DEFINING A TISSUE STEM CELL DRIVEN RUNX1/STAT3 SIGNALING AXIS IN EPITHELIAL CANCER

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1 Manuscript EMBO DEFINING A TISSUE STEM CELL DRIVEN RUNX1/STAT3 SIGNALING AXIS IN EPITHELIAL CANCER Cornelia Johanna Franziska Scheitz, Tae Seung Lee, David James McDermitt, and Tudorita Tumbar Corresponding author: Tudorita Tumbar, Cornell University Review timeline: Submission date: 01 May 2012 Editorial Decision: 31 May 2012 Revision received: 21 August 2012 Editorial Decision: 26 August 2012 Accepted: 26 August 2012 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 31 May 2012 Thank you very much for submitting your research paper that tries to generalize a role of Runx1 in human epithelial cancer for consideration to The EMBO Journal editorial office. Having received a full set of referee comments, I am in a position to reach a final decision to enable efficient proceedings for you and the paper. As you will learn from the enclosed reports, the referees generally judge the topic as an interesting one but are divided in their eventual recommendation for potential further pursuit. Major reasons for this are relatively broad claims with regard to various epithelial tumors that remain only experimentally substantiated in the epidermal system studied here in some detail. Thus, stronger evidence for a rather general role of RUNX1 (as remarked on figure 1 by essentially all referees) and in particular the intestine (ref#1's comment on figure3) would be needed to corroborate this claim. A second critical issue is the actual biological function of RUNX1 in this system. Both ref#2 and #3 speculate about a proliferative advantage of RUNX1-expressing cells, an issue that would need further attention as particularly ref#2 concludes based on this point that the paper seems overall not that surprising. As these major issues would have to be addressed by a fair amount of experimental work that seems however feasible based also on the constructive suggestions made by the referee, we would be willing to grant the opportunity to significantly extend and revise the current dataset paper. As this entails novel and time-consuming experimentation, we would understand if you might decide to seek potential rapid publication elsewhere. However, and in case you embark on revisions for our more general and strongly selective title, I urge you to take the specified demands into careful consideration to avoid disappointments much later in the process. Please do not hesitate to contact me with in case of further questions (preferably via to have this on record according to our transparency initiative). EMBO 1

2 I do hope that clearly communicating our demands and expectations facilitates efficient proceedings for this project and are looking forward assessing a revised study. Yours sincerely, Editor The EMBO Journal REFEREE REPORTS: Referee #1: In this well written manuscript Scheitz et al address the role of the Runx1 transcription factor in malignancy, using the epidermis as an in vivo model. Previous work by this group has established that Runx1 expression is induced by treatment of the epidermis with chemical carcinogens and that deletion of Runx1 reduces the number of tumors following carcinogen treatment (Hoi et al., MCB 2010). This study significantly expands our knowledge of the role of Runx1 in carcinogenesis, with implications for tumours beyond the epidermis. Specific points: Figure 1 examines Runx1 expression in a range of tumor types. The authors state "Runx1 was among the top 1% of all over-expressed genes in head and neck, and skin SCC, and was highly expressed in several other epithelial tumors such as esophageal SCC, cervical carcinoma, and colon adenocarcinoma". It would be helpful to clarify what is meant by "over-expressed", as for a tumor it is unclear what one can compare gene expression to. Does this mean than Runx1 is in the top 1% most abundant of all genes in the tumor, or that it is in the top 1% of genes whose expression is increased compared with a normal tissue control? Immunofluorescence is then used to examine Runx1 expression in a set of cell lines...a Western blot might be helpful to confirm the relative protein expression between lines. The images of tumors are difficult to interpret. Is the whole of each image within the tumor or is the interface between stroma and tumor shown? If the latter it would help to add some labels/arrows to orientate the reader. It would be most helpful to show an image of Runx1 staining in normal human epidermis and oral mucosa for comparison. In Figure 2 an inducible Cre driven by the basal layer specific promoter K14 is used to delete Runx1 at various stages of a chemical carcinogenesis protocol. The data show that deletion of Runx 1in a BL6 prior to initiation causes a substantial reduction in tumor number while deletion post initiation has minimal effect. Strikingly the tumors that do form after Runx1 deletion contain Runx1 positive cells, presumably deriving from cells that escaped Runx1 deletion, and is confirmation of the competitive disadvantage of cells lacking Runx1. This point would be more convincing if the image showing Runx1 and Krt10 staining was made clearer, perhaps by showing a version with the channels separated in supplementary and/or staining with a pan keratin antibody, to convince the reader that the Runx1 positive cells are indeed epithelial and sstromal or immune cells which would not be targeted by K14 cre. Impressively the authors also show the results apply in the more tumor prone FVB/n background. It would be helpful to add a line to explain exactly what the authors mean by a tumor stem cell and to summarise the evidence that these express CD34. Deletion of Runx1 is also shown to delay tumor formation in Ras mutant mice. When the authors mention rectal tumors, do they mean anal? The anal margin is targeted by K14cre, whereas the rectum proper is not. Anal tumors have been observed in ras mutant mice and this would seem to be more likely (Eg van de Weyden et al., J Pathol, 2011). In Figure 3 lineage tracing from Runx1 creer mice is used to track the fate of the progeny of Runx1 positive cells. Long lived Runx1 derived cells are found in hair follicles and oral mucosa. The authors also comment on the intestine. This data is relatively weak, as it is important to confirm that the labeled cells are indeed epithelial and not of haemopoietic origin, that they express stem cell markers, eg Lgr5 in the case of the crypt base cells and explain the disparate results seen with EMBO 2

3 different reporters. These findings seem peripheral to the main story and might perhaps be omitted from the manuscript without losing much. If the data remains in it needs to be reinforced significantly. Figure 4 combines lineage tracing with carcinogenesis. Tumors are found to derive from labeled cells which are found in the hair follicle bulge. To be able to make a definitive statement on the origin of the tumors it is essential to exclude the presence of rare Runx1 positive cells in the interfollicular epidermis. The authors should either provide strong evidence (eg from wholemounts, Ito et al. Nature Medicine 2005), or make sure this point is qualified in the text. Figure 5 is an impressive demonstration that deletion of Runx1 causes tumor regression, showing Runx1 is required for the growth of established tumors as well as their development. This result is supported by the effects of shrna knockdown of Runx1 in human SCC lines. The study concludes, Figure 6, with a demonstration that loss of Runx1 reduces Stat signaling via effects on SOCS. Overall this is a most impressive study with significance beyond the epidermal field. The experiments are well executed, clearly described and appropriately interpreted. The manuscript might be improved by considering the minor points above, but overall is of high quality and suitable for publication. Referee #2: The authors present interesting data that Runx1 plays a critical role in several epithelial cancer types, using a variety of previously characterized mouse models. In doing so they build on their previous studies, which provide a firm foundation and to some extent impact on the novelty of the message. In the current manuscript they investigate the potential stem cell role of Runx1 in murine tumours and complement these data with oncomine database mining and immunostaining of human pathological specimens. In its current form the paper is not particularly accessible to a non-expert audience but the message that Runx1 might be a novel therapeutic target in cancer as a result of its role in stem cell biology as opposed to merely conveying a proliferative signal is not clear to me. I have a general concern with the "stem cell" message of this paper. What is to say that Runx1 doesn't just convey a proliferative signal. If you lose it prior to DMBA induction then cells are less primed to proliferate and may be less sensitive to damage (DMBA is more toxic to actively dividing cells). However, if you don't lose it until the cells are exposed already to a strong proliferative influence (eg TPA treatment) then the loss has no effect due to the highly proliferative influence of TPA. Once TPA treatment is stopped then the cells once again become sensitive to removal of the Runx1 mediated proliferative signal. The novelty of the data is not too clear - their MCB paper (Hoi 2010) already shows the requirement for Runx1 in DMBA/TPA carcinogenesis and also describes the Stat3 link (although not the SOCS 3/4 intermediates) and their JCB paper (Osorio cited, but not included in references) describes the importance of Runx1 in HFSCs. Fig 1. The staining of human/mouse sections in fig1 is unremarkable and lacks comparison with normal tissue. Why do they show the non-scc cell lines only - I would like to see the data rather than just see images for the less relevant cell lines and a bar graph and table of the more interesting lines. Furthermore no mention of how staining was quantitated in 1D. Sup fig 2A and B should be reversed so that they are mentioned in order in the ms. White arrows don't seem to illustrate anything particularly convincing. Fig 2. Should the graphs be relabeled as papillomas rather than tumours? EMBO 3

4 The colours of the dotted lines in G don't match the key. The CD34 +ve cells look nothing like those in the Malanchi paper - they don't look to be in the epithelial compartment. Fig 4. The lines linking the bars with the picture in B don't make sense. Sup Fig 3. Not clearly described in the legend Sup Fig 4. Dashed line in A is not described? Labelling in B - Bu = bulge Sup Fig 5. A. What is fold change referring to? B. What is the significance of the colours? Where are the real data? The ChIP figure is a real shame given that this is potentially exciting data - need some evidence. I certainly think it is unacceptable to cite that the method will be published elsewhere (unpublished data)? C. What happens if you knock down both SOCS 3 and 4 together? Where is the quantitation? Or what happens if knockdown either in the absence of Runx1 knockdown? Referee #3: The manuscript of Scheitz and colleagues is built on previous publications showing that deletion of Runx1 in the mouse epidermis protects from tumour formation in chemically induced skin tumourigenesis assays. Scheitz et al. now show that Runx1 is over-expressed in various epithelial tumours and investigate whether Runx1 is needed for the initiation or maintenance of tumour formation. Expression of Runx1 seems mainly important for tumour initiation and the authors mechanistically explain this effect by the negative transcriptional regulation of the Stat3 inhibitors SOCS3 and SOCS4 by Runx1. How cancer and tissue stem cells are regulated during tumour development and maintenance is highly important to define novel therapeutic strategies in cancer. Thus, I consider this manuscript as a potential important contribution to the field. However, I do have some major concerns that need to be addressed before I can recommend publication in EMBO J. Although the authors provide a tremendous amount of data, the overall manuscript is at times quite superficial and some strong claims are not supported by the data provided. For instance, the relevance to human cancers as implicated in the abstract and the introduction is low and solely descriptive. In addition, the title implicates an important role of Runx1 in various epithelial tissues. However, the authors themselves exclude other than squamous epithelia from the analysis because the data are inconclusive. In my opinion the manuscript including the summary figure 7 needs to be revised and more carefully worded. Major concerns: 1. Aim of figure 1 was to determine Runx1 in human epithelial cancers. Except for skin cancers, the authors do not provide evidence that Runx1 protein is indeed expressed in primary epithelial tumour samples. 2. The authors do not determine Cre-recombinase efficiency throughput the epidermis in their different treatment regimes, which makes it very difficult to draw precise conclusions from the respective experiments. Cre-recombination should be achieved in all K14-positive layers and Runx1-positive tumours derive from non-recombined keratinocytes. It is unclear to me why the authors chose to treat only once instead of treating longer with Tamoxifen to achieve a more homogenous recombination. EMBO 4

5 3. On page 8 lines 21-25; the authors conclude that 'that Runx1-negative tumours are shortlived'. How can this be explained? Do Runx1-negative cells exhibit a proliferative disadvantage to Runx1-positive cells? If yes, are Runx1-negative tumours smaller in size? 4. To make the argument that Runx1 may be important for the maintenance of the CD34+ tumour stem cells, at a minimum the authors have to provide some quantification. 5. Figure 3B: LacZ staining at 7 month is not very convincing. 6. The statement that Runx1-expressing adult bulge cells are '... differentiating HFSCs' is not supported by data. At a minimum a co-expression of LacZ with differentiation markers of hair lineages need to be provided. 7. The relevance of the LacZ stainings in figure 3E is unclear to me. The authors do not specify the tamoxifen treatment protocol or mention after which time points the samples have been selected. For instance, LacZ marks scattered cells throughout the intestine. How is this possible? Given that the intestine has a relative high cellular turnover one would expect a LacZ-positive villus after tracing, if Runx1 is indeed expressed in crypt stem cells. In addition, figures S3B,C do not support the LacZ staining in figure 3E. Detecting endogenous expression of Runx1, at least in the oral epithelium should further support these data. 8. Figures 4A-C are confusing. The authors state in the text (page 13; line 1) that the bulge scheme resulted in 'no infundibular or other epithelial cell labelling'. However, in figure 4C 20% of infundibular cells were labelled under the bulge scheme. 9. Why are infundibular cells labelled in the first place? The authors do not provide evidence that Runx1 bulge cells can contribute to the infundibulum or for endogenous expression of Runx1 in the infundibulum. If rare cells of the infundibulum express Runx1, how can the authors exclude that there are also rare cells in the interfollicular epidermis expressing Runx1 contributing to their assay. 10. Figure 6 convincingly connects loss of Runx1 to impaired Stat3 signalling. However, in these assays normal keratinocytes and the cancer cell lines are similarly dependent on Runx1 in these in vitro assays. A comment on why there is a strict dependence on Runx1 in vitro but survival of normal and cancer cells in vivo does not depend on Runx1 would be helpful. Minor points: 1. I believe the header of figure 1F should say 'initiation'. 1st Revision - Authors' Response 21 August 2012 We thank the reviewers for their constructive criticism that have substantially improved out manuscript. Referee #1: We thank this reviewer for the positive evaluation of our manuscript. In this well written manuscript Scheitz et al address the role of the Runx1 transcription factor in malignancy, using the epidermis as an in vivo model. Previous work by this group has established that Runx1 expression is induced by treatment of the epidermis with chemical carcinogens and that deletion of Runx1 reduces the number of tumours following carcinogen treatment (Hoi et al., MCB 2010). This study significantly expands our knowledge of the role of Runx1 in carcinogenesis, with implications for tumours beyond the epidermis. Specific points: Figure 1 examines Runx1 expression in a range of tumour types. The authors state "Runx1 was among the top 1% of all over-expressed genes in head and neck, and skin SCC, and was highly expressed in several other epithelial tumours such as esophageal SCC, cervical carcinoma, and colon adenocarcinoma". It would be helpful to clarify what is meant by "over-expressed", as for a tumour it is unclear what one can compare gene expression to. Does this mean than Runx1 is in the top 1% most abundant of all genes in the tumour, or that it is in the top 1% of genes whose expression is increased compared with a normal tissue control? We changed the sentence from Notably, Runx1 was among to "Notably, comparing tumour to normal tissue Runx1 was among..." to clarify the comparison. Page 5 EMBO 5

6 Immunofluorescence is then used to examine Runx1 expression in a set of cell lines...a Western blot might be helpful to confirm the relative protein expression between lines. We added a new western blot showing Runx1 levels in the mouse cell lines and all human SCC associated cell lines in Figure 1B. The images of tumours are difficult to interpret. Is the whole of each image within the tumour or is the interface between stroma and tumour shown? If the latter it would help to add some labels/arrows to orientate the reader. The entire image is from the tumour, but within we can delineate tumour and stroma based on keratins. We added a dashed line to mark the interface between tumour and stroma and labelled the stroma with * in the appropriate images in Figure 1 and 2. It would be most helpful to show an image of Runx1 staining in normal human epidermis and oral mucosa for comparison. To clarify the difference in Runx1 level, we added a picture of Runx1 in a normal human hair follicle at equal exposure to Figure 1E. In Figure 2 an inducible Cre driven by the basal layer specific promoter K14 is used to delete Runx1 at various stages of a chemical carcinogenesis protocol. The data show that deletion of Runx 1 in a BL6 prior to initiation causes a substantial reduction in tumour number while deletion post initiation has minimal effect. Strikingly the tumours that do form after Runx1 deletion contain Runx1 positive cells, presumably deriving from cells that escaped Runx1 deletion, and is confirmation of the competitive disadvantage of cells lacking Runx1. This point would be more convincing if the image showing Runx1 and Krt10 staining was made clearer, perhaps by showing a version with the channels separated in supplementary and/or staining with a pan keratin antibody, to convince the reader that the Runx1 positive cells are indeed epithelial and Stromal or immune cells which would not be targeted by K14 cre. We show the separate channels in Supplementary Figure 2D and added a Keratin14 staining of a previous serial section. In 2D KO- (how can we make it clear that this was a tumour that escaped targeting is confusing if we just read the labels Impressively the authors also show the results apply in the more tumour prone FV B/nbackground. It would be helpful to add a line to explain exactly what the authors mean by a tumour stem cell and to summarise the evidence that these express CD34. We added this sentence In general, tumour SCs maintain tumours and cause their reoccurrence for extended periods of time, like CD34+ cells that initiate SCC formation upon serial transplantation for at least 3 generations (Malanchi et al., 2008). Page 9 Deletion of Runx1 is also shown to delay tumour formation in Ras mutant mice. When the authors mention rectal tumours, do they mean anal? The anal margin is targeted by K14cre, whereas the rectum proper is not. Anal tumours have been observed in ras mutant mice and this would seem to be more likely (Eg van de Weyden et al., J Pathol, 2011). This reviewer is right. We changed rectal to anal throughout the manuscript. In Figure 3 lineage tracing from Runx1 creer mice is used to track the fate of the progeny of Runx1 positive cells. Long lived Runx1 derived cells are found in hair follicles and oral mucosa. The authors also comment on the intestine. This data is relatively weak, as it is important to confirm that the labelled cells are indeed epithelial and not of haemopoietic origin, that they express stem cell markers, eg Lgr5 in the case of the crypt base cells and explain the disparate results seen with different reporters. These findings seem peripheral to the main story and might perhaps be omitted from the manuscript without losing much. If the data remains in it needs to be reinforced significantly. There seems to be a general misunderstanding with original Figure 3 E. This is not lineage tracing, but endogenous Runx1 expression as shown by a LacZ KI. The lineage tracing for the colon and intestine is presented in the supplement. We cannot explain the disparity between the 3 different lineage-tracing markers especially in the intestine. It is to note that in the skin they all show the same pattern. So the inconsistencies could have something to do with different protein stabilities in the different tissues and cells. Additionally, although all are in the same locus they are driven by different promoters, which could affect their expression in different tissues as well. Upon careful analysis it appears as though Runx1-CreER is extremely inefficient in the intestinal epithelium, which precluded labelling and EMBO 6

7 therefore consistent lineage analysis in this tissue. Since Runx1 has two promoters and an intricate isoform structure, it is possible that the insertion of CreER upstream of the 2 nd promoter does not favour expression of CreER in the intestine in a pattern that recapitulates Runx1 endogenous expression. Since addressing this problem, would require ~ 2 years to generate a new targeting vector and a new mouse model, it is therefore beyond our powers to move this project further in this direction. Therefore, we removed the intestinal and colon lineage tracing data that was presented in the supplementary. However, we were able to co-localize Runx1 immunofluorescence antibody signals with a subset of LGR5 expressing cells, which supports the idea that at least some intestinal stem cells may express Runx1. This was corroborated by Runx1-LacZ endogenous expression data, and co-localization with epithelial markers K8. (new Figure 6) We feel that with our expression data and co-localization with LGR5 we have further strengthened the relationship with a 3 rd stem cell population, which along with all of our other data justify our model. Figure 4 combines lineage tracing with carcinogenesis. Tumours are found to derive from labelled cells which are found in the hair follicle bulge. To be able to make a definitive statement on the origin of the tumours it is essential to exclude the presence of rare Runx1 positive cells in the interfollicular epidermis. The authors should either provide strong evidence (e.g. from whole mounts, Ito et al. Nature Medicine 2005), or make sure this point is qualified in the text. We now quantified IFE labelling resulting in 0 blue cells in appr IFEs. This information was added to the appropriate paragraphs. Figure 5 is an impressive demonstration that deletion of Runx1 causes tumour regression, showing Runx1 is required for the growth of established tumours as well as their development. This result is supported by the effects of shrna knockdown of Runx1 in human SCC lines. The study concludes, Figure 6, with a demonstration that loss of Runx1 reduces Stat signalling via effects on SOCS. Overall this is a most impressive study with significance beyond the epidermal field. The experiments are well executed, clearly described and appropriately interpreted. The manuscript might be improved by considering the minor points above, but overall is of high quality and suitable for publication. Referee #2: The authors present interesting data that Runx1 plays a critical role in several epithelial cancer types, using a variety of previously characterized mouse models. In doing so they build on their previous studies, which provide a firm foundation and to some extent impact on the novelty of the message. In the current manuscript they investigate the potential stem cell role of Runx1 in murine tumours and complement these data with oncomine database mining and immunostaining of human pathological specimens. In its current form the paper is not particularly accessible to a non-expert audience but the message that Runx1 might be a novel therapeutic target in cancer as a result of its role in stem cell biology as opposed to merely conveying a proliferative signal is not clear to me. We feel that data are already compelling in favour of our interpretation that Runx1 role in cancer is relevant in proliferation only in stem cells. Runx1 is not expressed in the rapidly dividing progenitor cells (matrix) of the hair follicle (our published work). Our lineage tracing showed that bulge stem cells but not infundibullar non-stem cells that express Runx1 contribute to tumorigenesis. The simple expression of Runx1 in a non-stem cell population (the infundibulum) was insufficient to render those cells into tumour producing cells by conferring them a growth advantage. Thus Runx1 must work in stem cells in the epithelial cancers to confer its essential function in tumour growth. I have a general concern with the "stem cell" message of this paper. What is to say that Runx1 doesn't just convey a proliferative signal. If you lose it prior to DMBA induction then cells are less primed to proliferate and may be less sensitive to damage (DMBA is more toxic to actively dividing EMBO 7

8 cells). However, if you don't lose it until the cells are exposed already to a strong proliferative influence (e.g. TPA treatment) then the loss has no effect due to the highly proliferative influence of TPA. Once TPA treatment is stopped then the cells once again become sensitive to removal of the Runx1 mediated proliferative signal. We are not doubting that there is a proliferative component to the phenotype, however as we mention above our lineage tracing shows that Runx1 acts in the stem cells to confer this advantage, and not in more differentiated short-lived cells. Additionally, we have shown that Runx1 protects from tumour formation in the Kras mutant background. This eliminates the use of DMBA and thus the questions of priming cells for damage. The novelty of the data is not too clear - their MCB paper (Hoi 2010) already shows the requirement for Runx1 in DMBA/TPA carcinogenesis and also describes the Stat3 link (although not the SOCS 3/4 intermediates) and their JCB paper (Osorio cited, but not included in references) describes the importance of Runx1 in HFSCs. We now included Osorio in the reference, thank you for pointing this out. Hoi et al only describes the general connection. Here we pinpoint the exact stage and the mechanism of Runx1 involvement. In Hoi et al., we only show that activated Stat3 is reduced in Runx1 KO tumours. This could be due to an indirect effect of Runx1 within keratinocytes on inflammation, which could explain our previous observation, without the need to have Runx1 as a direct upstream regulator of Stat3 signalling. In this report we take cells outside of the context of the skin and possible inflammation and show for the first time that indeed Stat3 is signalling is directly downstream of Runx1 in epithelial cells of the skin. Furthermore we extent these findings to mouse and human cancer cells: SCCs both of skin and the oral epithelium and the ovary. Moreover, we provide the mechanism of this action by regulating phosphorylation of Stat3 via SOCS3 and 4 transcriptional repression. In terms of tissue homeostasis, this is the first report showing by lineage tracing, which is the ultimate test, that Runx1is expressed in the adult HFSCs. Osorio et al only showed it found in early embryonic precursors. Moreover, Runx1 also marks putative stem cells in the oral tissue, which have been so far understudied although they likely generate the very invasive oral epithelium SCCs which are currently uncurable. Fig 1. The staining of human/mouse sections in fig1 is unremarkable and lacks comparison with normal tissue. To clarify the difference in Runx1 level, we added a picture of Runx1 in a normal human hair follicle at equal exposure to Figure 1E. Why do they show the non-scc cell lines only - I would like to see the data rather than just see images for the less relevant cell lines and a bar graph and table of the more interesting lines. We added a western blot for the SCC cell lines (Figure 1B) and added representative images for all cell lines (Figure 1C). Furthermore no mention of how staining was quantitated in 1D. We added the following statement to the Supplementary Experimental Procedures (Histology and Immunofluorescence): " Staining intensity of individual nuclei was quantified with Autoquant. Cells were manually marked underline the cell border and the integrated voxel intensity was recorded and averaged over all cells counted. Staining intensities of Ki67, caspase3, CD34 and Runx1 were quantified per tumour section with ImageJ." Sup fig 2A and B should be reversed so that they are mentioned in order in the ms. White arrows don't seem to illustrate anything particularly convincing. We re-designed Supplementary Figure 2 and removed arrows in the original Supplementary Figure 2A (now 2C). Fig 2. Should the graphs be relabelled as papillomas rather than tumours? Among the tumours are also SCC precursors. Since we need to cater to both, we feel that the general term tumour is more appropriate. EMBO 8

9 The colours of the dotted lines in G don't match the key. To avoid confusion we redesigned the key to show each colour for each linetype. The CD34 +ve cells look nothing like those in the Malanchi paper - they don't look to be in the epithelial compartment. To clarify this staining we now provide a zoomed in image of Runx1 and CD34 staining (Figure 2J) and also show Runx1 and CD34 channels separately in Supplementary Figure 2F. Additionally, we quantified CD34 and other proteins in the Runx1 tumours (Figure 2I). The epithelial compartment is more tightly packed with cells, as seen by nuclei density, and stains for Keratins. Here we are providing an example of a papilloma, similar to that used for CD34 staining, stained with K14. Fig 4. The lines linking the bars with the picture in B don't make sense. Thank you for paying close attention. Lines had been shifted and were now moved to connect to the right fragments. Sup Fig 3. Not clearly described in the legend We adjusted the legend to reflect the new figure. Sup Fig 4. Dashed line in A is not described? Labelling in B - Bu = bulge The dashed line represents the boundary between tumour and stroma where it is not apparent from the staining. The caption is now changed to reflect that (same for Bu = Bulge). Sup Fig 5. A. What is fold change referring to? This information was indeed missing. We had normalized the data to SCC13 and we now added this information to the axis label and the caption. B. What is the significance of the colours? Where are the real data? The ChIP figure is a real shame given that this is potentially exciting data - need some evidence. I certainly think it is unacceptable to cite that the method will be published elsewhere (unpublished data)? The colours represented the p-value associated with Runx1 binding to that fragment as seen in the ChIP-qPCR data, which was displayed in the original Figure 6I (now 7J). This scheme from Supplementary Figure 5B was placed to the main Figure (7I) to avoid confusion. We now added more details for our ChIP protocol to the Supplementary Experimental Procedures. C. What happens if you knock down both SOCS 3 and 4 together? Where is the quantitation? Or what happens if knockdown either in the absence of Runx1 knockdown? The quantification of the images shown in the original Supplementary Figure 5C is in original Figure 6K (now 7L). We now Knock down both SOCS's in the absence of Runx1 recovers cell growth similar to the single knockdown and we added quantification results for the double knockdown to the new Figure 7L. Knockdown of SOCS3 or 4 alone is not predicted to cause any defect. In normal keratinocytes SOCS3 and 4 are barely expressed. They get up-regulated upon Runx1 KO, which is how we discovered them as seen in Figure 7I (old 6H). All plates used for GFP quantification had equal confluency and given that GFP content was the same it shows that GFP- cells, which are either backbone or SOCS 3 or 4 transfected, all grow equally well. We now performed an additional control experiment comparing plko1-puro transfection to SOCS 3 or 4. All lead to equal confluency on a plate as we now show in the new Supplementary Figure 4C, confirming our previous data. EMBO 9

10 Referee #3: The manuscript of Scheitz and colleagues is built on previous publications showing that deletion of Runx1 in the mouse epidermis protects from tumour formation in chemically induced skin tumourigenesis assays. Scheitz et al. now show that Runx1 is over-expressed in various epithelial tumours and investigate whether Runx1 is needed for the initiation or maintenance of tumour formation. Expression of Runx1 seems mainly important for tumour initiation and the authors mechanistically explain this effect by the negative transcriptional regulation of the Stat3 inhibitors SOCS3 and SOCS4 by Runx1. How cancer and tissue stem cells are regulated during tumour development and maintenance is highly important to define novel therapeutic strategies in cancer. Thus, I consider this manuscript as a potential important contribution to the field. However, I do have some major concerns that need to be addressed before I can recommend publication in EMBO J. Although the authors provide a tremendous amount of data, the overall manuscript is at times quite superficial and some strong claims are not supported by the data provided. For instance, the relevance to human cancers as implicated in the abstract and the introduction is low and solely descriptive. In addition, the title implicates an important role of Runx1 in various epithelial tissues. However, the authors themselves exclude other than squamous epithelia from the analysis because the data are inconclusive. In my opinion the manuscript including the summary figure 7 needs to be revised and more carefully worded. We reworded the title, abstract, introduction and discussion to tone down the implications of Runx1 in human epithelial cancers. We also redesigned Figure 8 (original Figure 7) according to your feedback. We make it more clear what "other" tissues are and indicate that in these tissues the mechanism is only a prediction. However, we want to point out that our data extends to cancers beyond SCC, since ovarian human cancer cells, also required Runx1 for their normal growth. Major concerns: 1. Aim of figure 1 was to determine Runx1 in human epithelial cancers. Except for skin cancers, the authors do not provide evidence that Runx1 protein is indeed expressed in primary epithelial tumour samples. In addition to skin tumours, we also analysed oral squamous cell carcinoma. In existing Table 1 we supplement or images by summarizing the number and types of tumours analyse. Additionally, the majority of Oncomine results stem from comparisons of primary tumour to normal tissue, indicating overexpression of Runx1. We observe the same trend in human cancer cell lines. Obtaining frozen human tissue of high quality that is suitable for the Runx1 antibody is a big challenge and we could not add additional tumours besides skin and oral SCC. 2. The authors do not determine Cre-recombinase efficiency throughput the epidermis in their different treatment regimes, which makes it very difficult to draw precise conclusions from the respective experiments. Cre-recombination should be achieved in all K14-positive layers and Runx1-positive tumours derive from non-recombined keratinocytes. It is unclear to me why the authors chose to treat only once instead of treating longer with Tamoxifen to achieve a more homogenous recombination. In Figure 2B we did show (and maintain on the revised manuscript) the quantification of Runx1 expression after recombination, which was over 90%. We chose one injection of 115ug/ml because in our previous trials mice of this genotype would not survive a higher dose or more injections. 3. On page 8 lines 21-25; the authors conclude that 'that Runx1-negative tumours are short-lived'. How can this be explained? Do Runx1-negative cells exhibit a proliferative disadvantage to Runx1- positive cells? If yes, are Runx1-negative tumours smaller in size? This could be explained both by reduced proliferation and increased apoptosis. To address this issue we added a representative staining (Supplementary Figure 2F) and new quantification (Figure 2I) of Ki67 and caspase 3. While we do not see a difference in caspase 3 we see a clear EMBO 10

11 drop in Ki67 content in the tumours lacking Runx1. Additionally, indeed Runx1 negative tumours appear smaller in size, and we now quantified that in new Figure 2I and show an example image in Supplementary Figure 2E. 4. To make the argument that Runx1 may be important for the maintenance of the CD34+ tumour stem cells, at a minimum the authors have to provide some quantification. We quantified existing stainings for Runx1 and CD34 levels as shown in the new Figure 2I. Indeed, CD34 is only present in the Runx1 high samples. 5. Figure 3B: LacZ staining at 7 month is not very convincing. We added an inlay with a zoom-in of an independent hair follicle to show the different affected layers in Figure 3B. 6. The statement that Runx1-expressing adult bulge cells are '... differentiating HFSCs' is not supported by data. At a minimum a co-expression of LacZ with differentiation markers of hair lineages need to be provided. We stained 3 rd Anagen samples with beta-gal and K14 as well as AE13,AE15 (markers of the different layers) as shown in Figure 3E. Additionally, in the same figure we show a HF crosssection derived from X-gal staining of 3 rd Anagen. 7. The relevance of the LacZ stainings in figure 3E is unclear to me. The authors do not specify the tamoxifen treatment protocol or mention after which time points the samples have been selected. For instance, LacZ marks scattered cells throughout the intestine. How is this possible? Given that the intestine has a relative high cellular turnover one would expect a LacZ-positive villus after tracing, if Runx1 is indeed expressed in crypt stem cells. In addition, figures S3B,C do not support the LacZ staining in figure 3E. Detecting endogenous expression of Runx1, at least in the oral epithelium should further support these data. There is a misunderstanding here. Original Figure 3E did not show lineage tracing, but endogenous expression of Runx1 using a LacZ knock-in ( KI) in the different tissues. This data now rearranged in Figure 6 and we adjusted the labels to make this clearer. Our lineage tracing data for the colon and intestine was presented in Supplementary Figure 3, which we now removed. We cannot explain the disparity between the 3 different lineagetracing markers especially in the intestine. It is to note that in the skin they all show the same pattern. So the inconsistencies could have something to do with different protein stabilities in the different tissues and cells. Additionally, although all are in the same locus they are driven by different promoters, which could affect their expression in different tissues as well. Upon careful analysis it appears as though Runx1-CreER is extremely inefficient in the intestinal epithelium, which precluded labelling and therefore consistent lineage analysis in this tissue. Since Runx1 has two promoters and an intricate isoform structure, it is possible that the insertion of CreER upstream of the 2 nd promoter does not favour expression of CreER in the intestine in a pattern that recapitulates Runx1 endogenous expression. Since addressing this problem, would require ~ 2 years to generate a new targeting vector and a new mouse model, it is therefore beyond our powers to move this project further in this direction. However, we were able to co-localize Runx1 immunofluorescence antibody signals with a subset of LGR5 expressing cells, which supports the idea that at least some intestinal stem cells may express Runx1. This was corroborated by Runx1-LacZ endogenous expression data, and co-localization with epithelial markers K8. (new Figure 6) We feel that with our expression data and co-localization with LGR5 we have further strengthened the relationship with a 3 rd stem cell population, which along with all of our other data justify our model. 8. Figures 4A-C are confusing. The authors state in the text (page 13; line 1) that the bulge scheme resulted in 'no infundibular or other epithelial cell labelling'. However, in figure 4C 20% of infundibular cells were labelled under the bulge scheme. This was indeed a mistake in the text, and we thank the reviewer for pointing it out. We changed from no infundibular to "20% infundibular " Page 11 This does not change our conclusions, however, because when we specifically and strongly label those infundibular cells in nearly all hair follicles, they never contribute to homeostasis or tumorigenesis (Figure 4D, G). We corrected the text and clarified this point. EMBO 11

12 9. Why are infundibular cells labelled in the first place? The authors do not provide evidence that Runx1 bulge cells can contribute to the infundibulum or for endogenous expression of Runx1 in the infundibulum. If rare cells of the infundibulum express Runx1, how can the authors exclude that there are also rare cells in the interfollicular epidermis expressing Runx1 contributing to their assay. We showed that Runx1 is strongly expressed in the infundibulum upon injury (new Figure 5). It is very probable that micro-injury leads to low or intermediate levels of Runx1 in some areas, explaining the labelling that we observe in the bulge scheme. For our infundibular tracing schemes we induced Runx1 expression by ~5 TPA treatments. We now quantified IFE labelling resulting in 0 blue cells in appr IFEs. This information was added to the appropriate paragraphs. 10. Figure 6 convincingly connects loss of Runx1 to impaired Stat3 signalling. However, in these assays normal keratinocytes and the cancer cell lines are similarly dependent on Runx1 in these in vitro assays. A comment on why there is a strict dependence on Runx1 in vitro but survival of normal and cancer cells in vivo does not depend on Runx1 would be helpful. In cancer we do observe that cells die in vivo without Runx1 (Figure 2 and 5), however the presence of TPA and other proliferative factors can delay this process. This matches what we observe in vitro in Figure 7 (original Figure 6). In HFSCs the niche is likely providing some additional signals and protection from Runx1 loss. It is possible that Runx1- cells are more sensitive to oxidative stress, or have a different epigenetic status, etc. In vitro the cells are directly exposed to any environmental influence and there is no protective niche and as a result the cells die without Runx1. In vivo the niche is providing protection. To clarify the niche protection we added the following to the discussion "The tolerance of Runx1 loss in HFSCs is probably due to the unique niche environment." Additionally we have made minor adjustments to results and discussion to emphasize the similarity between in vivo and in vitro in cancer as opposed to the disparity in normal skin. Minor points: 1. I believe the header of figure 1F should say 'initiation'. Assuming that the reviewer means Figure 2F (there is no 1F), both Promotion and Initiation are analysed here and "Initiation" was added to the label. We thank the reviewer for pointing this out. 2nd Editorial Decision 26 August 2012 One of the original referees reassessed your work and despite finding the study in parts hard to follow, has no further objections against its publication. The only remark was that the labeling in figure 3B seems wrong (+oil/+tm), an issue that should be latest addressed while proofing the manuscript. I am thus really pleased to inform you that your manuscript entitled "Defining a Tissue Stem Cell Driven Runx1/Stat3 Signaling Axis in Epithelial Cancer" has been accepted for publication in the EMBO Journal. Yours sincerely, Editor The EMBO Journal EMBO 12