CCM3 is a gatekeeper in focal adhesions regulating mechanotransduction and YAP/TAZ signalling
Authors: Vinay Swaminathan
Ryu Kanzaki
Håkan Axelson
Mattia Saggioro
David Lindgren
Emelie Englund
Johannes Kumra Ahnlide
Shan Wang
Chris D Madsen
Carmen Rodriguez-Cupello
Christelle N Prinz
Zhen Li
Pontus Kjellman
Kristian Pietras
Journal: Nature Cell Biology (2021)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: YAP/TAZ; CCM3; mechanotransduction; tumour progression; stem cell differentiation
Affiliations: Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden., Division of Solid State Physics and NanoLund, Lund University, Lund, Sweden., Division of Oncology, Department of Clinical Sciences, Lund University, Lund, Sweden., Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden. .

Abstract:  The YAP/TAZ transcriptional programme is not only a well-established driver of cancer progression and metastasis but also an important stimulator of tissue regeneration. Here we identified Cerebral cavernous malformations 3 (CCM3) as a regulator of mechanical cue-driven YAP/TAZ signalling, controlling both tumour progression and stem cell differentiation. We demonstrate that CCM3 localizes to focal adhesion sites in cancer-associated fibroblasts, where it regulates mechanotransduction and YAP/TAZ activation. Mechanistically, CCM3 and focal adhesion kinase (FAK) mutually compete for binding to paxillin to fine-tune FAK/Src/paxillin-driven mechanotransduction and YAP/TAZ activation. In mouse models of breast cancer, specific loss of CCM3 in cancer-associated fibroblasts leads to exacerbated tissue remodelling and force transmission to the matrix, resulting in reciprocal YAP/TAZ activation in the neighbouring tumour cells and dissemination of metastasis to distant organs. Similarly, CCM3 regulates the differentiation of mesenchymal stromal/stem cells. In conclusion, CCM3 is a gatekeeper in focal adhesions that controls mechanotransduction and YAP/TAZ signalling. 

Retracted: No

PubPeer comments:
 Pointed out to me by a reader. The supplementary materials belonging to this paper show the uncropped blots, which is always appreciated. However, with this paper, not all blots look uncropped, and the samples selected for the figures in papers appear very random. I can imagine someone running 10 different samples and incubating them with 5 different antibodies, and then finally selecting only two samples for the paper, but I would expect those two samples to be run on the same position on each of those gels or blots. However, with most figures the selected samples appear to have been run on - what appears to be - random positions in the different blots. Can the authors please clarify how the lanes chosen for the paper appear to be run on different positions for each individual blot? For starters, here is **Figure 1C** from the paper (left) and its corresponding uncropped blots (right). * The uncropped blots are not not really uncropped; I do not see the edges of the blots, in particular in the case of the CCM3 blot. * The relative position of the lanes chosen for the main article switch. In particular, alpha-SMA, lanes 9 10; pT850-MYPT1,lanes 4 5; pS19-MLC2,lanes 7 8, pLIMK1 2, lanes 1 2; pS3-Cofilin, lanes 5 6; CCM3, lanes 1-2? (not clear), Tubulin, lanes 5 6. ![file] , 
 The **Figure 3a** panels appear to show a similar haphazardly arrangement of lane positions. * The uncropped blots are not not really uncropped. * In some cases (e.g., pY397-FAK) the V-CAF and Cer-CAF lanes were run on the same gel, in other cases these lanes were run on individual gels. The gels appear to differ in number of lanes and arrangements of the sample order. * The relative position of the lanes chosen for the main article switch. For example, the HN-CAF Tubulin samples were run in lanes 3 and 4, while the corresponding pY118-Paxillin samples were taken from lanes 2 and 3, the pY416-Src samples from lanes 5 and 6, and the Paxilin samples came from lanes 1 and 2. * The HN-CAF pY416-Src and Cer-CAF pY416-Src blots might be different exposures of the same blot. Shown with pink boxes. ![file] , 
 **Extended Figure 7f**. * The CCM3 and Tubulin blots appear to be the reincubations of the same blot membrane, as per shape of the marker lanes. The blot has 12 to 14 lanes, but the left lanes did not react with the Tubulin antibody. Can the authors please clarify if these should have shown a band? ![file] , 
 #1 Yes, we know that the order of the samples are not always consecutive, as we had to run many blots over the last 5 years including additional blots during the revision. Many of these blots included additional siRNA gene depletions that were examined in an siRNA-screen in the initial phase of the project; therefore, the positions may seem a bit random. The WBs in Fig 1c and 3a are therefore merely showing representative blots from different biological repeats, meaning that not all blots were done on the same cell lysate. Thus, the WB in Fig 1c and 3a demonstrate representative blots while the quantification truly represent the differences. In the ideal world, we would had been able to show the tubulin (loading control) for each of the blots; however, that would have taken up too much space in the already very condensed figures. We therefore only show one representative tubulin blot. As this might have confused the reader, we have therefore compiled all the blots from Fig 1c and 3a, including the tubulin control blots in an additional pdf file that we have attached. We hope the reader is less confused now about the order of the blots. It was clearly not our intension to confuse the reader, however, we could not show so many tubulin blots in the figure panels. It is very important for us to state that all WB experiments were repeated many times as illustrated by the quantification in Fig 1c. For instance, aSMA expression in CAFs, were quantified from 12 independent biological repeats, while pS19-MLC2 levels were quantified from 14 independent biological repeats. It should also be stated that multiple persons in the laboratory ran the different WB experiments throughout the paper, and we are very confident of the outcome. Finally, the CCM3 antibody is very difficult to develop by western blot. The exposure time needs at least 10-15 min and the blots are still not very nice. Here we used a Bio-Rad CCD camera with stronger substrate to get the blots. The protein ladder is not shown by Bio-Rad CCD camera. ![file] #2 As the supplementary materials, we showed the original data. In most of the blots, we ran gels and cut the membrane to incubate different antibodies. And we tried to show the necessary data in the supplementary materials and not to make readers confused. Again, in most of the gels, we ran many different samples and incubated them with several different antibodies. The results are therefore from different experiments with different samples. Again, we have many biological repeats for each antibody and have the quantifications, where each data point represents an independent biological experiment. Such as pY397-FAK and pY118-Paxillin expression in CAFs, we quantified 6 different biological repeats. We chose the representative blots from one of these results and showed them in the figures. Yes, the HN-CAF pY416-Src and Cer-CAF pY416-Src blots are different exposures of the same blot. Both HN-CAF (siCtr and siCCM3) and Cer-CAF (siCtr and siCCM3) were loaded in the same gel and were incubated by pY416-Src. Cer-CAF pY416-Src blots needs shorter exposure time but HN-CAF pY416-Src needs longer exposure time. The Cer-CAF pY416-Src blots were also showed in the longer exposure results together with HN-CAF pY416-Src but itu2019s over exposure. ![file] #3 Yes, the CCM3 and Tubulin blots are from the same blot membrane. The CCM3 antibody is very difficult to develop by western blot. The exposure time needs at least 10-15min and the blots are still not very nice. They are weak and with high background. Here the author took 18min to get the blots. The Tubulin blots only needs 10s. We found that the left lanes (from 1 to 10) were very very poor lysates that they had some strange bands when developed for 18min. They didnu2019t work at all. Thatu2019s why there was no bands when developed for 10s. , 
 This is interesting although I found it difficult to understand as all so-called uncropped blots are semi-cropped. I appreciate the discussion from both parties. It would be nice if the authors could explain a few more figures. Examples added. Extended figure 1c. Why pS19-MCL2 was cropped from a different place? ![file] Extended figure 1c. It seems that molecular weight markers were inserted in TAZ blots? pS109-YAP and pLASTS1 2 blots were from the same membrane, then are those logical cropping bands from a different place? ![file] Finally, in extended figure 9b, FAK, CCM3, and tubulin blots are from the same membrane, and aSMA, tubulin, and pS19-MCL2 blots are from the same membrane? Then how do authors motivate the selections? ![file] , 
 You are completely right; we should have chosen the same membrane with the same cell lysates for the pMLC2 blot. I donu2019t know why we ended up choosing the other membrane. This was an unintended mistake, but as you can see from the second experiments, the data are reproducible and loss of CCM3 induced increased pS19-MLC2. However, it would of course have been more appropriate to use the same membrane, which even demonstrate a higher induction as compared to the one we show in Ext. Fig 1c. We apologies for that. Just to give you more information on the two different membranes: In the first membrane with aSMA, CCM3, and tubulin blots, we loaded the following samples: siCtr, siCCM3_sp, siMST3, wt, siCtr, siCCM3_01, siCCM3_02, siCCM3_04, siCCM3_SP. On the second membrane with pS19-MLC2, we loaded samples from a different experiment. The tubulin for pS19-MLC2 now is attached in the image. The loading samples were: siCtr, siCCM3_sp, siMST3, siCtr, siCCM3_01, siCCM3_02, siCCM3_04, siCCM3_SP. As noticed by your sharp eyes, the same tubulin blot was mistakenly shown for both membrane 1 and 2. This is an obvious mistake from our side when we looked through all our WB scans. We have attached a new file with the two independent tubulin blots. We are truly sorry for this mistake and apologies for the confusion. ![file] Regarding the insertion of the molecular weight marker in the TAZ blots, this is a huge mistake from our side. I spoke with my technician about this and she explains that by mistake, the molecular weight markers were copied and overlaid the original scans. This resulted in a small shift of the molecular weight marker compared to the original membrane. We have attached the original membrane showing the same molecular weight marker but in its correct position, showing that the marker is slightly higher than the one in the Extended WB scans presented in the paper. We cannot express deeply enough how sorry, frustrated and disappointed we are by ourselves for making such a huge error in a paper. This kind of mistake should of course not happen and we are truly sorry for this. ![file] The pS109-YAP and pLASTS1 2 blots were from four independent biological repeats separated on the same gel and used for the quantification in the figure. One of the 4 biological repeats were then chosen for the figure as a representative sample. ![file] When we generated the CRISPR-Cas9-cell lines were began by testing different sgRNA targeting CCM3. First, we ran some CCM3 blots of different sgRNAu2019s and it seems that we forgot to add the tubulin blot for the 5x sample WB blot. We have now added the tubulin blot (see below). On a different membrane we then characterized the level of aSMA and pS19-MLC2 from two independent experiments (different dates of cell culture). We loaded both consecutive experiments on the same gel: sgControl, sgCCM3#1, sgCCM3#2, and sgControl, sgCCM3#1, sgCCM3#2 (lane 1-3: first experiment; lane 4-6, second experiment). In the Ext Fig 9b my technician ended up picking the aSMA and tubulin from the first set of lysates (lane 1-3), while she picked the pS19-MLC2 from the second set of lysates (lane 4-6). I donu2019t know why she did this, but itu2019s an honest mistake, and it is still clear that both independent sets of lysates (lane 1-3 vs lane 4-6) are showing the same results: both sgCCM3u2019s induce pS19-MLC2 expression. On a later time point, we then analyzed the expression of pY397-FAK in our favorite sgCCM3 cell line and compared it to the sgControl cells. On the same gel, we included other established CRISPR-Cas9-modifed cells (sgControl vs sgMST3 (targeting the MST3 kinase)). The results from the sgMST3 is not part of the paper. ![file] , 
 Author responses have many points to be discussed. As others also suggested, **please pay attention to uncropped raw blots**. It adds more confusion speculation if authors consider semicropped blots as uncropped raw blots (that is what happened in data posted by authors here). It is still not clear how samples were loaded in several figures. None of the blots were labeled with sample names. For example, both pT850-MYPT1 and pS3-Cofilin blots (Fig 1c) contain 9 samples, probably from 3 experiments? It can not be an excuse that the Bio-rad CCD camera does not detect molecular weight markers, it is the author s responsibility to document molecular weight markers. More importantly, the experimental setting is questionable. If all blots in the same figure panel are not from the same experiment that the author stated in response, showing only loading control is not valid for this experimental setting. The author used siRNA for several experiments. Therefore, it is important to consider the CCM3 knockdown efficiency for each sample in each experiment. There are several blots in the author s response that are confusing. Please see below-marked blots and comments on figures. ![file] ------------------------------------------------------------------------------------------------------------ ![file] ------------------------------------------------------------------------------------------------------------ ![file] ------------------------------------------------------------------------------------------------------------ ![file] **I think the author performed experiments is confused with the term uncropped blot, senior authors could offer help.** ![file] , 
 We apologies for not providing uncropped blots showing all four edges throughout the paper. This could have been done more elegantly, we agree. Thanks for pointing this out. I would like to state that we have repeated these experiments many independent times as demonstrated by the quantifications. We hope this can calm down the confusion and we point to the fact that the conclusions are supported by other assay types like IF, force measurements, point-mutations experiments, over-expression experiments including rescue experiments where siRNA is combined with overexpression of various different CCM3-constructs. The experiments were also conducted in many different fibroblasts from different tissue and MSCs. We are very sorry if the uncropped WBs may have caused confusions, but we ask the reader to look at all the data in a coherent manner. The conclusions are drawn on all the data in the paper, and not from single WBs. We think our findings are very important and we hope that these results will start preclinical and later on clinical testing of patients we the disease Cerebral Cavernous Malformation. These patients have no treatment options except surgery, so having new options would be very beneficial. So, in conclusion, we are very sorry for the confusion that the uncropped WBs may have brought to the reader, but please remember to consider the coherent picture, including all the experimental assays, the biological independent repeats, the use of many different fibroblast lines and all the rescue experiments conducted throughout the paper. We hope the reader still appreciate our hard work and the significant findings in our paper, despite the issue with the uncropped WBs? , 
 #8  ...we ask the reader to look at all the data in a coherent manner. The point of a scientific paper, I think you ll agree, is for the author(s) to present their data in a coherent manner, not leave it to the reader.  ...we hope that these results will start preclinical and later on clinical testing of patients we the disease Cerebral Cavernous Malformation. These patients have no treatment options except surgery, so having new options would be very beneficial. This illustrates even more why it is important to present your data in a coherent manner. If there is anything wrong with your work (and I don t think that s what anyone is alleging here), then it is important to catch it long before you start experimenting on some of the most vulnerable members of our society. ,