activated forms of other signaling molecules tested fail to restore cell proliferation! 2) I don't understand Fig 7 where RlfCAAX and PTEN shrna are shown to promote cell proliferation in Ras-less cells only when they are co-expressed with activated MEK. But activated MEK alone promotes cell proliferation so no conclusions about RlfCAAX or PTEN can be made from these findings. Referee #3 (Remarks to the Author): The manuscript from Drosten et al describes the analysis of MEFs lacking H, N and KRas European Molecular Biology Organization 2

3 individually and in combination, using an elegant system in which the essential KRas gene is floxed and cre-mediated deletion controlled by either adeno-cre, or a 4HT-driven expression of a Cre-ER transgene. Using this system they show that any individual Ras isoform is sufficient to maintain proliferation, but that deletion of all three causes loss of proliferation, decreased cell migration, and increased focal adhesions. Inducible expression of a KRas transgene showed that these phenotypes were reversible. Neither upstream activated receptor tyrosine kinases, nor closely related G proteins could substitute for lack of Ras in rescuing cell proliferation. However, activation of the Raf/MEK/ERK pathway, but not RalGDS or PI3K, was sufficient to rescue proliferation. Combined activation of RalGDS and PI3K also did not rescue proliferation, but combination of all three effector pathways rescued to a greater extent that Raf alone. The importance of inactivation of pocket proteins by Ras in cell proliferation was shown by the ability of Rb knockdown to rescue proliferation in cells lacking Ras. Etopic expression of a stable cyclin D1 mutant was also sufficient to rescue proliferation, despite an increase in cyclin D1 seen in the absence of Ras. This is an interesting, well-controlled study that will be of interest to the readers of the EMBO Journal. The model system used to generate Ras-less MEFs is very elegant, and the reconstitution experiments appear reliable and convincing to identify relevant downstream components of Ras signaling that are required for proliferation under these circumstances. There are some additional experiments that would further strengthen the manuscript before publication, which are described below. 1. The authors have shown that inducible expression of WT and mutant KRas rescues BrdU incorporation in the Ras-less cells. It would be nice to show the data on the rescue of the additional phenotypes such as migration and focal adhesion formation in the Supplementary data. In additional, it would be nice to determine whether HRas and NRas were also capable of reversing these phenotypes, as well as the relevant importance of KRas4a vs 4b. 2. I think it is important to show the signaling consequences of introducing the genes in figures 5-7, and compare this to the positive control used, which is WT and activated HRas. For example, how well do the Raf-CCAX constructs increase MEK and ERK phosphorylation relative to HRas, how well do the PI3K constructs and PTEN shrna increase Akt phosphorylation etc. Similarly, if the authors wish to claim that the reason craf-cca doesn't rescue proliferation due to increased p16 expression, they should analyze the expression of p16 in response to the Raf-CAAX constructs as well as the control KRas. I would be surprised if p16 induction was limited to craf-caax but it will be important to show either way. 3. As loss of Ras decreases expression of cyclin A2 and cyclin E but not cyclin D1, it would seem more logical to determine whether ectopic expression of one of both of these genes rescues the effects on proliferation, rather than cyclin D1. Related to this, I don't understand the final assertion from the authors that cyclind/cdk complexes are not sufficint to inactivate the pocket proteins - it seems to me that they may. They should assay the activities of the different cyclin/cdk complexes following Ras deletion. Minor points. Move Figure 4 to Supplementary data? I didn't think the observation about the focal adhesions being more dynamic in the Ras+ cells relative to the Ras-less cells was particularly convincing. Are there additional videos that could illustrate this point more convincingly? Or perhaps the authors can indicate particular focal adhesions to focus on during the video? Name the lanes in Fig. 5B like in Fig. 5C. Please describe the difference between ERK2MEK1 and ERK2MEK1LA, and why the latter but not the former rescues proliferation The results from Tanaka et al did not show that Ras proteins were essential for proliferation, only for transformation. European Molecular Biology Organization 3

4 1st Revision - authors' response 11 January 2010 Reviewer 1: 1. It would be interesting to see an analysis of how well B-Raf CAAX can induce transformed growth of RaslessMEFs (e.g. growth in soft agar) and whether or not this approaches what activated Ras can do. Agree: We have carried out transformation assays in NIH3T3 cells as well as in Rasless MEFs. As indicated in the revised manuscript (see page 13), B-Raf CAAX has rather limited transforming activity in NIH3T3 cells and cannot transform Rasless MEFs in the presence of E1A. Thus, the intensity of B-Raf CAAX signaling appears to be less potent than that of Ras oncoproteins, a result that correlates well with its more limited efficiency in inducing proliferation of Rasless MEFs. We have also added a short paragraph to the Discussion regarding this issue. 2. In figure 7A, an indication of the number of colonies formed by MEK1 Q56P alone would be very useful in determining the synergistic impact of RlfCAAX or PTEN shrna. Agree: We have added this information to Figure 7A in the revised manuscript. Reviewer 2: 1. The approach used to test whether a specific Ras effector can compensate for the lack of all Ras function has some serious weaknesses. Differing versions of activated signaling molecules were used without really knowing how active they were in these cells and whether secondary effects could mislead results. For example, it is known that too much activity of some signaling molecules actually shuts down its effectors so that a negative finding in these studies could be hard to interpret. We appreciate the concerns raised by the reviewer. Yet, we trust that the reviewer realizes that these limitations are inherent to the experimental nature of the issues addressed in this study since there is no universal mechanism to constitutively activate all Ras effectors. To minimize this potential problem, we have measured the activity of the activated signaling molecules involved in this study including the different Raf and PI3K mutants. This information has been included in Supplementary Figures 3 and 4 and is described in the Results section (see pages 13 and 14). 2. I don't understand Fig 7 where RlfCAAX and PTEN shrna are shown to promote cell proliferation in Ras-less cells only when they are co-expressed with activated MEK. But activated MEK alone promotes cell proliferation so no conclusions about RlfCAAX or PTEN can be made from these findings. As illustrated in Figure 7A, Rlf CAAX and PTEN shrna cooperate with constitutively active Mek1 Q56P to induce proliferation of Rasless cells as efficiently as H-Ras or K-Ras oncoproteins. Yet, they cannot sustain proliferation of Rasless cells either by themselves or in combination. We do not quite understand why the reviewer says that so no conclusions about RlfCAAX or PTEN can be made from these findings. The conclusion is that constitutive activation of the RalGEF and PI3K pathways either alone or in combination cannot sustain proliferation of MEFs that lack Ras proteins. We feel that this is one of the main conclusions of the study and is clearly substantiated by the data. In any case, we have modified the text, including the Abstract, in an attempt to make these conclusions more clear. Reviewer 3: 1-1. The authors have shown that inducible expression of WT and mutant KRas rescues BrdU incorporation in the Ras-less cells. It would be nice to show the data on the rescue of the additional phenotypes such as migration and focal adhesion formation in the Supplementary data. European Molecular Biology Organization 4

5 Agree. We have added the data to the new Figure In addition, it would be nice to determine whether HRas and NRas were also capable of reversing these phenotypes, as well as the relevant importance of KRas4a vs 4b. We agree with the reviewer that it would have been nice to have this data. But, we hope the reviewer appreciates the effort that represents to generate all the data included in this manuscript with the three Ras isoforms. We find that the comment regarding the K-Ras4A and 4B isoforms is important and should be addressed. We have tested the effect of K-Ras4A G12V expression in Rasless cells. The results have been added to Supplementary Figure 2A and C and are described in the Results section (see page 10) I think it is important to show the signaling consequences of introducing the genes in figures 5-7, and compare this to the positive control used, which is WT and activated HRas. For example, how well do the Raf-CCAX constructs increase MEK and ERK phosphorylation relative to HRas, how well do the PI3K constructs and PTEN shrna increase Akt phosphorylation etc. Agree. We have carried out the experiments requested by the reviewer. They are shown in Supplementary Figures 3 (Raf proteins) and 4 (PI3K constructs and PTEN shrna). These results are described in the Results section pages 13 and 14 (Raf proteins) and pages 16 and 17 (PI3K constructs and PTEN shrna) Similarly, if the authors wish to claim that the reason craf-cca doesn't rescue proliferation due to increased p16 expression, they should analyze the expression of p16 in response to the Raf- CAAX constructs as well as the control KRas. I would be surprised if p16 induction was limited to craf-caax but it will be important to show either way. Agree: We have carried out the experiments requested by the reviewer. Indeed, as predicted by the reviewer, ectopic expression of each of the three Raf CAAX proteins resulted in increased p16 INK4a expression (Supplementary Figure 3). Yet, knock down of p16 INK4 a had no effect on the ability of A-Raf CAAX or B-Raf CAAX proteins to induce cell proliferation. Indeed, we observed similar results with the PI3K constructs and with PTEN shrna. Thus, we have changed the text to indicate that the stress response induced by C-Raf CAAX may not be directly mediated p16 INK4 a but by specific C-Raf effectors (see Results section, page 14). Yet, our results clearly illustrate that such stress response can be ultimately bypassed by knock down of p16 INK4a expression As loss of Ras decreases expression of cyclin A2 and cyclin E but not cyclin D1, it would seem more logical to determine whether ectopic expression of one of both of these genes rescues the effects on proliferation, rather than cyclin D1. Agree: As requested by the reviewer, we have ectopically expressed Cyclin D1, Cyclin E1, Cyclin A2 and Cyclin B1 in Rasless MEFs. This data is now shown in Figure 8 and is described in the Results section (page 18) Related to this, I don't understand the final assertion from the authors that cyclind/cdk complexes are not sufficint to inactivate the pocket proteins - it seems to me that they may. They should assay the activities of the different cyclin/cdk complexes following Ras deletion. Agree: As suggested by the reviewer, we have assayed the presence and activity of the different Cyclin/Cdk complexes in Rasless cells. We now report in the revised manuscript that Rasless cells have normal levels of Cyclin D1/Cdk4 and Cyclin E2/Cdk2 complexes (see Figure 8B). However, these complexes are not capable of phosphorylating Rb in vivo (see Figure 8C) and have significantly reduced kinase activity in vitro (see Supplementary Figure 6). This data has been added to the Results section in pages 18 and 19. Minor points: European Molecular Biology Organization 5

6 4. Move Figure 4 to Supplementary data? We realize that this figure may be trivial to the reviewer but in our experience, this schematic representation is very helpful to other scientists that may not be so familiar with this type of assay. Thus, we prefer to leave this schematic diagram in the main body of the manuscript. 5. I didn't think the observation about the focal adhesions being more dynamic in the Ras+ cells relative to the Ras-less cells was particularly convincing. Are there additional videos that could illustrate this point more convincingly? Or perhaps the authors can indicate particular focal adhesions to focus on during the video? Agree: To clarify this issue, we have added arrows to Supplementary Movie 3. In the control cells, the arrow now indicates a region with rapid focal adhesion turnover and in Rasless cells stable focal adhesions are highlighted. 6. Name the lanes in Fig. 5B like in Fig. 5C. Agree. We have now designated these proteins as R-Ras, R-Ras2 and R-Ras3 throughout the manuscript. 7. Please describe the difference between ERK2MEK1 and ERK2MEK1LA, and why the latter but not the former rescues proliferation. Agree: We have included the requested information in the text (page 14, lines 5-8). Briefly, ERK2MEK1LA is a fusion protein between the rat Erk2 and the human MEK1 kinases in which the four lysine residues of the MEK1 nuclear export motif were mutated to alanine, resulting in constitutive nuclear expression. In contrast, the ERK1MEK1 fusion protein does not contain these mutations. We added this information to the text (see Results section, page 14) as requested by the reviewer. Additional information describing these proteins can be found in the original publication by Robinson et al, Curr Biol., 8, 1141, The results from Tanaka et al did not show that Ras proteins were essential for proliferation, only for transformation. Agree: We have removed this reference from the text. European Molecular Biology Organization 6