The ADAM17 sheddase complex regulator iTAP/Frmd8 modulates inflammation and tumor growth

  1. Marina Badenes
  2. Emma Burbridge
  3. Ioanna Oikonomidi
  4. Abdulbasit Amin
  5. Érika de Carvalho
  6. Lindsay Kosack
  7. Camila Mariano
  8. Pedro Domingos
  9. Pedro Faísca
  10. Colin Adrain

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Abstract

The metalloprotease ADAM17 is a sheddase of key molecules, including TNF and epidermal growth factor receptor ligands. ADAM17 exists within an assemblage, the “sheddase complex,” containing a rhomboid pseudoprotease (iRhom1 or iRhom2). iRhoms control multiple aspects of ADAM17 biology. The FERM domain–containing protein iTAP/Frmd8 is an iRhom-binding protein that prevents the precocious shunting of ADAM17 and iRhom2 to lysosomes and their consequent degradation. As pathophysiological role(s) of iTAP/Frmd8 have not been addressed, we characterized the impact of iTAP/Frmd8 loss on ADAM17-associated phenotypes in mice. We show that iTAP/Frmd8 KO mice exhibit defects in inflammatory and intestinal epithelial barrier repair functions, but not the collateral defects associated with global ADAM17 loss. Furthermore, we show that iTAP/Frmd8 regulates cancer cell growth in a cell-autonomous manner and by modulating the tumor microenvironment. Our work suggests that pharmacological intervention at the level of iTAP/Frmd8 may be beneficial to target ADAM17 activity in specific compartments during chronic inflammatory diseases or cancer, while avoiding the collateral impact on the vital functions associated with the widespread inhibition of ADAM17.

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  1. Version published to 10.26508/lsa.202201644
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    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    The metalloprotease ADAM17 is a key drug target for inflammatory diseases and tumors. The authors previously demonstrated that ADAM17 activity is controlled by iRhom proteins and by a largely uncharacterized protein called iTAP, which had been mostly investigated in vitro. The current manuscript extends the previous findings to in vivo functions, in particular in pathophysiological conditions. The authors demonstrate that iTAP-deficient mice are viable and largely without overt phenotypes under physiological conditions, but do show phenotypes upon inflammatory conditions and during tumorigenesis. During LPS-induced inflammation, iTAP-deficient mice are reported to show defects in epithelial repair functions. Upon lung tumor cell injection, iTAP-deficient mice revealed reduced tumor growth in a cell autonomous and a non-cell autonomous manner, raising the possibility to use iTAP as a potential new drug target for certain tumors.

    The study is novel, the manuscript is well written and easy to understand and the conclusions are largely justified by the data.

    My only major concern is the choice of the control LLC cells. The cells used are not the ideal control for the iTAP knock-out cells. Both wild-type and iTAP knock-out cells were transduced with a Cas9 expressing vector, as it should be. But only the knock-out cells were further transduced with a virus expressing the gRNAs against iTAP, whereas the control cells apparently were not transduced with a virus expressing control gRNAs. Three single cell clones of the iTAP ko cells were pooled for in vivo injection, whereas the parental pool (and not clones) where apparently used as a control. My concern is that the ko cells do not only differ from the wild-type cells due to the knock-out of iTAP, but potentially also due to other gene expression alterations resulting from the additional transduction of the knock-out cells with a gRNA virus and because of the selection of single cell clones. Such expression changes beyond the simple lack of iTAP may have a major influence on those tumor phenotypes in vivo, where these cells were used. Ideally, the authors would generate an additional, independent pool of iTAP knock-out cells and repeat one of the crucial in vivo experiments. As a time-saving alternative, the authors need to demonstrate that the iTAP knock-out cells are nearly identical to the control cells (with the exception of iTAP). This could be done by RNA sequencing or cell lysate proteomics or by blotting for several different proteins (at least 10 from different compartments) and demonstrating that there is no significant change in protein abundance - apart from iTAP.

    I do have a number of additional, but minor points.

    1. Indicate the concentrations of the used drugs (marimastat, PMA) in the figure legends.
    2. Indicate in the manuscript that LLC cells are of mouse origin.
    3. Page 6, top paragraph: it is not clear to me, whether there is an eye phenotype or not. Please rephrase this sentence.
    4. Figure legend 1: "...with 3 replicates per experiment". Indicate whether this refers to biological or technical replicates.
    5. Indicate in figure legends which statistical test was used.
    6. Fig. 2F. The y-axis label should be body weight and not body weight loss.
    7. Fig. 4C: the increase in the 75 kDa fragment upon iTAP OE is difficult to see. Can you quantify the increase? And also the reduction in the KO cells?

    Review Cross-commenting

    When reading the comments from reviewer 1 and 2, it is not always obvious to me which experiments must be done (as a requirement) and which ones are "just" nice to add. It would be great if this could be specified clearly in their reviews.

    Significance

    This study is exciting. It shows for the first time the pathophysiological role of iTAP in vivo and has major implications for ADAM17, which is a drug target in numerous diseases, in particular sepsis, inflammation and tumors. However, systemic ADAM17 inhibition induces severe side effects so that approaches are sought that allow a tissue-specific inhibition of ADAM17. One way to achieve this, is to block the protein iRhom2 which is a non-proteolytic subunit of an ADAM17-iRhom2 complex. Loss of iRhom2 allows a tissue-specific inhibition of ADAM17 specifically in immune cells, because other tissues express iRhom1 that can largely (but not fully) compensate for loss of iRhom2. Thus, iRhom2 inhibition is currently pursued in drug development. The current manuscript demonstrates an additional way (through iTAP) of selectively blocking pathophysiological functions of ADAM17 in tumors (and potentially sepsis), while maintaining physiological functions. This study is an important step towards the use of iTAP as a drug target. Thus, this study will be of interest to basic scientists studying ADAM17, its regulation, its substrate specificity and its physiological functions. The study will also be of interest to translational scientists in academia and pharma/biotech studying the numerous ADAM17-dependent diseases. A clear strength of the study is the inclusion of different disease models, where iTAP plays a role (protective or non-protective), and the demonstration that iTAP contributes to tumors both in the tumor niche and in the tumor itself. A limitation of the study is that the underlying mechanisms remain unclear apart from reduced ADAM17 activity. In particular, it remains open which substrate(s) contribute on the tumor side or the niche side. This lack of mechanistic insight is addressed in the discussion section, where a number of future follow-up experiments are suggested. Another central open mechanistic point is the question of why iTAP, that binds to both iRhom1 and iRhom2, apparently only affects iRhom2 function in vivo. Maybe iTAP only acts on iRhom2-dependent ADAM17 substrates? Despite these mechanistic weaknesses that need to be addressed in future studies, the study is exciting. I have expertise in ADAM17 and iRhoms, but cannot fully judge the tumor histology.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In "The ADAM17 sheddase complex regulator iTAP modulates inflammation, epithelial repair, and tumor growth" the authors investigate the role of iTAP (FRMD8) in regulation of the ADAM17 sheddase complex. This manuscript is a follow-up to a previous study (Oikonomidi et al. 2018) in which the same group (and another group, Künzel et al. 2018) defined iTAP and generated an iTAP-deficient mouse model via CRISPR. The current work uses in vivo models of sepsis (LPS injection), colitis (DSS administration), and tumor growth and metastasis (LCC subcutaneous and IV transfer models) to further investigate the role of iTAP during disease. The authors find that mature ADAM17 levels were decreased in immune cells from iTAP-deficient mice and shedding of L-selectin was impaired. They next showed that iTAP KO mice have reduced TNF serum levels in a sepsis model. During experimental colitis, iTAP KO mice had higher levels of intestinal disease indicators. In a subcutaneous tumor model, iTAP KO mice showed decreased tumor burden. Furthermore, the authors observed tumor cell autonomous and cell-non-autonomous roles for iTAP in subcutaneous and IV LLC transfer models. While this study nicely adds to our knowledge about in vivo effects of iTAP-deficiency, it is largely descriptive with little investigation into the mechanisms by which iTAP/ADAM17 promote disease. Despite these limitations, the authors make claims about mechanism in the title, abstract, and text without data to support these statements. For example, one of the conclusions of the manuscript is that iTAP influences tumor growth via control of cell proliferation yet there are no data to support this claim. Therefore, I have serious reservations that need to be addressed before I could consider publication of this work.

    Major comments:

    Figure 1: The findings regarding L-selectin shedding are very clear and perhaps meaningful. However, a discussion putting these findings in context with the disease models used later in the manuscript is warranted. Currently, inclusion of these data does not add much to the story if not discussed or referenced later in the manuscript.

    Figure 2: The authors observe that iTAP KO mice have worse outcomes following DSS-colitis. In the text, they mention that iRhom2 KO mice do not phenocopy the iTAP KO mice following DSS-colitis, yet no explanation is offered. If the mechanism of iTAP is proposed to be through iRhom2 activity and ADAM17 shedding, you would expect the iRhom KOs to demonstrate similar intestinal phenotypes. The authors should comment on this discrepancy.

    Additionally, the conclusion about the importance of iTAP in intestinal repair would be better supported if the DSS colitis experiments were continued to later time points to include the recovery phase (once the mice return to original body weight), rather than just ending the experiment at peak repair.

    Figure 3: The authors make the statement that "...although inflammatory infiltrates were modest in the lungs of mice..." Is this based on histology alone? If the authors want to make this claim, they must assess immune infiltrates directly (e.g. using flow cytometry).

    The authors evaluate lung metastasis in the LLC subcutaneous model but any conclusion about metastasis cannot be made in this model without looking at primary tumors of a similar size. Metastasis tends to be associated with the size of the primary tumor so smaller primary tumors usually mean lower levels of metastasis (without being able to parse apart direct effects on the metastatic process). I assume that the data in Fig 3H are from mice with different tumor sizes--in order to properly evaluate this, the authors need to euthanize WT and KO animals with similar tumor burdens and compare metastatic burden.

    Including total mRNA levels of cytokines does not add to this figure. First, bulk levels of mRNA are not a good way to evaluate the state of a tumor (immune cell phenotype/activity would be better). Second, TNF and IL-6 were used in previous figures as readouts of ADAM17 activity (or not) and here are just markers of inflammation? This is confusing/contradictory. If included, this should be moved to the supplement.

    Figure 4: Claims about proliferation cannot be made here because the results as shown are not significant (Fig 4K). Additional readouts for proliferation should be used to support this conclusion.

    Similar to Figure 3, claims about metastasis cannot be made from these experiments without comparing mice with similar primary tumor burden. The metastasis data in Figure 5 are much more solid and convincing.

    Figure 5: The authors use Fig 5 K & L as evidence that tumor cells proliferated more or less rapidly, depending on expression levels of iTAP. The data do not support this statement. If I understand the methods correctly, this assay involves plating of 500K tumor cells and then harvesting after several days. Upon harvest, there were 100 fold fewer cells (~5K). To me this indicates effects on survival, not proliferation. Proliferation was never measured in this assay. Without these data, the authors can make no claim regarding the mechanisms of tumor cell autonomous functions of iTAP.

    Minor comments:

    The language regarding any results that are not statistically-significant need to be softened in the text. In several places, there are statements about non-significant results that are much too definitive and somewhat misleading. Non statistically-significant results can be useful to include to show trends (as in Fig 5 G-I), but the interpretation should not be overstated.

    The title is overstated. In this manuscript, the authors do not show clear mechanistic links for iTAP promoting epithelial repair (worse outcomes after DSS are not just caused by decreased repair). The strongest data in the manuscript are those regarding tumor growth. This should be highlighted in the title.

    Significance

    This work adds additional data to support the importance of iTAP/sheddase complex/ADAM17 in disease development. Most importantly, it suggests a role for iTAP in tumor progression. However, the mechanisms leading to increased tumor growth still remain unknown. Additional work is required to elucidate the molecular mechanisms underpinning these observations.

    The target audience of this work would include cancer biologists and experts studying growth factors and metallopeptidases. For context, my background is in tumor immunology and immune-stromal interactions.

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    Referee #1

    Evidence, reproducibility and clarity

    The study analyzes the function of the FREM domain containing protein Frmd8/iTAP, which the authors have identifies as a binding partner of iRhom2. This rhomboid pseudoprotease has earlier been identified as a binding partner of the membrane-bound metalloprotease ADAM17. The iRhom2 protein is necessary for trafficking of ADAM17 through the ER/Golgi network to eventually reach the cell surface. Apparently, the proteins Frmd8/iTAP, iRhom2 and ADAM17 form a sheddase complex. In the present study, the authors have used knock-out mice for the gene coding for Frmd8/iTAP to analyze the role of the Frmd8/iTAP protein in vivo. The authors found that maturation of ADAM17 in hematopoietic cells was impaired and that shedding of ADAM17 substrates was strongly reduced. In a DSS inflammatory bowel disease model, Frmd8/iTAP knock-out mice were slightly more affected than WT mice. When tumor cells were injected into WT or Frmd8/iTAP knock-out mice, tumors were smaller in the absence of the Frmd8/iTAP protein. The deficiency of Frmd8/iTAP in tumor cells resulted in less tumor growth whereas the overexpression of Frmd8/iTAP in tumor cells led to more tumor growth. I.v. injection of tumor cells deficient for Frmd8/iTAP led to significantly less metastases, tumor volume and tumor burden. Th authors suggest that therapeutic intervention at the level of Frmd8/iTAP might be helpful during inflammatory diseases or cancer.

    This is an interesting study, which addresses the important role of ADAM17 and the pathways controlled by this protease. There are, however, some points the authors should address.

    Major points:

    1. Although the Frmd8/iTAP protein was identified as a binding partner of the iRhom2/ADAM17 complex, it remains unclear whether this protein also serves as a binding partner of other proteins. When analyzing Frmd8/iTAP knock-out mice, this might be an important aspect, which is not addressed in the manuscript. Is Frmd8/iTAP always co-expressed with iRhom2 and ADAM17?
    2. It has been shown that iRhoms have additional clients apart from ADAM17. For instance, the adaptor protein STING has been reported to be constitutively associated with iRhom2. Therefore, it is possible that Frmd8/iTAP also plays a role in the STING pathway. This point needs to be addressed.
    3. All Western blots shown in the figures and supplemental figures should be quantified by a suitable software such as Image J.
    4. In Fig. 2C,D, the authors use a sepsis model and they show that Frmd8/iTAP knock-out mice have lower TNFa levels than WT mice. Is this also true for sIL-6R levels? Was survival of the mice affected by the absence of Frmd8/iTAP?
    5. In Fig. 2E-J, the authors employ a DSS-driven inflammatory bowel disease model. It has been shown before (Chalaris et al, 2010; cited in the manuscript) that the higher susceptibility of hypomorphic ADAM17 mice was related to reduced shedding of EGF-R ligands in this model. Therefore, the authors should address shedding of these ligands in Frmd8/iTAP knock-out mice.
    6. In the experiment shown in Fig. 5, the authors inject parental and Frmd8/iTAP knock-out LLC tumor cells into WT mice. The note that the number of metastases, tumor volume and tumor burden is dramatically decreased. In the study by Bolik et al, 2022 (cited in the manuscript) it has been shown that in hypomorphic ADAM17 mice, metastasis formation by LLC tumor cells was dramatically reduced. In this study it was also shown that ADAM17 activity in endothelial cells was responsible for this effect, which was at least in part mediated by TNF-RI and TNF-RII. This mechanistic difference should be addressed in the manuscript.
    7. Along the same line: when tumor cells are injected i.v., the cells need to extravasate before they can form tumors. The authors need to mechanistically address whether the effects of Frmd8/iTAP are on extravasation or on tumor growth (or both).

    Minor points:

    1. The authors name the protein Frmd8/iTAP sometimes as Frmd8 and sometimes as iTAP. This is confusing for the reader. Since the protein has been characterized under both names, the authors should stick to Frmd8/iTAP.
    2. Along the same line: the authors should stick to the name ADAM17 and not sometimes switch to the older name TACE.
    3. The authors use Frmd8/iTAP knock-out mice. It is not clear from the statement of p5, whether they use the mice described in Künzel et al, 2018 or the mice described in Oikonomidi et al, 2018. This should be clarified.
    4. Some references (e.g. Dong et al, 1999 and Gschwind et al, 2003) are incomplete.

    Significance

    Nature and significance of the advance:

    Knowledge about the susceptibility of Frmd8/iTAP knock-out mice to some disease models of inflammation and cancer.

    Compare to existing published knowledge:

    It was known before that Frmd8/iTAP plays a role in ADAM17 maturation and that the absence of Frmd8/iTAP leads to lower shedding of several substrates.

    Audience:

    ADAM17 governs important pathways such as TNFa, IL-6R, EGF-R and others and therefore, the regulation of ADAM17 activity is of interest to many readers.

    Your expertise:

    I work on the cytokine IL-6 and the IL-6 trans-signaling pathway, which relies on the soluble IL-6R, generated by ADAM17. Therefore I feel competent to review the manuscript.

  10. Version published to 10.1101/2022.04.11.487842v1 on bioRxiv