The histone H2B ubiquitin ligase RNF40 is required for HER2-driven mammary tumorigenesis

  1. Florian Wegwitz
  2. Evangelos Prokakis
  3. Anastasija Pejkovska
  4. Robyn Laura Kosinsky
  5. Markus Glatzel
  6. Klaus Pantel
  7. Harriet Wikman
  8. Steven A. Johnsen

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Abstract

The HER2-positive breast cancer subtype (HER2 + -BC) displays a particularly aggressive behavior. Anti-HER2 therapies have significantly improved the survival of patients with HER2 + -BC. However, a large number of patients become refractory to current targeted therapies, necessitating the development of new treatment strategies. Epigenetic regulators are commonly misregulated in cancer and represent attractive molecular therapeutic targets. Monoubiquitination of histone 2B (H2Bub1) by the heterodimeric ubiquitin ligase complex RNF20/RNF40 has been described to have tumor suppressor functions and loss of H2Bub1 has been associated with cancer progression. In this study, we utilized human tumor samples, cell culture models, and a mammary carcinoma mouse model with tissue-specific Rnf40 deletion and identified an unexpected tumor-supportive role of RNF40 in HER2 + -BC. We demonstrate that RNF40-driven H2B monoubiquitination is essential for transcriptional activation of RHO/ROCK/LIMK pathway components and proper actin-cytoskeleton dynamics through a trans-histone crosstalk with histone 3 lysine 4 trimethylation (H3K4me3). Collectively, this work demonstrates a previously unknown essential role of RNF40 in HER2 + -BC, revealing the H2B monoubiquitination axis as a possible tumor context-dependent therapeutic target in breast cancer.

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  1. Version published to 10.1038/s41419-020-03081-w
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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this study by Wegwitz et al, the authors examine the tumour promoting properties of RNF40 (and the H2B monoubiquitinylation catalysed by it) in Her2 driven breast cancer.

    They report, using publicly available data, that increased RNF40 expression is associated with reduced overall and disease-free survival.

    Using a mouse model, where they crossed the Erbb2 (mouse Her2) under the control of the MMTV promoter with conditional Rnf40 deletion constructs, the authors found that deletion of Rnf40 simultaneous to Her2 overexpression resulted in a prolonged tumour-free survival, somewhat reduced tumour growth kinetics and tumour incidence.

    siRNA silencing of Rnf40 in two Her2 positive breast cancer cell lines resulted in reduced proliferation, clonogenicity and tumour sphere formation and cellular motility.

    Transcriptome analysis revealed pathways that could explain the phenotype, like increased apoptosis and actin cytoskeleton regulation. The authors then took further some candidates in the later pathway to investigate the mechanism. They find that Rnf40 loss impacts on actin cytoskeletal dynamics. They also investigate the impact on focal adhesion signalling integrity.

    Finally, they investigate the relationship between the transcriptome and H3K4me3 and H2Bub1 landscape in the presence or absence of Rnf40.

    The manuscript is convincing regarding the tumour promoting roles of Rnf40, but the key claim that H2B monoubiquitinylation is essential for activation of the Rho/Rock/Limk pathway, where genes are down regulated upon Rnf40 loss resulting in decreased tumourigenicity of cells, is so far not convincing.

    "Together, these findings support the hypothesis that the actin regulatory gene network is dependent on direct epigenetic regulation by RNF40 through modulation of H2Bub1 and a trans-histone cross-talk with H3K4me3 levels in HER2-positive BC cells."

    Although the correlation is apparent, at this point it's unclear if the phenotype is dependent on the catalytic activity of Rnf40 or it's a non-catalytic effect. Generating a catalytic mutant RNF40 and test it at least in the cell lines studied would be desirable.

    We thank the reviewer for this comment and agree that the addition of data with a catalytic mutant RNF40 could strengthen our findings and further clarify mechanisms involved. Thus, in a resubmission we will directly address this point by performing knockdown/rescue experiments with either wildtype or a RING finger mutant RNF40. This will be done by transfecting cells with expression constructs for either wildtype or mutant RNF40 proteins followed by knockdown of endogenous RNF40 using siRNAs targeting the 3’ UTR. Experiments central to our take-home message will be performed (e.g., cell migration, target gene expression, Western blot for H2Bub1, F-actin formation). Together, we hope these experiments will help significantly solidify the message of this paper and further clarify the individual role of RNF40 within the RNF20/40 heterodimer.

    **Other comments that need a response:**

    1."we investigated RNF40 expression and H2Bub1 levels by immunohistochemical staining of 176 primary BC tumors and 78 brain metastases."

    In Fig 1 I can only count 41 primary BC tumours and 73 brain metastases. Numbers don't add up. Also, how is "low" defined as opposed to negative? What is used as controls?

    We apologize for this mistake. We corrected the numbers of primary and metastatic HER2-positive specimens used in this study.

    2."Moreover, HER2-positive metastatic BC samples showed a particularly high expression of RNF40 compared to primary tumors"

    Figure 1 or Fig S1A does not contain data on HER2-positive metastatic BC

    We think there might have been a confusion regarding this point. The manuscript does provide information about RNF40 and H2Bub1-staining in primary HER2-positive breast cancer lesions as well as HER2-positive brain cancer metastasis specimens in Fig.1A-C as well in Fig.S1A (representative brain metastases are shown in IHC pictures). This is stated both in the main text as well as in the respective figure legends. However, if for some reason this remains unclear, we would certainly be open to suggestions as to how we can modify the respective sections to improve their clarity.

    3."tumors did not display a loss of either RNF40 or H2Bub1 (Fig. 1H) when compared to the adjacent normal mammary epithelium (Fig. S1F)."

    I don't understand what I see in Fig S1F, where is the tumour, what is adjacent?

    We agree with the reviewer that splitting tumor staining in the main Fig 1 and normal tissues in Fig S1 makes a comparison difficult. We will therefore edit the Fig S1F and provide there an overview of tumor and surrounding normal tissues together with magnifications of the respective areas. This should significantly ease a comparison of both RNF40 and H2Bub1 in tumor and adjacent normal tissues.

    4."homozygous loss of Rnf40 (Rnf40fl/fl) resulted in dramatically increased tumor-free survival of MMTV-Erbb2 animals (Fig.1E)." This is overinterpretation of the data, I would not call it dramatic, just significant.

    The MMTV-Erbb2 mouse model is a very reliable mouse model for the induction of HER2-positive lesions. In our hands, the tumor incidence in these animals was 100% with a median tumor free survival of 166 days. In comparison, approx. 20% of the Rnf40fl/fl animals (3 out of 14) never developed the disease during the 18 month observation. The animals that still developed lesions had a median tumor free survival of 241 days, which represents a delay of 75 days (45% delay). In light of this, it seems to us that the effect of RNF40 loss on HER2–positive lesions is, indeed, remarkably strong. However, we do not wish to give an impression of over-interpreting or misrepresenting the data. For that reason we modified the wording in the main manuscript according to the reviewer’s suggestion (line 140: “dramatically” was replaced with “pronounced”).

    5."loss of Rnf40 led to strongly reduced tumor growth kinetics (Fig.1G)." Is this result significant, I did not see an evaluation of statistical significance in this data.

    As suggested by the reviewer, we have performed additional analyses to examine the statistical significance. We have now included the results of these tests in the respective figure.

    6."Rnf40fl/fl lesions displayed a heterogeneous pattern of RNF40 expression (Fig.1H), suggesting that the few tumors that did develop in this model were largely caused by an incomplete loss of the Rnf40 allele." If this conclusion is suggested, the authors should check if the "escaper" cells have failed to flox the Rnf40 allele on the genetic/protein level. Otherwise it's not conclusive.

    The reviewer brings up an interesting and important point about the heterogeneous loss of RNF40 in “escaper” tumors. Very important to note is that these “escaper” tumors developed significantly later and three animals never developed tumors. Thus, the “escaper” phenotype is rare (at the cellular level) and is likely similar to the selective process that occurs in human tumorigenesis and tumor progression. It is well established through a number of publications that deletion of genes essential for tumorigenesis via Cre-based systems frequently results in a specific selection for the rare instance that the Cre-mediated excision is ineffective. These “escaper” cells can then grow out and proliferate because they do not suffer from deletion of the floxed allele. This effect has also been established when combining MMTV-HER2 and MMTV-Cre. For example, analogous findings were recently published by Costa, et al., in Nature Communications (doi: 10.1038/s41467-019-11510-4) where the MMTV-Cre-mediated deletion of Pak4 resulted in impaired MMTV/HER2 or MMTV-PyMT-driven tumorigenesis, but occasional tumors did appear, which all retained some degree of PAK4 expression. This effect, which we have also seen in our system, was also reported by Miao, et al. in Cancer Research (doi: 10.1158/0008-5472.CAN-11-1015) in 2011. In their work the authors observed that deletion of the Myb gene also impaired MMTV-HER2-driven tumorigenesis and those tumors that developed in Myb flox/flox mice displayed a late onset and invariably retained MYB expression. Similar findings have been reported in a number of other tumor types and with various Cre drivers. Thus, we posit that these findings provide further support for the essential role of RNF40 in HER2-driven tumorigenesis to the extent that rare, RNF40/H2Bub1-expressing “escaper” cells are positively selected for during tumorigenesis and tumor progression.

    In order to definitively establish this, we propose performing dual immunofluorescence staining of Rnf40 flox/flox tumors to verify that H2Bub1 is exclusively and universally lost together with RNF40 and that each case of a complete loss of RNF40 also results in a complete loss of detectable H2Bub1 staining. Additionally, we will assess the efficiency of the cre mediated deletion of Rnf40 exons 3 and 4 in Rnf40fl/fl animals by detecting their presence using a conventional PCR approach.

    1. Fig S4D - is this clonogenic assay? How many replicates were done, biological technical?

    We apologize for the imprecise description of this figure. We edited the manuscript accordingly and included details about the number of replicates.

    8."Additionally, treatment with either CYM-5441 (Fig.4J) or …"

    Fig 4J is missing! It makes this section rather hard to follow. Fig S4F-G, how many replicates were done, biological technical?

    We thank the reviewer for noticing this error. The figures were indeed inappropriately referenced in the text. This error has been corrected.

    9."Consistent with our analyses based on changes in H3K4me3 occupancy, genes downregulated upon RNF40 silencing displayed the most prominent decrease in H3K4me3 in the gene body (the 3' end of the peak)"

    The impact of these mods changes is hard to judge because they are rather small (I would not use the wording prominent).

    As implied by the reviewer, we will replace the word “prominent” with “noticeable”.

    Also, are there many other "peak narrowing" genes but they are not downregulated?

    The point mentioned here is very interesting. The bioinformatic analyses performed in this study solely focused on the relationship of significantly regulated genes and the H3K4me3 peak narrowing at their TSSs. However, we did not analyze the global regulation of genes showing H3K4me3 peak narrowing near the tSS. As this information might be of high relevance for this study, we propose to investigate this interesting aspect in the revised version of the manuscript.

    In fact, our analyses have revealed that a large fraction of genes with H3K4me3 narrowing peaks do not show an appreciable decrease in expression. To better understand the epigenetic features determining the sensitivity of genes to H3K4me3 peak narrowing, we studied the occupancy of several histone marks at differently behaving genes. We discovered that sensitive genes globally present lower occupancy of histone modifications which are known to positively influencing gene transcription. We therefore propose that the epigenetic context (i.e., presence of additional histone modifications) strongly determines whether the loss of H2Bub1, and ensuing narrowing of H3K4me3 near the TSS, results in decreased transcription.

    Statistical analysis missing: for example in Fig 2C, Fig 2E, Fig 3G what is n=?, how many technical, biological replicates were analysed?

    This information has been added to the revised manuscript.

    Fig 4E seems to be a partial duplication of Fig 3D!

    The samples in Fig. 3D and 4E originate from independent experiments. In Figure 4D, we indeed provide again PARP and Casp3 signal for siRNF40 samples in order to allow a direct comparison of the effect magnitude between RNF40 depletion and ROCKi treatment.

    **Minor:**

    Figure referencing: it can be quite confusing to see a different ordering of figures compared to the referencing in the manuscript, for example Fih 1H is referenced in the text before Fig 1F, G. The authors should change the order in the main figures....

    We thank the reviewer for pointing this out. We have updated the figure order in the revised manuscript accordingly.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this study, Wegwitz et al propose that the E3 ubiquitin ligase RNF40 is highly expressed in HER2+ breast cancer tumours and correlates with poorer survival, using their own and TCGA data. Contrary to observations suggesting a tumour-suppressive role in other cancers, authors show using RNF40-knockout breast cancer mouse models and in vitro data shat RNF40 promotes tumour growth. RNF40 depletion impairs proliferation, survival and sphere formation by inducing apoptosis. In addition, RNF40 promotes cell migration by upregulating expression of cytoskeletal proteins (ROCK1, VAV3, LIMK2) and their effectors such as phosphorylated cofilin. Authors show elegant partial rescue experiments of the effect of RNF40 depletion on apoptosis and survival.

    Given that RNF40 function seems to be context-dependent, findings from this study could have broad significance for other cancers with high RNF40. It also provides some mechanistic data (that should be improved as suggested below) linking this ubiquin ligase to the cytoskeletal machinery and, therefore, control of migration and also proliferation and survival.

    Data are well presented and most conclusions are supported by the data. However, there are some gaps at the mechanistic level. Since migration is controlled by RNF40 in vitro, evaluation of metastatic ability in vivo (local invasion for example as suggested below) should be evaluated and would strengthen this part too.

    **Major comments**

    1. Fig.1A-B, S1A. Specificity of RNF40 antibody should be shown, which could be done quite easily in the tumours from the knockouts. From the datasheets, antibodies recognize human protein only.

    We thank the reviewer for this suggestion and apologize for this mistake. The antibody utilized in the IHC studies is actually from Abcam (ab191309) and, in fact, recognizes both species. Table S5 has been corrected accordingly.

    It is unclear when the murine tumours were analysed, at endpoint? This should be stated.

    We thank the reviewer for this comment. Indeed, all IHC analyses were performed after dissection (endpoint). This information will be added to the manuscript. Kinetic analyses of tumor growth (i.e., Fig. 1G) were performed on the same mouse cohort.

    Could authors establish cell lines from the mouse tumours (knockout, partial knockout escapers..)? These could be very useful tools to evaluate key in vitro findings from the study.

    The reviewer makes an interesting suggestion. Unfortunately, we were not able to establish cell lines from this model and have since stopped breeding this mouse line (due to the relocation of the principle investigator). However, we did try to generate RNF40-deficient breast cancer cell lines using the CRISPR/Cas9 technology. Interestingly, all attempts failed, supporting the fact that the loss or RNF40 is lethal for the cancer cells. However, to further establish this, for the revision we propose to transfect HCC1954 cells with CRISPR/Cas9 constructs targeting exons 3 and 4, similar to our mouse model. We will then assess the evolution of RNF40-negative cells population over time (i.e., via immunofluorescence staining for H2Bub1). This assay should inform about the expected growth “disadvantage” following RNF40 loss.

    Fig.1F-G: since RNF40 controls the cytoskeletal machinery and therefore, migration (Fig. 2G) in the RNF40 knockout tumours, was metastasis (if observed) affected? Or if there was no growth in distant organs detected in the time frame of these experiments, was invasion (and/or pattern of invasion or mode of invasion (morphology of invading cells)) into adjacent tissues affected upon RNF40 depletion? This would add in vivo relevance to the in vitro mechanistic findings, especially since the authors later showed that p-cofilin was also decreased in the RNF40-depleted mouse tumours (Fig.4D).

    We agree that the metastasis data from our mouse genetic tumor model would significantly help solidify our findings. Unfortunately, the MMTV-Erbb2 mouse model (overexpressing wildtype Erbb2 gene) only rarely develops distal metastases. In our analyses, we only ever observed two macroscopically visible metastases (one wt/wt and one in an Rnf40flox/flox mouse). However, we feel that the reviewer’s suggestion is a one and will follow this suggestion and attempt to examine possible changes in local invasion of primary tumors into adjacent tissues.

    Fig.3: most results using HCC1954 cell line. Key findings should be validated in other cell lines.

    We agree with the reviewer about the importance of cross validation of findings using different cell lines. For this purpose, we have now generated data with an additional HER2-positive cell line. These data using the SKBR3 cell line were performed for several of the key experiments. Key findings about phenotypic changes (growth kinetics and colony formation), Ki67 protein levels differences and mRNA regulation of identified regulators of actin cytoskeleton (VAV3, ROCK1, LIMK2 and PFN2) will be included in the revision for both cell lines. Furthermore, as seen in the HCC1954 cell line, an increase of the apoptosis marker cleaved PARP as well as a loss of VAV3 and ROCK1 protein levels was also observed upon RNF40 knockdown in SKBR3 cells. These data will be included in the revised manuscript.

    Fig.3A: authors state "both pathways remained intact following RNF40 depletion". However, from those blots, siRNF40 clearly increases pERK and slightly pAKT, which would be unexpected according to previous data in Fig.2. Authors could show quantifications of different blots, or show a more representative blot if increase in pERK was not consistently observed. Was this also seen in SKBR3 cell line?

    We thank the reviewer for this comment. Initially, we had anticipated that oncogenic signaling may be decreased in the Rnf40 conditional knockout model. However, much to our surprise, the activity of the downstream signaling actually appears to be increased. In fact, the increase in AKT and ERK1/2 phosphorylation following RNF40 silencing was consistent across different experiments and replicates. While this finding is also consistent with our previous results in an ER-positive system (e.g., see Prenzel, et al., 2011), we do not understand the mechanistic underpinnings of this finding. Importantly though, while consistent, we do not feel that this increase explains the observed phenotype. Nevertheless, to more precisely show the overall change of p-ERK/ERK and p-AKT/AKT, in the revision we will provide a densitometry quantification for both cell lines. We will also modify the sentence to more precisely describe this finding and make the point that since these pathways are not reduced/impaired, they are unlikely to be responsible for the increased apoptosis observed upon RNF40-KD. Western blots assessing p-ERK/ERK and p-AKT/AKT levels in SKBR3 upon RNF40 knock-down will also be added into the supplementary data of the revised manuscript (Fig.S3).

    For Fig.3G and Fig.S3A, authors selected genes from this set, how was this done (fold change?). Was expression of the other family members (ROCK2, LIMK1, etc) or of Rho GTPases regulated too?

    This information was indeed missing in the manuscript. We have modified the figure legend and the main text accordingly in order to provide the information about the cutoff used in the Enrichr analysis. Regarding the expression of other family members of the actin regulatory network, in the past we performed a more, in depth and focused analysis of our RNA-seq data, broadening our view to other members of the RHO/RAC/CDC42 pathways. While we did identify a few further potentially regulated target genes (e.g. ROS1 or PAK6), these genes were either only weakly expressed or weakly regulated. For this reason, we presumed that these factors could only play a marginal role in the observed phenotype and have focused our attention on the robust part of the signature.

    Fig.4B: this may not help, decrease of p-cofilin by Vav3 knockdown is way less dramatic compared to RNF40 depletion or ROCK inh treatment. See comment below regarding other effectors such as Myosin.

    Indeed, the consequence of VAV3 loss on p-cofilin levels is less pronounced than the effect observed upon RNF40 knockdown or ROCK1i treatment. Given the fact that RNF40 loss not only affects VAV3 expression, but also has additional direct effects on the expression of other pathway members, this may be expected. We do, however, feel that the VAV3 regulation is likely one component of the effects of RNF40 loss. In addition, it has also been shown that VAV3 is not the only GEF regulating the activity of RHO kinases upstream of ROCK1. Therefore, we would also expect that VAV3 loss only partially reduces ROCK1 activity and therefore only partially phenocopies the effects observed. We will expand the description of these findings in the revised manuscript to reflect these views.

    Fig.4C: does ROCK inh reduce RNF40 levels? It may from the immunofluorescence picture.

    We thank the reviewer for this comment. In fact, we have examined this possibility. However, no significant changes in RNF40 protein levels were observed upon RKI-1447. If helpful, we can provide Western blot data demonstrating this in the supplemental figures.

    Fig.4H-I: the sphingosine 1-phosphate receptor-3 agonist (CYM-5441) partially rescued the effects of RNF40. Since S1P signalling involves Rho GTPase activation -presumably downstream of VAV3 -which is a GEF for Rho, Rac and Cdc42- and upstream of ROCK, LIMK, was activity of these Rho GTPases affected upon RNF40 depletion? This would strengthen the mechanism.

    The reviewer points at an interesting aspect of the actin regulation. Indeed we expect that the reduction of VAV3 levels upon RNF40 loss would significantly influence the activity of the downstream client GTPases. However, the measurement of RHO-GTPase activity is tricky and expensive. Furthermore, as mentioned in the previous comment (#7, part 1) VAV3 is only one component of the four major genes encoding critical actin cytoskeleton regulatory proteins regulated upon RNF40 loss, and the only factor upstream of RHO-GTPases. The reduction of downstream ROCK1, LIMK2 and PFN2 levels also influence the activity of this pathway downstream of RHO-GTPase activity. We therefore focused our efforts on assessing F-actin and p-cofilin levels as these may provide more sensitive readouts about the consequence of RNF40 loss on this signaling cascade. However, if the reviewer considers this information as indispensable, we would attempt to investigate changes in Rho-GTPase activity by commercially available Active Rho Detection Kits, although this will significantly delay the resubmission of a revised manuscript.

    Related to this, was Myosin II activity (phosphorylated MLC2) affected -since its upstream regulators, especially ROCK are controlled by RNF40?

    We thank the reviewer for this insightful suggestion. To address this possibility, we will test this hypothesis for the revised manuscript as suggested by performing Western blot analysis for phosphorylated MLC2.

    Fig. S5E:

    Authors should consider presenting data of decreased histone methylation of cytoskeleton regulators in main Fig. 5, since this is an important conclusion of this part.

    As suggested we will shift the information currently presented in figure S5E to the main figure 5.

    Statistics should be revised throughout the manuscript. Comparisons of more than 2 groups should be performed with ANOVA or similar multiple comparison test (instead of t-test).

    We thank reviewer for this comment. We replaced statistical tests with the appropriate ANOVA in the respective graphs and updated the legends accordingly.

    **Minor comments**

    Statement of significance mentions "Anti-HER2-therapy resistance", but this is a misleading since the paper does not deal with therapy resistance. Or are the cell lines used in the study resistant to anti-HER2?

    We thank the reviewer for this suggestion. While resistance to anti-HER2 therapy remains one of the major clinical challenges in the treatment of HER2 positive BC lesions, we agree that our data do not strictly address this point. Thus, we have modified the sentence accordingly.

    In line with this, authors could add some lines of thought on how RNF40 could be targeted in the clinical/pre-clinical context, which could inform further translational studies.

    This is a great suggestion. In the revised manuscript we will include additional text to specifically address this point.

    Line 117 "Moreover, HER2-positive metastatic BC samples showed a particularly high expression of RNF40 compared to primary tumors".

    Perhaps rephrase, was it that the expression level (intensity) was higher or that the % of positive cells/tumours was higher in the brain mets?

    This is a critical point that we will consider in the revised manuscript. We have modified the sentence accordingly to read, “Moreover, the incidence of RNF40-high specimens was higher in HER2-positive brain metastases compared to primary tumors (Fig.1A-B)”.

    Fig.1D and S1C. while S1C shows TCGA data, it is unclear which set of patients is Fig.1D (since text says publicly available data, line 118-119), are these their own set of patients (used in Fig.1A-B)? This should be specified in the text, legend.

    The origin of the data shown in Fig.1D for relapse free survival of RNF40high and RNF40low patients (KM plotter) is mentioned in the figure legend (kmplot.com) and in the material and method section. However, since this was not apparent, to increase the readability, we have now added a statement about the publically available database of origin for every output graph in the main text as well in the legend and supplementary material.

    Line 122. Authors should be careful with this conclusion so far, a correlation between expression and cancer stage/survival does not necessarily mean a tumor suppressive/supportive role.

    We thank the review for this comment. We agree with this statement. Therefore, we have carefully rephrased this sentence as following, “In summary, these data demonstrate that RNF40 expression is maintained in HER2-driven primary metastatic BC and that its expression correlates with poor prognosis in these patients.”

    Fig. S4E: there are missing labels in graph (control, siRNF40).

    The labels have been added.

    Panels in some figures are discussed in text randomly and not following same order. For example, Fig.1 (after panel D, then panel H, then back to E, F, G), S3, 4A-C,

    We reorganized the order of the different panels of Fig1 to increase the readability. We further screened the main text for similar problems and modified the respective figures accordingly.

    Fig.1E: I would suggest changing the line colours, so Rnf40wt-wt line is red and the fl-fl is black, therefore it is similar to panel D (high Rnf40 red, low in black).

    We thank the reviewer for this suggestion. Accordingly, we have now indicated low RNF40 expression in red (Figure 1D, 1E and S1B) in the same way that we have indicated RNF40 expression throughout the rest of the study.

    Supp Videos: for reviewers and readers, it would help that video has a label while it plays, otherwise after downloading it, video name does not tell whether it is control or RNAi.

    As suggested we have renamed the video files of for each condition and added a label informing about the identity of the sample while the video plays.

    Reviewer #2 (Significance (Required)):

    Given that RNF40 function seems to be context-dependent, findings from this study could have broad significance for other cancers with high RNF40, or even in other pathological contexts -if any- that cursed with high RNF40.

    It also provides some mechanistic data (that should be improved as suggested in comments) linking this ubiquitin ligase to the cytoskeletal machinery and, therefore, control of migration and also proliferation and survival. This will also advance the field.

    Area of expertise

    Actin-myosin cytoskeleton, Rho GTPases-ROCK, cancer, metastasis, cell signalling

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    **Summary:**

    Wegwitz and colleagues present extensive and detailed data focussed on the role of the E3 ubiquitin ligase and ring finger protein RNF40 in HER-2 associated breast cancer. It is clear that the role of RNF40 and its major substrate histone H2B (monoubiquitination of histone H2B at lysine 120; H2Bub1) as part of a complex with RNF20, is not a simple one in the context of malignancy. This group, and others, have previously reported on the intriguing role of RNF40 that can in certain circumstances function to suppress tumorigenesis, and in other circumstances function to support tumorigenesis. While H2Bub1 has been shown to be lost in many different malignancies, these investigators show that in HER-2 associated breast cancer, this is not the case. In fact, the results presented in this study show that RNF40-mediated H2Bub1 is important for the expression of genes involved in the actin cytoskeleton and the downstream FAK signalling cascade. Supporting this, mining of a public database showed that RNF40 mRNA was high in HER-2 associated breast cancers and was correlated with a worse prognosis (overall and RFS, relapse free survival). The investigators also used a mouse model (MMTV-Erbb2) generating a tri-transgenic (MMTV-Erbb2; MMTV-Cre; Rnf40flox) that allowed breast tissue specific overexpression of HER2 at the same time as KO of Rnf40, so mimicking the human disease. In fact, mouse tumours recapitulated the human results, including in disease free survival (lower with higher Rnf40), and with less tumours seen when there was less Rnf40 (the Rnf40 floxed tumours appeared heterogenous in staining patterns for Rnf40 and H2Bub1, supporting the concept of "escaper" cells, positive for both Rnf40 and H2Bub1 that would be positively selected during tumorigenesis).

    The authors also took a cell biology approach to studying HER2 positive breast cancer and RNF40 using two HER2 positive cell lines (HCC1954 and SKBR3). RNF40 was down-regulated using siRNA and numerous functional studies showed that targeting RNF40 suppressed behaviours consistent with tumorigenesis (proliferation, migration, clonogenic survival, spheroid formation, growth kinetics). Furthermore, down-regulation of RNF40 in the HCC1954 cell line followed by GSEA identified gene signatures associated with apopotosis and the actin cytoskeleton regulatory pathway (e.g. ROCK1, VAV3, LIMK2, PFN2). They further showed that phospo - cofilin (that occurs downstream of ROCK1) was reduced in RNF40 down-regulated cells, also implicated in regulation of the actin cytoskeleton. Phalloidin staining for F-actin showed disruption of the cytoskeleton in RNF40 down-regulated cells. Additionally, the ROCK1 inhibitor, RKI-1447, showed similar effects to depletion of RNF40.

    The authors then sought to determine whether the RNF40 associated gene expression in HER2 positive cells was in fact happening through H2Bub1 and the active histone mark H3K4me3 it has been reported to cross-talk with. RNF40 regulated genes (up or down-regulated) showed lower levels of H2Bub1 occupancy compared to non-regulated genes. H3K4me3 was lost in most genes influenced by RNF40 down-regulation, including genes associated with the actin regulatory pathway. The overall conclusion is that RNF40 is a major epigenetic regulator of the actin regulatory gene network in HER 2 positive breast cancer and could be a therapeutic target.

    **Major comments: major issues affecting the conclusions.**

    (1) What is happening at the gene level for both H2Bub1 and H3K4me3 in the context of RNF20 down-regulation is complex and would benefit from inclusion of a schematic, or a series of schematics describing different scenarios, as the text is quite difficult to follow.

    This is a very constructive proposition. We will attempt to follow this suggestion in order to simplify the message of the respective section by providing to schematic illustrations depicting the cascade of events occurring upon RNF40 loss in the cancer cells.

    It is not entirely clear that the changes seen in H3K4me3 are a direct result of cross-talk with H2Bub1 (some literature reports that there is no cross-talk between these histone marks for instance). It is also not entirely clear how the other histone marks investigated support the main discoveries of the paper. The authors need to consider this in the way that they present the data and their interpretation of it.

    The reviewer addresses an important point about the mechanistic aspect of the RNF40-dependent epigenetic regulation. We and others have shown that RNF40-mediated H2B monoubiquitination is a central step for activation of the COMPASS complex and the TSS-proximal broadening of H3K4me3 (PMID:31733991, 19410543, 22505722, 28209164). However, the situation certainly is not as straight forward as it is in yeast, where the vast majority of H3K4 trimethylation is H2Bub1-dependent. To what degree global H3K4me3 levels are dependent upon the H2B ubiquitin ligases RNF20 and RNF40 appears to vary, depending upon the investigated system (probably the variation in the literature referred to by the reviewer). However, in our work, we reproducibly see widespread H3K4me3 peak narrowing specifically on RNF40-dependent genes, in a context-dependent manner (i.e., genes displaying these effects are different according to the system investigated). To support and consolidate the central function of the H2Bub1-H4K4me3 crosstalk in our system, we propose to perform rescue experiments: siRNAs targeting the 3’UTR of RNF40 will be co-transfected with an expression construct encoding for either a wild type or a ΔRING (catalytic inactive) form of RNF40 lacking the endogenous 3’UTR. The ability of ectopically expressed wild-type, but not catalytic inactive RNF40, to rescue the expression of the identified actin cytoskeleton genes and downstream signaling should provide a solid argument to support the hypothesis of our study. We will also include additional discussion about the potential different H3K4 methyltransferases that may potentially be involved.

    __(2) __RNF40 is known to work in a complex with RNF20 to monoubiquitinate histone H2B at lysine 120 (H2Bub1). In experiments where RNF40 has been down-regulated, did the authors also note down-regulation of RNF20 (as has been previously reported).

    This is an interesting question from the reviewer. We indeed observed a consistent reduction of RNF20 protein levels upon RNF40 knockdown (and vice versa) in different cell systems, including the HER2-positive cell lines HCC1954 and SKBR3.

    Is the data presented likely to be the result of abrogation of the complex rather than RNF40 specifically?

    Although particularly difficult to answer, the use of a catalytic mutant in key experiments should at least partially shed light on this aspect (as proposed in the answer to Reviewer #3’s question 1). In that case, the complex integrity can be maintained while specifically abrogating RNF40 ubiquitin ligase activity.

    While I am not asking for experiments to be repeated with down-regulation of RNF20, some consideration of this needs to be included in the Discussion. Is RNF20 also highly expressed in HER2 positive breast cancer (TCGA, KM Plotter data).

    We absolutely agree with the reviewer’s point of view. As an obligate binding partner of RNF40, RNF20 indisputably plays an important function in the phenotype caused through RNF40 loss. We will therefore carefully further discuss this aspect in the revised manuscript. Preliminary analyses based on the TCGA dataset point at a high expression of RNF20 in HER2-positive lesions. Furthermore, survival analysis of HER2+ BC patients based on the same dataset showed that patients with high RNF20 expression harbor an unfavorable prognosis, similar to what we have seen with RNF40. We may therefore implement these expression and survival data in the revised manuscript.

    **Minor comments: important issues that can confidently be addressed.**

    __(3) __It would appear that immunohistochemistry for RNF40 and H2Bub1 on human samples is only reported as "low" or "high". This is perhaps not dealing with the full spectrum of IHC scores, such as completely absent, although the methods do note a "null" value (no detectable staining). Were there no "null" results? Please define the criteria for "low" or "high".

    Indeed, specimens lacking H2Bub1 or RNF40 staining were attributed the “null” scoring. However, while we have observed null staining in other BC subtypes (e.g., see Bedi, et al., 2015), none of the HER2 positive BC samples were found to be negative for either RNF40 or for H2Bub1. However, for the revision, we will provide representative examples of null-stained tumor specimens (from other BC subtypes) for RNF40 and H2Bub1 from the same tissue microarray.

    __(4) __I think there might be some confusion in labelling of Fig 1A and B as the legend states that all breast cancers are on the left and the HER-2 positive on the right, for each of primary tumours and brain mets, but I think one is under the other? Labelling should be checked in this figure.

    We apologize for this mistake. This has been corrected in the figure legend accordingly.

    (5) What this IHC data doesn't show is whether RNF40 and H2Bub1 levels are always correlated in individual tumours (i.e. RNF40:H2Bub1, high:high OR low:low OR null:null). Can the authors please include and comment on this data.

    The reviewer has made a very interesting point here. We will comment on this point in the revision.

    (6) Please include overall survival data (KM Plotter) as a panel in figure 1, alongside RFS for RNF40 expression levels (currently in Supplementary).

    We added the OS as well the RFS data from the same database next to each other in the main figure.

    (7) Spheroid formation looks to only be shown in a single cell line (HCC1954). Was the other cell line not suitable for spheroid studies? Some comment should be made and care taken not to "overclaim" as text notes two cell lines.

    SKBR3 are unfortunately not suitable for tumor sphere formation assay. We may provide instead a soft agar assay with SKBR3 cells upon. If needed, we may replace the SKBR3 cell line with BT474 for this specific experiment

    (8) It would have been interesting to see results of a GSEA in the mouse mammary tumours as a complement to human. Is there a reason why this wasn't undertaken?

    Rnf40fl/fl tumors present a large fraction of “escaper” cancer cells retaining RNF40 expression. For this reason, bulk sequencing of such tumors would likely only provide a “diluted” molecular signature consequent to RNF40 loss. For this reason this experiment has not been done.

    (9) Conclusions are made about RNF40 in HER2 positive cells only in the context of H2Bub1 and H3K4me3. Without having conducted similar experiments in HER2 negative breast cancer cell lines / models, it is difficult to draw the conclusion that this is HER2 positive specific. Can the authors either soften some of their conclusions along this line, or consider repeating some of their data in HER2 negative models.

    The scope of the study has deliberately been set on HER2-positive malignancies, because former studies already extensively studied the impact of H2Bub1 loss in TNBC and Luminal BC (PMID 28157208, 18832071). We will therefore modify the manuscript text accordingly and soften the appropriate sections as suggested by the reviewer.

    (10) RNF40 likely has substrates other than histone H2B. There is a report describing interactions with RNF40 (STARING) and syntaxin for e.g., (Chin et al., 2002 J Biol Chem 277:35071-9). Can the authors please comment on other potential substrates of RNF40 in light of their data that focuses only on its epigenetic role as a regulator of the actin cytoskeleton.

    Our study was mainly focused only on the gene expression program driven by RNF40 in HER2+ BC. We therefore do not know nor have we focused on other novel non-histone substrates. We will, however, allude to this possibility in a revised manuscript.

    Reviewer #3 (Significance (Required)):

    Nature and Significance of the Advance:

    Clinically, this work provides a significant advance in that it is zeroing in on HER2 positive breast cancer and generating fundamental data that could underpin development of a new therapy for this malignancy. Conceptually, it is expanding knowledge of how the E3 ubiquitin ligase RNF40 is functioning as an epigenetic modifier of a specific type of malignancy by being important for the actin cytoskeleton.

    Work in Context of Existing Literature:

    As acknowledged by the authors, this work builds on a previous publication of theirs (Xie et. al., 2017 "RNF40 regulates gene expression in an epigenetic context-dependent manner." Genome Biol). They have other recent papers on RNF40 (Schneider et al., 2019 "The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells", Clin Epigenetics; Kosinsky et al., 2019 "Loss of RNF40 decreases NF-kappaB activity in colorectal cancer cells and reduces colitis burden in mice", J Crohns Colitis). H2Bub1 is one of the least well studied histone modifications and as such, this study of one of its key histone writers, RNF40, is significant in elucidating the significance of this histone mark.

    Audience:

    This paper will suit a discovery-based science audience interested in epigenomic regulation of malignancy. Further, it will suit those looking for new drug development strategies for malignancy.

    My Field of Expertise:

    Basic scientist with expertise in epigenetic/epigenomic regulation in malignancy; cell and molecular biology. I felt capable of reviewing all aspects of this paper.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Wegwitz and colleagues present extensive and detailed data focussed on the role of the E3 ubiquitin ligase and ring finger protein RNF40 in HER-2 associated breast cancer. It is clear that the role of RNF40 and its major substrate histone H2B (monoubiquitination of histone H2B at lysine 120; H2Bub1) as part of a complex with RNF20, is not a simple one in the context of malignancy. This group, and others, have previously reported on the intriguing role of RNF40 that can in certain circumstances function to suppress tumorigenesis, and in other circumstances function to support tumorigenesis. While H2Bub1 has been shown to be lost in many different malignancies, these investigators show that in HER-2 associated breast cancer, this is not the case. In fact, the results presented in this study show that RNF40-mediated H2Bub1 is important for the expression of genes involved in the actin cytoskeleton and the downstream FAK signalling cascade. Supporting this, mining of a public database showed that RNF40 mRNA was high in HER-2 associated breast cancers and was correlated with a worse prognosis (overall and RFS, relapse free survival). The investigators also used a mouse model (MMTV-Erbb2) generating a tri-transgenic (MMTV-Erbb2; MMTV-Cre; Rnf40flox) that allowed breast tissue specific overexpression of HER2 at the same time as KO of Rnf40, so mimicking the human disease. In fact, mouse tumours recapitulated the human results, including in disease free survival (lower with higher Rnf40), and with less tumours seen when there was less Rnf40 (the Rnf40 floxed tumours appeared heterogenous in staining patterns for Rnf40 and H2Bub1, supporting the concept of "escaper" cells, positive for both Rnf40 and H2Bub1 that would be positively selected during tumorigenesis). The authors also took a cell biology approach to studying HER2 positive breast cancer and RNF40 using two HER2 positive cell lines (HCC1954 and SKBR3). RNF40 was down-regulated using siRNA and numerous functional studies showed that targeting RNF40 suppressed behaviours consistent with tumorigenesis (proliferation, migration, clonogenic survival, spheroid formation, growth kinetics). Furthermore, down-regulation of RNF40 in the HCC1954 cell line followed by GSEA identified gene signatures associated with apopotosis and the actin cytoskeleton regulatory pathway (e.g. ROCK1, VAV3, LIMK2, PFN2). They further showed that phospo - cofilin (that occurs downstream of ROCK1) was reduced in RNF40 down-regulated cells, also implicated in regulation of the actin cytoskeleton. Phalloidin staining for F-actin showed disruption of the cytoskeleton in RNF40 down-regulated cells. Additionally, the ROCK1 inhibitor, RKI-1447, showed similar effects to depletion of RNF40. The authors then sought to determine whether the RNF40 associated gene expression in HER2 positive cells was in fact happening through H2Bub1 and the active histone mark H3K4me3 it has been reported to cross-talk with. RNF40 regulated genes (up or down-regulated) showed lower levels of H2Bub1 occupancy compared to non-regulated genes. H3K4me3 was lost in most genes influenced by RNF40 down-regulation, including genes associated with the actin regulatory pathway. The overall conclusion is that RNF40 is a major epigenetic regulator of the actin regulatory gene network in HER 2 positive breast cancer and could be a therapeutic target.

    Major comments: major issues affecting the conclusions.

    (1) What is happening at the gene level for both H2Bub1 and H3K4me3 in the context of RNF20 down-regulation is complex and would benefit from inclusion of a schematic, or a series of schematics describing different scenarios, as the text is quite difficult to follow. It is not entirely clear that the changes seen in H3K4me3 are a direct result of cross-talk with H2Bub1 (some literature reports that there is no cross-talk between these histone marks for instance). It is also not entirely clear how the other histone marks investigated support the main discoveries of the paper. The authors need to consider this in the way that they present the data and their interpretation of it.

    (2) RNF40 is known to work in a complex with RNF20 to monoubiquitinate histone H2B at lysine 120 (H2Bub1). In experiments where RNF40 has been down-regulated, did the authors also note down-regulation of RNF20 (as has been previously reported). Is the data presented likely to be the result of abrogation of the complex rather than RNF40 specifically? While I am not asking for experiments to be repeated with down-regulation of RNF20, some consideration of this needs to be included in the Discussion. Is RNF20 also highly expressed in HER2 positive breast cancer (TCGA, KM Plotter data).

    Minor comments: important issues that can confidently be addressed.

    (3) It would appear that immunohistochemistry for RNF40 and H2Bub1 on human samples is only reported as "low" or "high". This is perhaps not dealing with the full spectrum of IHC scores, such as completely absent, although the methods do note a "null" value (no detectable staining). Were there no "null" results? Please define the criteria for "low" or "high".

    (4) I think there might be some confusion in labelling of Fig 1A and B as the legend states that all breast cancers are on the left and the HER-2 positive on the right, for each of primary tumours and brain mets, but I think one is under the other? Labelling should be checked in this figure.

    (5) What this IHC data doesn't show is whether RNF40 and H2Bub1 levels are always correlated in individual tumours (i.e. RNF40:H2Bub1, high:high OR low:low OR null:null). Can the authors please include and comment on this data.

    (6) Please include overall survival data (KM Plotter) as a panel in figure 1, alongside RFS for RNF40 expression levels (currently in Supplementary).

    (7) Spheroid formation looks to only be shown in a single cell line (HCC1954). Was the other cell line not suitable for spheroid studies? Some comment should be made and care taken not to "overclaim" as text notes two cell lines.

    (8) It would have been interesting to see results of a GSEA in the mouse mammary tumours as a complement to human. Is there a reason why this wasn't undertaken?

    (9) Conclusions are made about RNF40 in HER2 positive cells only in the context of H2Bub1 and H3K4me3. Without having conducted similar experiments in HER2 negative breast cancer cell lines / models, it is difficult to draw the conclusion that this is HER2 positive specific. Can the authors either soften some of their conclusions along this line, or consider repeating some of their data in HER2 negative models.

    (10) RNF40 likely has substrates other than histone H2B. There is a report describing interactions with RNF40 (STARING) and syntaxin for e.g., (Chin et al., 2002 J Biol Chem 277:35071-9). Can the authors please comment on other potential substrates of RNF40 in light of their data that focuses only on its epigenetic role as a regulator of the actin cytoskeleton.

    Significance

    Nature and Significance of the Advance:

    Clinically, this work provides a significant advance in that it is zeroing in on HER2 positive breast cancer and generating fundamental data that could underpin development of a new therapy for this malignancy. Conceptually, it is expanding knowledge of how the E3 ubiquitin ligase RNF40 is functioning as an epigenetic modifier of a specific type of malignancy by being important for the actin cytoskeleton.

    Work in Context of Existing Literature:

    As acknowledged by the authors, this work builds on a previous publication of theirs (Xie et. al., 2017 "RNF40 regulates gene expression in an epigenetic context-dependent manner." Genome Biol). They have other recent papers on RNF40 (Schneider et al., 2019 "The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells", Clin Epigenetics; Kosinsky et al., 2019 "Loss of RNF40 decreases NF-kappaB activity in colorectal cancer cells and reduces colitis burden in mice", J Crohns Colitis). H2Bub1 is one of the least well studied histone modifications and as such, this study of one of its key histone writers, RNF40, is significant in elucidating the significance of this histone mark.

    Audience:

    This paper will suit a discovery-based science audience interested in epigenomic regulation of malignancy. Further, it will suit those looking for new drug development strategies for malignancy.

    My Field of Expertise:

    Basic scientist with expertise in epigenetic/epigenomic regulation in malignancy; cell and molecular biology. I felt capable of reviewing all aspects of this paper.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Wegwitz et al propose that the E3 ubiquitin ligase RNF40 is highly expressed in HER2+ breast cancer tumours and correlates with poorer survival, using their own and TCGA data. Contrary to observations suggesting a tumour-suppressive role in other cancers, authors show using RNF40-knockout breast cancer mouse models and in vitro data shat RNF40 promotes tumour growth. RNF40 depletion impairs proliferation, survival and sphere formation by inducing apoptosis. In addition, RNF40 promotes cell migration by upregulating expression of cytoskeletal proteins (ROCK1, VAV3, LIMK2) and their effectors such as phosphorylated cofilin. Authors show elegant partial rescue experiments of the effect of RNF40 depletion on apoptosis and survival.

    Given that RNF40 function seems to be context-dependent, findings from this study could have broad significance for other cancers with high RNF40. It also provides some mechanistic data (that should be improved as suggested below) linking this ubiquin ligase to the cytoskeletal machinery and, therefore, control of migration and also proliferation and survival.

    Data are well presented and most conclusions are supported by the data. However, there are some gaps at the mechanistic level. Since migration is controlled by RNF40 in vitro, evaluation of metastatic ability in vivo (local invasion for example as suggested below) should be evaluated and would strengthen this part too.

    Major comments

    1.Fig.1A-B, S1A. Specificity of RNF40 antibody should be shown, which could be done quite easily in the tumours from the knockouts. From the datasheets, antibodies recognize human protein only.

    2.It is unclear when the murine tumours were analysed, at endpoint? This should be stated. Could authors establish cell lines from the mouse tumours (knockout, partial knockout escapers..)? These could be very useful tools to evaluate key in vitro findings from the study.

    3.Fig.1F-G: since RNF40 controls the cytoskeletal machinery and therefore, migration (Fig. 2G) in the RNF40 knockout tumours, was metastasis (if observed) affected? Or if there was no growth in distant organs detected in the time frame of these experiments, was invasion (and/or pattern of invasion or mode of invasion (morphology of invading cells)) into adjacent tissues affected upon RNF40 depletion? This would add in vivo relevance to the in vitro mechanistic findings, especially since the authors later showed that p-cofilin was also decreased in the RNF40-depleted mouse tumours (Fig.4D).

    4.Fig.3: most results using HCC1954 cell line. Key findings should be validated in other cell lines.

    5.Fig.3A: authors state "both pathways remained intact following RNF40 depletion". However, from those blots, siRNF40 clearly increases pERK and slightly pAKT, which would be unexpected according to previous data in Fig.2. Authors could show quantifications of different blots, or show a more representative blot if increase in pERK was not consistently observed. Was this also seen in SKBR3 cell line?

    6.For Fig.3G and Fig.S3A, authors selected genes from this set, how was this done (fold change?). Was expression of the other family members (ROCK2, LIMK1, etc) or of Rho GTPases regulated too?

    7.Fig.4B: this may not help, decrease of p-cofilin by Vav3 knockdown is way less dramatic compared to RNF40 depletion or ROCK inh treatment. See comment below regarding other effectors such as Myosin. Fig.4C: does ROCK inh reduce RNF40 levels? It may from the immunofluorescence picture.

    8.Fig.4H-I: the sphingosine 1-phosphate receptor-3 agonist (CYM-5441) partially rescued the effects of RNF40. Since S1P signalling involves Rho GTPase activation -presumably downstream of VAV3 -which is a GEF for Rho, Rac and Cdc42- and upstream of ROCK, LIMK, was activity of these Rho GTPases affected upon RNF40 depletion? This would strengthen the mechanism.

    Related to this, was Myosin II activity (phosphorylated MLC2) affected -since its upstream regulators, especially ROCK are controlled by RNF40?

    9.Fig. S5E: Authors should consider presenting data of decreased histone methylation of cytoskeleton regulators in main Fig. 5, since this is an important conclusion of this part.

    10.Statistics should be revised throughout the manuscript. Comparisons of more than 2 groups should be performed with ANOVA or similar multiple comparison test (instead of t-test).

    Minor comments

    1.Statement of significance mentions "Anti-HER2-therapy resistance", but this is a misleading since the paper does not deal with therapy resistance. Or are the cell lines used in the study resistant to anti-HER2? In line with this, authors could add some lines of thought on how RNF40 could be targeted in the clinical/pre-clinical context, which could inform further translational studies.

    2.Line 117 "Moreover, HER2-positive metastatic BC samples showed a particularly high expression of RNF40 compared to primary tumors". Perhaps rephrase, was it that the expression level (intensity) was higher or that the % of positive cells/tumours was higher in the brain mets?

    3.Fig.1D and S1C. while S1C shows TCGA data, it is unclear which set of patients is Fig.1D (since text says publicly available data, line 118-119), are these their own set of patients (used in Fig.1A-B)? This should be specified in the text, legend.

    4.Line 122. Authors should be careful with this conclusion so far, a correlation between expression and cancer stage/survival does not necessarily mean a tumor suppressive/supportive role.

    5.Fig. S4E: there are missing labels in graph (control, siRNF40).

    6.Panels in some figures are discussed in text randomly and not following same order. For example, Fig.1 (after panel D, then panel H, then back to E, F, G), S3, 4A-C,

    7.Fig.1E: I would suggest changing the line colours, so Rnf40wt-wt line is red and the fl-fl is black, therefore it is similar to panel D (high Rnf40 red, low in black).

    8.Supp Videos: for reviewers and readers, it would help that video has a label while it plays, otherwise after downloading it, video name does not tell whether it is control or RNAi.

    Significance

    Given that RNF40 function seems to be context-dependent, findings from this study could have broad significance for other cancers with high RNF40, or even in other pathological contexts -if any- that cursed with high RNF40. It also provides some mechanistic data (that should be improved as suggested in comments) linking this ubiquitin ligase to the cytoskeletal machinery and, therefore, control of migration and also proliferation and survival. This will also advance the field.

    Area of expertise Actin-myosin cytoskeleton, Rho GTPases-ROCK, cancer, metastasis, cell signalling

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    Referee #1

    Evidence, reproducibility and clarity

    In this study by Wegwitz et al, the authors examine the tumour promoting properties of RNF40 (and the H2B monoubiquitinylation catalysed by it) in Her2 driven breast cancer. They report, using publicly available data, that increased RNF40 expression is associated with reduced overall and disease-free survival. Using a mouse model, where they crossed the Erbb2 (mouse Her2) under the control of the MMTV promoter with conditional Rnf40 deletion constructs, the authors found that deletion of Rnf40 simultaneous to Her2 overexpression resulted in a prolonged tumour-free survival, somewhat reduced tumour growth kinetics and tumour incidence. siRNA silencing of Rnf40 in two Her2 positive breast cancer cell lines resulted in reduced proliferation, clonogenicity and tumour sphere formation and cellular motility. Transcriptome analysis revealed pathways that could explain the phenotype, like increased apoptosis and actin cytoskeleton regulation. The authors then took further some candidates in the later pathway to investigate the mechanism. They find that Rnf40 loss impacts on actin cytoskeletal dynamics. They also investigate the impact on focal adhesion signalling integrity. Finally, they investigate the relationship between the transcriptome and H3K4me3 and H2Bub1 landscape in the presence or absence of Rnf40.

    The manuscript is convincing regarding the tumour promoting roles of Rnf40, but the key claim that H2B monoubiquitinylation is essential for activation of the Rho/Rock/Limk pathway, where genes are down regulated upon Rnf40 loss resulting in decreased tumourigenicity of cells, is so far not convincing. "Together, these findings support the hypothesis that the actin regulatory gene network is dependent on direct 271 epigenetic regulation by RNF40 through modulation of H2Bub1 and a trans-histone cross-talk with H3K4me3 272 levels in HER2-positive BC cells." Although the correlation is apparent, at this point it's unclear if the phenotype is dependent on the catalytic activity of Rnf40 or it's a non-catalytic effect. Generating a catalytic mutant RNF40 and test it at least in the cell lines studied would be desirable.

    Other comments that need a response:

    1."we investigated RNF40 expression and 114 H2Bub1 levels by immunohistochemical staining of 176 primary BC tumors and 78 brain metastases." In Fig 1 I can only count 41 primary BC tumours and 73 brain metastases. Numbers don't add up. Also, how is "low" defined as opposed to negative? What is used as controls?

    2."Moreover, HER2-positive metastatic BC samples showed a117 particularly high expression of RNF40 compared to primary tumors" Figure 1 or Fig S1A does not contain data on HER2-positive metastatic BC

    3."tumors did not display a loss of either 132 RNF40 or H2Bub1 (Fig. 1H) when compared to the adjacent normal mammary epithelium (Fig. S1F)." I don't understand what I see in Fig S1F, where is the tumour, what is adjacent?

    4."homozygous loss of Rnf40 (Rnf40fl/fl134 ) resulted in 135 dramatically increased tumor-free survival of MMTV-Erbb2 animals (Fig.1E)." This is overinterpretation of the data, I would not call it dramatic, just significant.

    5."loss of Rnf40 led to 139 strongly reduced tumor growth kinetics (Fig.1G)." Is this result significant, I did not see an evaluation of statistical significance in this data.

    6."Rnf40fl/fl 142 lesions displayed a 143 heterogeneous pattern of RNF40 expression (Fig.1H), suggesting that the few tumors that did develop in this 144 model were largely caused by an incomplete loss of the Rnf40 allele." If this conclusion is suggested, the authors should check if the "escaper" cells have failed to flox the Rnf40 allele on the genetic/protein level. Otherwise it's not conclusive.

    7.Fig S4D - is this clonogenic assay? How many replicates were done, biological technical?

    8."Additionally, treatment with either CYM-5441 (Fig.4J) or 225" Fig 4J is missing! It makes this section rather hard to follow. Fig S4F-G, how many replicates were done, biological technical?

    9."Consistent with our analyses based on changes in H3K4me3 occupancy, genes downregulated upon RNF40 256 silencing displayed the most prominent decrease in H3K4me3 in the gene body (the 3' end of the peak)" The impact of these mods changes is hard to judge because they are rather small (I would not use the wording prominent). Also, are there many other "peak narrowing" genes but they are not downregulated?

    10.Statistical analysis missing: for example in Fig 2C, Fig 2E, Fig 3G what is n=?, how many technical, biological replicates were analysed?

    11.Fig 4E seems to be a partial duplication of Fig 3D!

    Minor:

    Figure referencing: it can be quite confusing to see a different ordering of figures compared to the referencing in the manuscript, for example Fih 1H is referenced in the text before Fig 1F, G. The authors should change the order in the main figures....

    Significance

    It's an interesting study that associates epigenetic regulation of actin cytoskeletal dynamics in Her2 driven breast cancer.

  6. Version published to 10.1101/2020.02.24.962902v1 on bioRxiv