The small GTPase ARF3 controls invasion modality and metastasis by regulating N-cadherin levels

  1. Emma Sandilands
  2. Eva C. Freckmann
  3. Erin M. Cumming
  4. Alvaro Román-Fernández
  5. Lynn McGarry
  6. Jayanthi Anand
  7. Laura Galbraith
  8. Susan Mason
  9. Rachana Patel
  10. Colin Nixon
  11. Jared Cartwright
  12. Hing Y. Leung
  13. Karen Blyth
  14. David M. Bryant

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Abstract

ARF GTPases are central regulators of membrane trafficking that control local membrane identity and remodeling facilitating vesicle formation. Unraveling their function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. Through a functional genomic screen of three-dimensional (3D) prostate cancer cell behavior, we explore the contribution of ARF GTPases, GEFs, GAPs, and interactors to collective invasion. This revealed that ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of turnover of N-cadherin. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumor transplants and ARF3/N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organize during invasion and metastasis.

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  1. Version published to 10.1083/jcb.202206115
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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    The manuscript is dedicated to a study of functional roles of a panel of cell migration regulatory factors, and notably the highly homologous family of ARF GTPases. The chosen model is a prostate cancer cell line, used in a number of assays in culture, as well as in a study of primary tumor formation and metastasis in mice. The authors apply a rigorous quantitative approach to their assays of 2D and 3D migration in culture, and use artificial intelligence for the analysis of results, thus upgrading their work from mere phenotypic observations, and gaining statistically significant results. The main finding of the study is the discovery of a unique role of ARF3, a regulatory protein that is shown to control a switch between individual and collective cell migration depending on its abundance. In fact, a depletion of ARF3 leads to an increased individual cell migration and invasion, and to increased metastasis formation in mice, whereas an overexpression of ARF3 favors a sheet-like collective cell migration, which is also more efficient than control in culture, but does not induce metastasis in vivo. This phenomenon appears to depend on the levels of cellular N-cadherin, that is shown to be positively regulated by ARF3 on the protein level, by a mechanism that remains unclear. Finally, the authors analyze the expression of ARF3 and N-cad in a variety of tumors of different origins and grades, and attempt to show the prognostic value of these factors for progression-free survival.

    Major comments:

    • Are the key conclusions convincing?

    The majority of conclusions are convincing, with the exception of the observations I will address in the next paragraph.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    In Fig. 2B, the phenotypes 1 (Spread, pink line) and 5 (Spindle, yellow line) are described in the text as showing a "modest but robust increase". No such increase is evident by eye, and if it is statistically robust, the information should be presented in the Figure, or the statement should be modified/removed from the article.

    In Fig. 2E, the lower middle panel shows a dramatically increased quantity of acini, whereas the authors specifically say that proliferation is not impacted by the KD of ARF3, and indeed, the KD2 looks very much like the control in this respect. It is misleading, and a more typical panel should be presented for ARF3_KD1.

    In Fig. 3, the authors study the effects of simple versus double KD of ARF1 and ARF3, and conclude that a double KD leads to a phenotype "midway" between the two simple KDs. However, with regard to the 3D invasion assays (Figs. 3DEJ), it looks like a double KD is less efficient than either of the simple ones, as if ARF1 and 3 were partially mutually dependent in this regulation. It is not clear what "midway phenotype" the authors are talking about.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    The authors make a strong case for physical interaction and mutual stabilization of ARF3 and N-cad, though the negative regulation of N-cad following ARF3 depletion is not obvious from Fig. 5B (positive regulation is very clear). Moreover, it is difficult to understand why a total disappearance of ARF3 has such a discreet effect on N-cad, whereas a very modest overexpression of ARF3 leads to such a dramatic increase of N-cad. Perhaps, some experiments with proteasome inhibition (using MG132, for example) could substantiate the authors' claim about the mutual stabilization of the two proteins.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Yes, the experiments are realistic and should not take more than a month.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes.

    • Are the experiments adequately replicated and statistical analysis adequate?

    I am not sufficiently qualified in artificial intelligence algorithms to judge this part of the study. In general, as mentioned above, all differences characterized as "significant" or "robust" should have a statistical basis for this statement, which was not always the case in the manuscript.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    Please see above.

    • Are prior studies referenced appropriately?

    To the best of my knowledge, yes.

    • Are the text and figures clear and accurate?

    Yes, with the exceptions described before.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Please see above.

    Referees cross-commenting

    I fully agree with the detailed and careful analysis made by the reviewers 1 and 2. I do not have any additional comments.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    The study shows, for the first time and in a very clear way, that the small GTPase ARF3 has a unique function in determining the pattern of human cancer cell migration, that this function depends on the C-terminal domain of ARF3 and on N-cadherin (by mechanisms that remain to be elucidated), and that this phenomenon is important for metastases formation in vivo.

    • State what audience might be interested in and influenced by the reported findings.

    The study will be interesting for the field of cell migration, but also for specialists in cancer and metastasis formation.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Cell migration 2D 3D, acini assay, RAC-WAVE-ARP2/3 pathway. I am not sufficiently qualified to evaluate the robustness of the artificial intelligence algorithms, nor to judge the relevance of the analysis presented in Fig. 7.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Sandilands et al. analyze the role of ARF GTPases ARF1 and ARF3 in human prostate cancer cell setting with specific focus on 3D versus 2D growth and invasion. The study connects to previous work where the authors conducted similar studies with ARF GTPase exchange factor IQSEC1 acting as an invasion promoting factor. Now, the authors interrogate the "ARFome" in prostate cancer cell line PC3 by use of a lentiviral shRNA library. The authors conduct detailed 3D and 2D cell culture analyses to identify specific differences in cell morphology between the different single knockout clones, showing loss of ARF1 and ARF3 as key switches of a spindle like morphology that is associated with enhanced migration and invasion. Interestingly, the authors find ARF3 functioning significantly dependent on the C-terminal region, and, further, that ARF3 is a direct companion of N-cadherin levels, whose downregulation leads to enhanced migratory capability. The main findings of the manuscript are: (1) ARF1 and ARF3 knockdown elicits key-differences in 3D morphology and migratory capacity, (2) specifically ARF3 is associated with maintenance of N-cadherin levels via PSD and RAB11FIP4 effectors, (3) whose downregulation leads to enhanced metastatic spread in a orthotopic xenograft mouse model, and (4) lowered N-cadherin protein / ARF3 mRNA levels identify more aggressive human prostate tumors. Analyses and experiments conducted in the manuscript are highly extensive, they follow robust methods and are well controlled. Results described are highly detailed and are impressively visualized and presented. Key data are highlighted, and the manuscript is clearly structured. However, some data could be described and argued more concisely, which would strongly support the results shown. I recommend publication after some minor but important changes.

    Major comments:

    1. The entire results are based on studies of a single cell line PC3 derived from a highly aggressive metastasic lesion. To infer such an essential principle of tumor invasion and migration from this may be a bit precarious, and perhaps this principle should also be demonstrated in another cell line. The choice of PC3 cells and its implications should at least be discussed.
    2. Results showing cell morphology page 6 / Fig.2: end of paragraph: stating "normally suppressing invasion": seems too far at this state of the manuscript, as these experiments are shown in the next section. maybe better "involved in preservation of a rounded phenotype". Results Suppl.Fig.3f: please use the same colors for the morphologies as in Fig. 2 etc. (round - red, spindle - green, spread - blue).
    3. Conclusive sentences should not be put at the beginning of an experiment, before one can know its outcome: Results page 8, Fig.4g, bottom: "This revealed that the C-terminus of ARF3 is required for sheet type invasive activity" maybe put that rather as a conclusion of the whole section.
    4. Results showing 2D migration and 3D invasion: In the illustrations of migration and invasion assays shown throughout Figs 3-5 and Suppl. Figs 3 and 4, please clearly state and indicate for each case whether this is 2D migration or 3D invasion, as these two assays are very similar, which is a little confusing throughout the manuscript. Suppl.Fig.3e,k,l: is this 2D or 3D? Results Fig.4b+d and Fig.5g: please amend "3D invasion".
    5. Summarizing sketch Results Fig.5k: in the scheme on the right side indication of respective presence / absence of ARF3/N-cadherin is missing. Which state induces which condition? Please amend.
    6. ARF3 suppresses PC3 xenograft metastasis shown in Fig.6: N-cadherin stainings of mouse xenografts and metastases are missing.
    7. Patient data ARF3 mRNA correlations Fig.7 and Suppl.Fig.6ab / Results page 11: the whole section describing ARF3 mRNA levels in diverse tumor types is too long and a little bit confusing: maybe shorten the text, put Fig.7f+g supplemental, and please indicate the combined GENT2 database in Fig. S6b.
    8. Patient data CDH2 mRNA/protein correlations: Fig.7 and Suppl.Fig.6cd / Results page 12: one should also shorten and sharpen this section. Please also exchange Suppl. Fig. panels 6 dc to have the same order as Suppl. Fig.6ab. Regarding the detailed analyses of CDH2 mRNA levels, make a too long story short, essential are N-cadherin protein levels, and these results shown in Fig. 7 hik and Fig. 7rst should be enlarged and highlighted. All other data (Fig. 7jl) and the right bars of panels Fig. 7mno, as well as Fig. 7pq are rather supplemental.
    9. The finding of elevated N-cadherin levels correlated with reduced invasion/migration and according better tumor outcome is surprising, particularly with regard to the quite established "EMT" dogma of N-cadherin driven single cell migration. Could you go into more detail about this property of N-cadherin driven mode of reduced tumor spread in the discussion?

    Minor comments:

    1. Manuscript title: maybe rephrase and reverse order of events: first invasion, then metastasis?
    2. Results Suppl.Fig.1ij: which cells are shown? Please amend RWPE-1 and PC3.
    3. Results page 5: although already described in Nacke et al 2021, please explain the term "acinus".
    4. Results page 6: maybe introduce an additional subchapter? Some subchapter titles could be more explicative.
    5. Results page 6, 2nd paragraph: please describe what was done: "revealed that knockdown of ..."
    6. Results page 7: please explain more detailed class I ARFs, which ARFs are included in this class?
    7. Results page 7, Fig.3f-j and 2nd paragraph: maybe better switch: first 3D, then 2D? Results Fig.3j: maybe amend indication of knockdown "KD" as indicated in Figure 3b-e.
    8. The conclusion on page 8 top "this suggests that a function of ClassI ARFs may be to regulate molecules that control collective bahaviours" is quite broad, please be more specific.
    9. Suppl.Fig.4n: should be named "l"?
    10. Results page 8, introducing sentence: please formulate more clearly and following the previous results.

    Significance

    Contents/Level of interest/merit:

    This study by Sandilands et al. analyzes cellular models of ARF-GTPase linked changes of cell morphology and migration to detect altered prostate cancer cell metastatic behaviour. Understanding the contribution of specific ARF GTPases in cancer cell shape and movement might help to identify markers of disease progression and metastasis.

    Strengths/Conclusions:

    The authors perform whole ARF-compendium knockdown and conduct detailed data analysis and visualization. The authors perform morphological analyses and conduct migration and invasion studies. Mechanistically, they confirm expression changes of N-cadherin, the key adhesive protein that is regulated. This study underlines the importance of analyzing subtle GTPase pathway differences by detailed morphological observations and methods.

    Comment/Weakness:

    The authors show extensive and detailed data that have been thoroughly analyzed, and results are presented and described fluently. There is a clear sequence of results description that is presented detailed. Form and contents of the paper is sound. The experiments are highly connected to previous experiments and data and this is also the major drawback of this manuscript: there is a lack of clear description of what is shown because it is already presupposed. Therefore, some sections should be worded and presented in more detail to present results more explicitly. The manuscript can be accepted with minor but essential revisions.

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    Referee #1

    Evidence, reproducibility and clarity

    This study represents a tour de force in advancing our understanding of the Arf family and it's associated regulators/effectors in controlling the morphology of individual cells and collective behaviours in 3D ECM. The authors use shRNA-mediated knockdown in high throughput imaging and AI approaches to define the influence of the ARFome on the morphology of prostate cancer PC3 cells growing as acini structures in 3D ECM. This allowed classification of ARFs and effectors/regulators on the basis of associated phenotypes, and this in itself is a useful resource for the community. It also drove the authors to focus on ARF3 and its regulator ESD and effector Rab11-FIP4. Analysing cell motility in elegant 2D and 3D assays showed that ARF3 levels control the collective migration of PC3 cells in 3D invasion, and the authors relate these findings to an interaction between Arf3 and N-cadherin. Using a mouse model of intra-prostatic injection of PC3 cells they demonstrate clear links between ARF3 levels and metastasis in vivo, and patient data further supports the link between Arf3, N-cadherin and metastasis. Experiments are well controlled and complex data are beautifully presented. In general data support the conclusions, where this is not as clear is highlighted below.

    Major comments:

    Figure 4: The use of the Arf3/4 chimeras is an interesting approach, used to show that the ARF3 C-terminus is important for its function related to migration/invasion. However the effect this has is not clear- it is not GTP loaded efficiently and may therefore act as a dominant negative. Furthermore the authors do not indicate which intracellular compartment ARF3 associates with, or if this is altered when the ARF3 C-term is replaced by that of ARF4 (ARF3N/1C).

    Figure 5: Links to N-cadherin are clearly interesting, but the model proposed in Figure 5K is a little speculative. Clearly it is possible that ARF3/Rab11-FIP4 regulate N-cadherin trafficking such that loss of the pathway leads to degradation and gain promotes stability, but it is also possible that expression levels are controlled at the level of transcription. This could be assessed by a simple surface labelling experiment in wt, overexpressing and knockdown cells, and/or by analysing localisation of N-cad with respect to ARF3 and late endosomes/lysosomes when ARF3 levels are manipulated. Does ESD knockdown similarly impact N-cad?

    Figure 6: The metastasis experiments are highly relevant to metastatic prostate cancer. Essentially the overall conclusion that high Arf3 (overexpression) suppresses and low Arf3 (knockdown) supports metastasis are well supported by the data, and the wildtype Arf3 levels sit in between (hence trends are observed but aren't statistically significant). Here it would be interesting to compare ARF3 levels in patient tumours with those in wt, overexpressing and knockdown PC3 cells in the mouse model (if sections are available) to give confidence that the overexpression is within the physiological range. If it were also possible to analyse N-cadherin levels in tumours or metastases that would provide an even stronger case for the mechanism proposed.

    Minor comments:

    Figure 2: The classification of phenotypes into groups is interesting, but the trends in some groups (eg Group4, 6 and 7) seem very similar. It wasn't clear to me if these are AI generated? Also, knockdown of individual ARFs is often in different groups- is this a reflection of knockdown level?

    Significance

    The manuscript provides a very significant advance in our understanding of the function of the ARFome with respect to cell morphodynamics. The first two figures and supporting data represent a fantastic resource for the field, and the remaining figures provide new insight into the function of ARF3 in collective cell movement and metastasis in mouse models and patients. Whilst ARF6 and its function in cell migration/invasion/metastasis is well studied, ARF3 has received relatively little attention. This study is therefore of broad interest to the trafficking community, and the new links between ARF3 and invasion/metastasis are broadly of interest to the cell biology and cancer communities. The mechanistic link between N-cadherin and ARF3 is fairly well defined and the fact that high/low levels of both correspond to improved/poor outcomes is a major strength of the study. Expertise: Vesicle trafficking/cell migration/invasion/cancer

  6. Version published to 10.1101/2022.04.25.489355v1 on bioRxiv