Oxytocin alleviated colitis and colitis-associated colorectal tumorigenesis by targeting fucosylated MUC2

  1. Xia Wang
  2. Dawei chen
  3. MengNan guo
  4. Yao Ning
  5. Jing Guo
  6. Jiahui Gao
  7. Xiaoran Xie
  8. Dong Zhao
  9. Lixiang Li
  10. Shiyang Li
  11. Yanqing Li
  12. Xiuli Zuo
  13. Jingxin Li

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Abstract

Colon cancer is commonly regarded as hormone-independent. However, there have been reports suggesting the involvement of sex hormones in colon cancer development. Nevertheless, the role of hormones from the hypothalamus-hypophysis axis in colitis-associated colorectal cancer (CAC) remains uncertain. In this study, we observed a significant reduction in the expression of the oxytocin receptor (OXTR) in colon samples from both colitis and CAC patients. To investigate further, we generated mice with an intestinal epithelium cell (IEC)-specific knockout of OXTR. These mice exhibited markedly increased susceptibility to dextran sulfate sodium (DSS)-induced colitis and DSS/Azoxymethane (AOM)-induced CAC compared to wild-type mice. Our findings indicate that OXTR depletion impaired the inner mucus of the colon epithelium. Mechanistically, oxytocin was found to regulate MUC2 maturation through B3GNT7-mediated fucosylation. Interestingly, we observed a positive correlation between B3GNT7 expression and OXTR expression in human colitis and CAC colon samples. Moreover, the administration of oxytocin significantly alleviated tumor burden. Hence, our study unveils oxytocin’s promising potential as an affordable and effective therapeutic intervention for individuals affected by colitis and CAC.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Oxytocin (OXT) is a neuro-hypophysial hormone and exerts its effects through binding to the oxytocin receptor (OXTR). OXTR is expressed by various types of cells, including leukocytes and gastrointestinal cells. Previous studies demonstrated that OXT alleviates experimental colitis and regulates anti-inflammatory response. The authors' group reported that conditional deletion of OXTR in macrophages and dendritic cells exacerbated dextran sulfate sodium (DSS)-induced colitis. In the present study, they aimed to uncover the essential function of OXT signaling in colonic carcinogenesis and colitis by using intestinal epithelium cell (IEC)-specific OXTR knockout (KO) mice. IEC-specific KO mice exhibited markedly increased susceptibility to DSS-induced colitis and Azoxymethane (AOM)/DSS-induced colitis-associated colorectal cancer (CAC) compared to wild-type mice. Mechanistically, OXTR depletion in IECs impaired the inner mucus of the colon epithelium. Furthermore, oxytocin was found to regulate MUC2 maturation through B3GNT7-mediated fucosylation. In human colitis and CAC colon samples, there was a positive correlation between B3GNT7 expression and OXTR expression. Moreover, the administration of oxytocin significantly alleviated tumor burden. These results suggested oxytocin's promising potential as an effective therapeutic intervention for individuals affected by colitis and CAC.

    Major comments:

    1. Figure 1: The expression levels of many genes are altered in cancer cells. It is unclear whether decreased OXTR expression is the cause or the consequence of CAC in both human cases and mouse experiments. In Figure 1B, the background staining on the control tissue is very high. On the CAC tissue section, the staining appears uneven and OXTR staining appears high where the background is high. Thus, the result is not convincing.
    2. OXTR is expressed by many types of cells, including leukocytes, and OXTR expressed by leukocytes is reported to have an anti-inflammatory activity (Mehdi et al., Front Immunol, 2022, 13:864007. doi: 10.3389/fimmu.2022.864007). In this study, the importance of OXTR expressed by leukocytes is not considered.
    3. Oxytocin is usually administered by injection. It is unclear how it was administered. Oral administration is probably not effective. There is also no description about the source of oxytocin.
    4. The effects of oxytocin could be different between males and females. It might be interesting to present data of males and females separately and comment on the finding. It was previously shown that OT plasma levels (pg / ml, mean {plus minus} SD) were significantly higher in women than in men (4.53 {plus minus} 1.18 vs 1.53 {plus minus} 1.19, p ˂ 0.001), and such differences might be related to behaviors, attitudes, as well as susceptibility to stress response, resilience and social emotions specific of women and men (Marazziti et al., Clin. Pract. Epidemiol. Ment. Health. 2019; 15: 58-63). Male mice are more susceptible to DSS-induced colitis and this could be due to different oxytocin levels.
    5. Figure S3: Is the antibody directed to sugar? In the absence of OXTR, MUC2 is almost absent. It is hard to believe that the expression/production of MUC2 is almost completely dependent on oxytocin.
    6. Figure 5: The authors indicated that reduced fucosylation was due to the decreased B3GNT7 expression. Addition of L-fucose may not result in increased fucosylation.
    7. The effects of fucose supplementation was studied using a colitis model, whereas the effects of oxytocin supplementation was studied using a colon cancer model. Thus, the effects by these two agents cannot be compared.
    8. Figure 6: Oxytocin treatment could activate OXTR expressed on both leukocytes and epithelial cells. There is no comment on this subject.
    9. Figure 6I: A few mice died after 20 days. Is it correct? What does "day 0" mean in this figure?
    10. Figure 7B: Again, the result is not convincing. Leukocytes are reported to express OXTR and many leukocytes are in the colon tissues, especially in colitis tissue. But, they are not positive.
    11. Figure 7M: It is good to have a summary figure; however, it appears not accurate. There is no data showing the floxed mice have "Tolerant immune response" and KO mice have "dysregulated immune response".
    12. The authors' group previously reported that OT activated IECs to release prostaglandin E2 that was required for the repair of intestinal epithelium after injury (Ref. 11 in this manuscript). What happened to this mechanism?

    Minor comments:

    1. Mice: There is no reference for the floxed mice. It would be also helpful to add the strain number.
    2. Figure 1D: What was the expression level "1"? There is only a small difference between 6.4 (Control) and 6.1 (AOM/DSS).
    3. Figure 1J: What caused the increase in spleen weight? Is this a marker for increased inflammatory responses or cancer cell growth?
    4. Figure 1K: What was the meaning of the increased expression of each cytokine?
    5. Figure 1L: The photos are too small to see the detail.
    6. Figure 1M: What was the end point?
    7. Page 3: "OXTR Deficiency in IEC Facilitates CAC Depends on Inflammation" This is not a sentence.
    8. Figure 2A and I: "% weight loss" were less then 1%. Is it correct?
    9. Figure 2N: Hard to see any differences. Too small.
    10. Fig 5K: Labels are not complete.
    11. Figure 5: Silver color is used for lines and columns but difficult to see.
    12. It is unclear at which time point samples were prepared.

    Significance

    General assessment:

    The role of OXT and its receptor OXTR in DSS-induced colitis was previously reported by using systemic or myeloid cell-specific OXTR KO mice. Here, the authors used IEC-specific OXTR KO mice and found that OXTR expressed by IEC cells plays an important role in inflammation-associated colitis and CAC by promoting the post-transcriptional modification of MUC2 via B3GNT7. This is a strength. The major imitation is that they used only IEC-specific KO mice and the relative importance of OXTR on IECs is unclear. In addition, important information necessary to understand the results is missing throughout the manuscript. There are other questions as listed in the comments.

    Advance:

    Interaction of OXT and OXTR has been demonstrated to protect mice from inflammation-associated colitis. This study went one step further by demonstrating that OXTR expressed by IEC cells plays a protective role in inflammation-associated colitis and CAC by promoting the post-transcriptional modification of MUC2 via B3GNT7.

    Audience:

    Basic research and translational/clinical.

    Field of expertise:

    The reviewer is expertized in the field of "cancer and inflammation" but not in "MUC2" or "glycosylation". Keywords: inflammation, chemokines, leukocyte trafficking, tumor microenvironment.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: In their paper "Oxytocin alleviated colitis and colitis-associated colorectal tumorigenesis by targeting fucosylated MUC2" the authors describe the contribution of oxytocin (OXT) to colonic mucus formation, and its protective contribution to colitis and colon cancer. The authors also describe the mechanism by which OXT execute its alleviating effects on the colon mucus; enhanced B3GNT7-mediated fucosylation. The findings are demonstrated in cell cultures, various mice models, and samples from human patients.

    Major comments:

    1. A conceptually confusing issue in this work is whether a decrease in OXTR expression is a predisposition, or a result of colonic illness. On one hand, the experiments with OXTR KO mice and cultured cells suggest that pre-existing lower levels of the receptor sensitize the tissue. However, in the AOM/DSS model the control mice present normal OXTR levels whereas mice that received AOM/DSS had lower expression, suggesting that changes in OXTR levels are not a predisposition but a result of the treatment/illness. Additionally, when tissue from CAC patients were analyzed, decreased levels of OXTR were found in sites of wounds but not in adjacent healthy tissue, implying that this decrease is not a genetic treat but a result of external cue. This inconsistency must be sorted out and clearly demonstrated.
    2. The study describes a new regulatory pathway for colonic mucin 2, and colon related conditions. Why did the author choose to generate mice lacking OXTR in the entire intestine (small+large) and not a large-intestine specific deficiency? And is there any way to demonstrate that the absence of OXTR in the small intestine does not interfere with the results presented here?
    3. The commonly used fixative for mucus and secreted mucins is Carnoy fixative (can be found in many of Hannson G.C and in Johansson M.E.V papers, and many other papers describing colon staining), while the use of formaldehyde and glutaraldehyde is less preservative for mucus layer. This raises a concern regarding the data obtained from aldehyde-fixed mucus samples.
    4. The authors found that mice lacking OXTR have lowered levels of B3GNT7, which leads to a decrease in mucin 2 fucosylation and to further damage in the colon. What is the mechanism by which supplementation with L-fucose alleviates these outcomes given that the enzyme that regulates the addition of the fucose to mucin 2 is downregulated?

    Minor comments:

    1. Some IHC images don't show comparable or similar areas. Specifically, Figure 1 B, Figure 7 F, I.
    2. There is a discrepancy between the dosage of DSS used to induce chronic colitis in the text (2%) and in the methods (2.2%). In addition, the difference between concentrations of DSS used to induce chronic and acute colitis (2.2% vs. 2.5%, respectively) is significantly smaller than what is reported in many other papers using these models.
    3. In Figure 2 A, I, Y-axis labeling doesn't seem right (compare with Figure 5 G). It looks like the decimal point is a mistake.
    4. All Western blots presented in this study lack the molecular weight of the proteins. In many cases it would have been more convincing to see a larger portion of the membrane.
    5. Mucin 2 in a large protein (more than 5000 amino acids in human mucin 2), and many disulfide bonds. The authors do not mention if any reducing or denaturing agents were added to the lysis buffers, and whether any other special conditions were employed to separate this huge globular protein on SDS-PAGE gel.
    6. The following sentence should be revised: " To examine the effects of fucosylation regulated by OXT on LS174T cells and colonic organoids, we found that..."

    Significance

    Though the concept of OXT-mediated suppression of colon cancer has been reported (For example: PMID: 34528509, and 31920487), the regulatory pathway by which it exerts its alleviating effect, and all the mechanistic components described in this paper, were not known before. This pathway may be a potential target for therapeutic intervention in various colonic diseases. Moreover, additional mucins may be regulated by OXT in a similar manner, which can extend the importance of these findings to other organs and disease-conditions. This type of findings is of interest to the broad audience of general cell biology as well as to GI clinicians. However, as stated in my comments there are some major issues with the hypothesis, the way data is presented, and in key methods that fundamentally limits my ability to evaluate this paper.

    Key words for my field of expertise: Disulfide catalysis, Golgi, mucin

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In this study, Wang and colleagues present data linking oxytocin signaling to protection against colitis and colitis associated cancer development. To this end, they utilize a Villin-Cre line to specifically remove OXTR only from intestinal epithelia and make use of AOM and/or DSS treatment to induce colitis, colorectal cancer or CAC. OXTRdeltaIEC mice consistently develop worse symptoms and more severe colitis/CAC compared to non-Cre expressing littermates, which appears to be associated with defects in the mucus layer. Using RNA-Seq, they identify the glycosyltransferase B3GNT7 as a differentially expressed gene. Due to its role in O-linked glycosylation, they investigate whether B3GNT7 is involved in mucin production and are able to show that OXT-induced upregulation of MUC2 protein is abolished when B3GNT7 is knocked down. In vivo, co-treatment with oxytocin reduces experimental CAC, which is an interesting that OXT may present a potential treatment option in CAC. The study is quite interesting in that it provides a potential treatment option where the mucus layer, which is often disturbed in IBD, can be impacted in a positive way. Still, there are some things missing to really be able to interpret the full picture and these should be experimentally addressed.

    Major comments

    • The staining for OXTR in Fig 1B is very strong (especially as others have reported that they were not able to demonstrate OXTR in human samples, Ohlsson et al. PMID: 16678285) - it would be beneficial to confirm OXTR distribution in steady state in mice, especially as you have a great negative control were OXTR staining should be absent from the IECs. Given the observations further in the study, I would also be very curious to see whether OXTR expression is specific to goblet cells, so co-staining with Muc2 would be interesting to include in later figures. The information for the OXTR antibody is also missing from the supplementary methods.
    • Fig 1D, Fig S2 - is this whole colon RNA or specifically epithelial cells? This can have major impact on observed expression levels as the relative amounts of epithelial vs. other cell types can drastically change, thereby falsely giving the impression that expression levels in the epithelial cells change, so this really needs to be analyzed in purified epithelial cells. In Fig S2 there is significant OXTR expression remaining in the deltaIEC mice, so this suggests to me that non-IEC cell types are also included.
    • The interpretation of the study would benefit from including some steady state/untreated data for the OXTRdIEC mice. For example in Fig 2 the researchers report increased spleen size, increased cytokines etc upon CRC in these mice, but it is important to also show steady state data as these parameters may already be significantly increased in basal conditions in these OXTR deficient mice (especially seeing as Fig 3 claims that under basal conditions the mucus layer is extensively damaged you would expect some phenotype in these mice).
    • Special care needs to be taken to preserve the mucus layer during fixation, and from the methods it is not clear whether the authors have taken these technical difficulties into account. Only PFA fixation is mentioned, but it is well-established that the golden standard for imaging the mucus layer is to fix tissues in water-free Carnoy's fixative, as the mucus layer tends to collapse using formaldehyde (see also Johansson & Hansson, PMID: 22259139).
    • In Fig 5, the message and conclusions become a bit more fuzzy. Overall fucosylation is measured, but it is unclear whether MUC2 itself is increasingly fucosylated due to OXTR signaling, or that this represents more global changes in the secretory pathway that eventually lead to more efficient MUC2 production. Perhaps an IP using fucose-specific lectin combined with western blotting for MUC2 may be an option to demonstrate whether MUC2 itself becomes increasingly fucosylated due to OXTR signaling?
    • The title broadly claims that OXT "alleviates" colitis and CAC through MUC2 fucosylation and Fig 6 indeed shows that OXT treatment affects the outcome of mice in a CAC model, which is very promising, but it also loses the link with the mechanistic insights surrounding MUC2 fucosylation in previous figures. To really definitively make the claim in the title, it's important to investigate whether these OXT treated mice indeed have restored B3GNT7 levels and a thicker mucus layer after AOS/DSS regimen compared to non-OXT treated mice (as one would expect based on the in vitro data using LS174T cells and organoids). Studying the effect of OXT treatment in the regular DSS colitis model would also provide additional support for this claim.
    • Optional as I am not a specialist in OXT signaling: I would assume that there are quite some differences between males and females when it comes to OXT and OXTR. Have the authors ever observed differences in staining pattern or expression levels between males and females? The methods state that all groups are sex matched, but I wonder if it may be necessary to include gender as a variable in the analysis?

    Minor comments

    • I would suggest to include another reference in the introduction and/or discussion, as MUC2 deficient mice are also known to develop colorectal cancer (Velcich et al. PMID: 11872843) and this serves as additional support for why it is important to discover how we can positively impact the mucin layer in IBD patients.
    • In Fig 1A GEO data is reanalyzed, but it's not immediately clear what the original samples were (i.e. colon biopsies). At first glance, the figure itself adds to this confusion with the titles 'hypothalamus' and 'hypophysis' - it's not very clear that these labels indicate synthesis location of the respective hormone and not the tissue where expression was measured.
    • Fig 1C - I could not find in the methods what software was used for these quantifications.
    • Fig 1C - N=5 is mentioned in the figure legend and there are 10 datapoints in each group. Were 2 biopsies quantified per patient then? Please state this more clearly.
    • Fig 3A, B and all other western blots - please include molecular weight indications in the figure
    • Several figures use light grey bars and datapoints, but this color was very hard to see after printing the manuscript.
    • The conclusion statement for Fig 3 should be revisited, as the expression of Muc2 mRNA is not affected at all by OXTR genotype (Fig S2F). Conclusion should make it clear that specifically (mature) protein levels are affected.
    • Fig 4A-D - would be nice to include the full list of DE genes in supplement, it's an important resource. For example, there are other factors known that influence the mucus layer (such as AGR2), so I would be interested to see how these are behaving in the knockout mice.
    • In Fig 4 H-J it would be informative to show the MUC2 mRNA expression level in these cultures as this could provide support for the mice data - i.e. do the cultures also display normal MUC2 mRNA levels, with a specific defect in the mucin maturation (as appears to be the case in mice)?
    • Fig S3H-K - this figure and the validation of the siRNA is not mentioned in the main text
    • It is interesting that L-fucose seems to partly reverse the effect of DSS, but I wonder whether mechanistically this is explained by restoration of B3GNT7 expression?
    • Please check the accession codes for the reanalyzed datasets, figure legends mention two accessions, while the Data availability statement mentions three accessions.
    • The number of repeats for each experiment is a bit unclear. It is now buried in the statistics statement in the methods, but it may be more clear if it is included in each figure legend.

    Significance

    This study shows a -for me- quite unexpected link between oxytocin and protection against colitis and colitis-associated cancer development. Disturbances in the mucin layer are a very common phenomenon in IBD and colitis and there has been a great interest in this in the scientific community for quite some years (Johansson, PMID: 25025717, Yao et al. PMID: 34902790, and many others). Current IBD treatment options are generally aimed at reducing inflammation, but this does not necessarily restore the mucin layer quality. It is therefore quite interesting to see that this is apparently heavily influenced by oxytocin (which already has applications in human medicine), and this provides significant advance to our current fundamental understanding of mucin barrier regulation.

    As mentioned in the comments, the study can be further improved. To me, a more detailed investigation into the steady state phenotype of these mice, and a more detailed confirmation of where the oxytocin receptor is expressed is necessary to fully put the results into a broader framework. Also Fig 6, where the actual interventional effect of oxytocin is evaluated, no longer demonstrates whether this indeed happens through the same mechanism as outlined in the previous figures and this should be developed more.

    I expect this study to be of interest primarily to a basic research audience, though I assume that a more clinical audience would be intrigued by the findings as well.

  9. Version published to 10.1101/2023.08.17.553684v1 on bioRxiv