The roles of distinct Ca 2+ signaling mediated by Piezo and inositol triphosphate receptor (IP3R) in the remodeling of E-cadherin during cell dissemination

  1. Alejandra J.H. Cabrera
  2. Barry M. Gumbiner
  3. Young V. Kwon

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Abstract

Given the role of E-cadherin (E-cad) in holding epithelial cells together, the inverse relationship between E-cad levels and cell invasion has been perceived as a principle underlying the invasiveness of tumor cells. In contrast, our study employing the Drosophila model of cell dissemination demonstrates that E-cad is necessary for the invasiveness of Ras v12 -transformed cells in vivo . Drosophila E-cad/β-catenin disassembles at adherens junctions and assembles at invasive protrusions—the actin- and cortactin-rich invadopodia-like protrusions associated with breach of the extracellular matrix (ECM)—during cell dissemination. Loss of E-cad attenuates dissemination of Ras v12 -transformed cells by impairing their ability to compromise the ECM. Strikingly, the remodeling of E-cad/β-catenin subcellular distribution is controlled by two discrete intracellular calcium signaling pathways: Ca 2+ release from endoplasmic reticulum via the inositol triphosphate receptor (IP3R) disassembles E-cad at adherens junctions while Ca 2+ entry via the mechanosensitive channel Piezo assembles E-cad at invasive protrusions. Thus, our study provides molecular insights into the unconventional role of E-cad in cell invasion during cell dissemination in vivo and describes the discrete roles of intracellular calcium signaling in the remodeling of E-cad/β-catenin subcellular localization.

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  5. Review Commons

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    I thank the Referees for their...

    Referee #1

    1. The authors should provide more information when...

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    1. Figure 6: Why has only...

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    Referee #2

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    Referee #3

    Evidence, reproducibility and clarity

    This study addresses the newly appreciated role of E-cadherin in tumor cell invasion. A strength of this study is their inducible RasV12 Drosophila model of transformation, which allows them to follow cell dissemination at the basal midgut. The authors demonstrate, convincingly, that depletion of E-cadherin in this model impairs cell dissemination. However, their efforts to demonstrate a role for two Ca2+ signaling pathways mediated by IP3R and Piezo channels are not convincing given the use of blunt and non-specific pharmacological tools, lack of Ca2+ monitoring, and generally descriptive observations.

    A significant weakness in this study is the sole and unsubstantiated use of Arm (the Drosophila b-catenin ortholog) as a proxy for e-cadherin localization and abundance. This leads to unsubstantiated statements such as, "disassembly of adherens junctions rather than E-cad levels" is involved in dissemination of cells. The role of E-cad is complicated by the observation that both knockdown and overexpression of E-cad perturb cell dissemination in their model.

    In Figure 4, the role of Piezo in Arm distribution is presented in confocal images of a few cells. Is this statistically significant? This experiment needs to be quantified with more cells to substantiate the claim that Arm signals redistribute from cell boundary to cytosol in Piezo and Calpain knockdowns. Piezo channels are non-selective for cations. This means that the results of the knockdown cannot be assigned to changes in cytosolic Ca2+. At the least, cytosolic Ca2+ levels should be monitored. The authors suggest that calpains work with Piezo through Ca2+ signaling on the basis of a single experiment in which calpain knockdown has a similar effect on Arm localization to Piezo knockout. This conclusion seems speculative and is not adequately supported.

    Thapsigargin is a blunt tool that causes a large, non-physiological and irreversible increase in cytosolic Ca2+. The observation that high Ca2+, resulting from Tg treatment, mediates disassembly of cadherins junction is not new, and indeed has been known for at least 20 years. Therefore, the effect of Tg in causing relocation of Arm signals from the adherens junctions is not insightful.

    Use of GdCl3 is a non-specific inhibitor of many ion channels, including voltage gated Ca2+ channels, TRP channels, leak K+ channels, etc. It cannot be used to infer the role of Piezo when used as described in this study. The observation that Tg + GdCl3 somewhat preserves basal/junction ratios of Arm, relative to Tg alone, is interesting but uninterpretable without additional experiments, including measurements of cytoplasmic Ca2+ levels. None of the findings related to Figure 5 are mechanistically insightful and the imputed role of Piezo is not convincing.

    Significance

    Overall, although this study addresses a topic of high significance and interest, the limitations in experimental approach do not allow mechanistic insights that advance our understanding of the role of E-cadherin in tumor invasion.

    Referee Cross-commenting

    Dear fellow reviewers,

    My expertise is in calcium signaling and ion transporters/channels. Although I was excited to review this work based on the abstract, I do not think that the link to calcium signaling (figs 4-5) is insightful or mechanistic, as I explain in my review. I realize that experimental approaches may be limited by the fly model, but the conclusions made by the authors would not be persuasive in other models.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Cabrera et. al. examine the role of DE-cadherin in cell dissemination, using a model of RasV12-transformed cells in the Drosophila hindgut. They show that E-cad relocates in the transformed cells from cell-cell junctions to basal invasive protrusions, and this relocation depends on calcium release from the ER by the PLC-IP3R-CAMK pathway. DE-cad is not required for the formation of actin-rich protrusions. However, upon RNAi-mediated DE-cad depletion (or depletion of the PLC-IP3R pathway), the basement membrane is not degraded and cell dissemination is inhibited. Furthermore, piezo and calpain are also shown to be required for DE-cad to assemble at invasive protrusions.

    Major comments:

    The data in figures 1-3 is overall convincing, although some of the points are missing quantification. The data supporting a role for piezo in regulating DE-cad assembly at adherens junctions is weaker. The data derived from ex-vivo pharmacological treatments (figure 5) is difficult to interpret or draw conclusions from.

    Specific comments:

    1. The data on DE-cad overexpression is puzzling. When DE-Cad is overexpressed in Ras-transformed cells it relocates from AJ to invadopodia and the ECM is degraded just like in Ras-transformed cells, yet cell dissemination is reduced by 50%. Why? The authors don't offer any explanation and in the absence of one I don't see how the DE-cad overexpression data adds to the story.
    2. DE-cad is knocked down by RNA interference using two different sequences. Admittedly, I am not a fly person, but isn't there a way for fly geneticists to show how well the KD worked? What percentage of WT DE-cad is left? This same question can be applied to all the RNAi experiments.
    3. Arm redistribution and co-localization with F-actin should be quantified. While the "representative" images are of high quality and their presentation in XY and XZ or basal, middle, ortho views is very helpful, the data from multiple cells, from multiple flies, from at least 3 different experiments must be quantified to show by how much does the level of E-cad decrease at AJ and by how much does it increase at the basal protrusions. Also, the degree of co-localization of F-actin and E-cad at invadopodia should be quantified. Perhaps F-actin is not the best marker for invadopodia since it is also present in other structures.
    4. The data presented in Figure 3c-h also needs to be properly quantified for level of DE-cad at lateral sides of cells and on basal side from multiple cells in multiple animals and at least 3 experiments.
    5. The connection made between piezo and DE-cadherin (figure 4) is tenuous. Piezo is known (based on the authors previous work as well as others) to be upstream of invadopodia formation, so it is not surprising that E-cad does not localize to invadopodia when they don't exist. The authors do not provide any evidence to directly link piezo activity or calcium entry to DE-cadherin localization at invadopodia or at AJ and therefore their claim that "calcium signaling mediated by the Piezo-calpain pathway plays a distinct role in the DE-cad/Arm remodeling process" is purely speculative. The images of fuzzy DE-cadherin in figure 4b are hardly proof.
    6. The experiment in fig 5a,b showing that calcium release from the ER leads to AJ disassembly, nicely complements the in vivo data in fig 3c-h. However, the remaining of figure 5, dealing with piezo, is entirely unconvincing. The images in 5f don't resemble invadopodia and have nothing to do with them. The data in 5i regarding apical delamination is interesting, but does not support their point about piezo and AJ. They don't even show DE-cadherin.

    Minor comments:

    Have a figure "for the reviewer" should be avoided. The authors must write the paper and include diagrams or images so that any reader is able to understand the model system just as well as the reviewer.

    Significance

    In my assessment of significance I am only taking into account the conclusions that I feel are well supported. Depending on when the preprint they cite (ref. 52) will be published, this could be the first report of E-cad localizing in invadopodia and, importantly, being essential for ECM degradation and cell dissemination. Also of significance is the identification of intracellular calcium release as a signal for AJ disassembly and relocation of E-cad to invasive protrusions. While the molecular mechanism through which E-cad regulates ECM degradation is not elucidated, the paper still represents a significant advance for the fields of cancer cell biology and cell and developmental biology and would be of interest to a wide audience.

    I am a cell and developmental biologist with expertise in cell adhesion and morphogenesis. I am not an expert on Drosophila.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This interesting manuscript examines how intracellular calcium signaling and cation channels (piezo) may affect the remodelling of adherens junctions in tumor cells. The authors use the Drosophila midgut model to examine the dissemination of RasV12 cells. Authors show that at day 2 of Ras expression, DE-cadherin/b-catenin complex redistributes from adherens junction to basal invasive protrusions. Depletion of DE-cadherin attenuates the dissemination and local invasion of RasV12 cells, as manifest in damage to underlying visceral muscles. The authors attribute disassembly of cadherin from cell-cell junctions to IP3R-dependent release of calcium from ER stores and subsequent activation of CaMKs. Surprisingly, the authors found an apparently separate role for Piezo (potentially mediating calcium signaling) to support the assembly of DE-cad at the invasive protrusions.

    Overall, this is a well-presented report that outlines the basic features of the phenomena with data of good quality. Over the years, there have been intermittent efforts to characterize the impact of calcium signaling on cell-cell junctions but much remains to be learnt. This manuscript is informative for focusing attention on a model of cadherin downregulation (and redistribution) in early tumors. However, it is somewhat descriptive and lacks depth of mechanistic analysis. For example, how might the cadherin in invasive protrusions be contribution to matrix degradation? What are the targets of either the IP3/CamK pathway or piezo/calpain signaling? Clearly, these foundational observations could be taken in many different directions. But some development would enrich the current package. Some suggestions follow.

    Major comments:

    1. What is the role of E-cadherin in invasive protrusions? The authors use DE-cadherin depletion to support a role for this relocated pool of cadherin in tumor dissemination. However, this doesn't distinguish between a direct action of the cadherin complex at the protrusions from some indirect effect. Further analysis of the "protrusion" cadherin could be informative. For example, one possibility is that E-cadherin is essential to localise other proteins to the invasive protrusion. Authors have previously reported (Lee et al. 2020) that cortactin is essential for dissemination of the RasV12 cells. Considering that the phenotype of cortactin KD (presented in Lee et al. 2020) and DE-cad KD mostly overlap, and literature suggests that E-cadherin and cortactin colocalise and can influence each others' localisation in cultured cells (e.g. Ren et al. 2009; PMCID: PMC2707247). Could KD of DE-cad perturb cortactin localisation at the invasive protrusions? This could be doable in a reasonable time, as according to Lee et al. 2020 authors already have a relevant Drosophila strain, that lets visualisation of cortactin.
    2. Another route to explore in greater depth is the impact of DE-cadherin depletion on the protrusion. The authors show that upon DE-cad KD, some actin-rich puncta are still able to form at the basal surface of the Ras cells. This should be characterised in more details to assess whether DE-cad KD affects the number, size and dynamics of forming protrusions. At current stage, it's not obvious why DE-cad depleted Ras cells do not disseminate, even though they are able to form the invasive protrusions. As authors may potentially use movies/images they already have, this should be doable within reasonable time frames.
    3. Authors could expand the discussion section to address the issue of the unusual DE-cad localisation. The open, basic question that the authors haven't addressed at any stage is "what does the DE-cad bind to at the invasive protrusions?". Do authors speculate that DE-cad is able to interact with some components of ECM/VM (as would be suggested by the cartoon Fig S5)? Have authors considered that E-cadherin transinteractions forming eg. between small adjacent protrusions may serve to stabilise the initial structures and let them develop into more prominent invasive protrusions?
    4. Given the rapid intracellular diffusivity of calcium, it is interesting that two different sources of calcium have such different effects on the cadherin pool. Why does calcium released from ER by IP3R is enough to disassemble the cadherin complex at AJs, but signaling from Piezo is needed to regulate assembly of the cadherin complex at the invasive protrusions. Can authors comment on why calcium released by ER can't control both processes? Is it possible to visualize the dynamic subcellular distribution of calcium in these cells to test if there are different pools involved?
    5. Further, the discrepancy between the influence of the piezo1 KD and GdCl3 treatment on the localisation of the DE-cad/Arm is quite striking. As literature suggests an existence of a physical interaction between Piezo channel and E-cadherin, blocking Piezo vs depleting it may potentially affect E-cadherin differently. To address this, can authors e.g. add GdCl3 treatment (covering same timeframes as the Piezo KD) to data presented in Fig 4b (this would also eliminate the ambiguity caused by possible differences between in vivo vs ex vivo conditions - Fig 4 vs Fig 5). Also, is the phenotype of loss of junctional DE-cad/Arm upon Piezo KD limited to the Ras cells or does it also happen in GFP control cells?
    6. Is the observed phenotype (dissemination and involvement of DE-cad) limited to RasV12 oncogene? Do authors have any experience with other oncogenes (e.g. Src)? If an adequate Drospihila strain is available to authors, it would be worth confirming that the presented findings are not limited to only one oncogene.
    7. Finally, the paper feels like a small, follow up story that is hugely dependent on author's 2020 paper (Lee et al. 2020). It may be quite hard to follow if the reader is not familiar with the 2020 paper, as eg. the current paper does not describe what the "dissemination process" actually is, instead it directs the reader to the 2020 paper (similarly the figure for reviewers consists of images taken from the 2020 paper, rather than the current one).

    Minor comments:

    1. How was junctional intensity of proteins quantified in samples that don't have obvious junctional staining (eg. Fig5b images and quantification for TG/ TG+GdCl3 in c and d or Image 5f and quantification in h). Do authors use an additional junctional marker to define where the junction is? If not, how is the position of junctions determined when the measured protein is dispersed from junctions?
    2. Is the calculation for IP3R-iGD1676 in Fig S3a correct? It doesn't match representative images presented in Fig3a or data in Fig3b, where IP3R-iGD1676 is not any different from other IR3R knock downs. If the quantification in Fig S3a is correct, the image presented in Fig3a is not representative of the majority of phenotype in this condition.
    3. Can delamination of Ras cells (Fig S1b and S3b) be quantified? It's quite challenging to assess the number of delaminated cells (especially in S3b).
    4. It would be good to quantitate the change in Arm (Fig 3d-h), in addition to showing representative images.
    5. Can authors describe the names used for labelling graphs in the figure captions? Some of the names used are not obvious for readers not already familiar with them eg. labels presented FigS3c - it's not obvious what norpA is, and the figure captions don't help at all.
    6. Is the antibody used for DE-cad able to distinguish between DE-cad and other cadherins (especially N-cadherin, which is commonly upregulated during EMT).

    Significance

    We believe that after addressing the above concerns, the paper may provide an informative advance to current knowledge about dissemination of early cancer cells leading to cancer metastasis. As authors mentioned, literature is not clear about involvement of E-cadherin in cancer metastasis. It was first suggested that loss of E-cadherin increases the metastatic potential of tumourigenic cells (Berx et al. 1995; Bogenrieder et al. 2003), however, more recent work indicated that the loss of E-cadherin, while increasing invasiveness, decreases metastatic potential, as well as cell proliferation and survival of circulating tumour cells (Padamanaban et al. 2019), indicating that the role of E-cadherin in cancer is much more complicated than previously appreciated. Thus, assessing the role of E-cadherin during cancer cell delamination in vivo may be a powerful and informative tool to bring some clarity to the cancer biology field.

  10. Version published to 10.1101/2021.11.10.467957v1 on bioRxiv