Ral GTPases promote metastasis by controlling biogenesis and organ colonization of exosomes

  1. S Ghoroghi
  2. B Mary
  3. A Larnicol
  4. A Klein
  5. N Osmani
  6. I Busnelli
  7. F Delalande
  8. N Paul
  9. S Halary
  10. F Gros
  11. L Fouillen
  12. AM Haeberle
  13. C Royer
  14. C Spiegelhalter
  15. G André-Grégoire
  16. K Murphy
  17. P Timpson
  18. R Carapito
  19. M Blot-Chabaud
  20. J Gavard
  21. C Carapito
  22. N Vitale
  23. O Lefebvre
  24. JG Goetz
  25. V Hyenne

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Abstract

Cancer extracellular vesicles (EVs) mainly exert pro-tumoral functions by changing the phenotypes of stromal cells to the benefit of tumor growth and metastasis. They shuttle to distant organs and fertilize pre-metastatic niches facilitating subsequent seeding by circulating tumor cells. The levels of tumor secreted EVs correlate with tumor aggressiveness, however, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Here, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and thereby tune the biogenesis and secretion of pro-metastatic EVs. RalA and RalB promote lung metastasis in a syngeneic mouse model. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivo and, as a consequence, are less efficient in promoting lung metastasis. RalA or RalB modulate the EV levels of the adhesion molecule MCAM/CD146, which mediates lung colonization. Finally, RalA and RalB, but also MCAM/CD146, are factors of poor prognosis in human breast cancer patients. Altogether, our study identifies Ral GTPases as central molecules linking the mechanisms of EVs secretion, cargo loading to their capacity to disseminate and induce pre-metastatic niches.

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    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on September 9 2020, follows.

    Summary

    The role of extracellular vesicles (EVs) such as exosomes as factors potentiating metastasis by solid tumors has attracted considerable recent interest. The article by Ghoroghi et al is a very complete and thorough study of the role of 2 small GTPases, RalA and RalB, in extracellular vesicle (EV) release and breast carcinoma progression. The main advance of this manuscript is to describe a signaling role for the GTPases RalA and RalB in regulating phospholipase D (PLD) and multivesicular body function to regulate exosome biogenesis. They also show that RalA and RalB regulate expression of MCAM/CD146 on EVs, and that reduced levels of CD146 on EVs affects efficiency of lung cancer metastasis. Finally, they show RalA, RalB and CD146 levels are indicators poor prognosis for breast cancer patients. Overall these are interesting observations with clinical relevance. The study is extremely carefully performed, in general, with appropriate controls and conclusions. There are a limited number of weaknesses that need to be addressed.

    Essential Revisions

    1. Figure 3C shows that RALA and RALB have functions where they do not always act in series with each other. They may for exosome excretion, but not necessarily for proliferation. For Supp. Fig. 4C, this experiment needs to be longer than 72 hours for a proliferation assay to be truly convincing. While interesting that proliferation results differ between mice and growth on plastic, a 10 day growth experiment would lead to greater conviction that this is a sustained difference. Figure 3D better agrees with Supp. Fig 4C, where loss of RALB gives greater proliferation over control, albeit not as great as RALA loss. Was Fig. 3D performed at 12 days? The variable time points make it difficult to assess the role of RALA/B on growth.

    2. Figure 4 carefully describes why RALA/B deficient EVs fail to prime regions for metastasis. As the EVs are not directed to those locations, the contents fail to increase permeability of the regions. However, statistically significant does not mean biologically meaningful; specifically, looking at Fig 4f and 4g, knockdown of RALA/B had little effect on EV internalization. One question that is not answered is whether RALA/B are acting in series or parallel. Concurrent depletion of both RALA and RALB in the types of experiment performed in Figure 4 would help answer this question. If depletion of both isoforms is greater than either alone, then it implies they act in parallel.

    3. How are RALA/B activated in the context of 4T1 cells? The use of the RAL inhibitor in Figure 1B is most effective in cell lines without RAS mutations. Panc1 and MD231 cells have KRAS mutations, yet RALGEF inhibitors are least effective. At the concentrations used, all RALGEF activity should be inhibited. Is RAL expression levels similar between cell lines?

    4. The logic for targeting PLD1 seems rushed. There are several RAL effectors, and while the data on PLD1 are compelling, it is unclear why only this effector was selected. Were other effectors tested? The Hyenne paper, which is cited throughout, provided stronger logic for connecting these pathways. The statement in the discussion, "Our work further identifies PLD as the most likely effector acting downstream of RAL to control exosome section" is incorrect as stated. This work identified PLD1 as A downstream effector important for exosome secretion. To state that it is the most likely effector is an overstatement as no other RAL effectors were tested.

    5. The use of either PLD inhibitor at 10 uM will have significant inhibition of both PLD1 and PLD2 regardless which inhibitor is being tested, as well as additional off-target effects. Given both inhibitors have nanomolar IC50 values for their specific PLD isoform and low micromolar IC50 values for the related isoform, these experiments are inconclusive as presented. This section of the text should be reworked to account for the lack of isoform selectivity at the concentrations of inhibitor used. a. siRNA or shRNA could be used to validate the PLD1/2 inhibitor data.

    6. In all cases when using inhibitors, there are no western blots or additional validation showing target inhibition in any cell line. In this case, the use of such high concentrations of inhibitor likely resulted in inhibition of the target proteins. However, such high concentrations often result in off-target effects. Determining the lowest efficacious dose to inhibit target function should have been performed and used throughout the experiments.

    7. Would loss of MCAM expression, via direct knockdown, result in EVs that are unable to permeabilize HUVEC cells, as in Fig 4A/B? Alternatively, does loss of MCAM expression (or blocking via anti-CD146) result in decreased metastasis, similar to RALA/B knockdown? While Fig. 5g shows a decrease in EV localization to lungs on treatment with Anti-CD146, the rate of metastatic lesion formation was not assessed when CD146 was blocked.

    8. In a previous study (Hyenne et al, 2015) the authors already demonstrated a role for RalA and RalB in controlling MVB and EV secretion in the 4T1 breast cancer model, in a process evolutionarily conserved through nematodes. They also wrote a follow-up review article describing considerable evidence in the literature for RalA controling PLD and ARF6 function to control EV secretion. This diminishes the novelty and interest of the first part of the study, which could be reduced. The more novel and thought-provoking parts of the study are in the definition of the relationship between the RAL proteins and CD146, and the identification of unique properties of the exosomes produced in cells with manipulated RAL proteins (for instance, in regard to influencing exosome permeability). These could be better emphasized.

    9. Almost the entire mechanistic model is based on the work of a single breast cancer cell line, 4T1, which has been described by some as triple negative. This is a significant weakness, as there may be features of that line that make it uncharacteristic of breast cancer in general. At least some of the key conclusions should be functionally confirmed in an additional cell model.

    10. In addition, breast cancer cells fall into multiple different subtypes, which have different metastatic propensities and gene expression patterns. Is the functional relationship between the expression of RALA/B and CD146 in exosomes observed in just triple negative cells, or in other subtypes?

    11. In Fig 5h, K-M analysis of TCGA shows a weak relationship between MCAM expression and survival. Were the tumors analyzed segregated by tumor subtype? Were data corrected for tumor stage? This is important, as the MCAM staining pattern may reflect a propensity for MCAM expression in tumors at a late stage, or subtypes with a poorer prognosis.

    12. The authors provide observations suggesting that RalA and B control primarily the EV secretion pathway involving Multivesicular bodies, hence leading to exosome secretion. This is mainly demonstrated by observation of a decrease in MVBs upon knock-down of RalA/B, demonstrated by thorough electron microscopy analyses. This is correlative, rather than truly demonstrative, but the best one can do so far. In most experiments, the authors use EVs isolated by a relatively crude method, ultracentrifugation, that co-isolates non-specific components, and they do not analyse larger EVs that can be recovered at lower centrifugation speed, thus an effect of RalA/B on these non-exosomal EVs cannot be excluded. The EVs are only characterized by their number (NTA counting), which is not very precise, and not consistent with guidelines of ISEV (MISEV 2018, J Extracell Vesicles 2018, 7: 1535750).

    Maybe the authors can argue that they did perform more complete analyses of their 4T1 EVs in a previous article? Did they use the EV-TRACK website to verify their experimental EV isolation and characterization set up? Of note, the authors also perform quantitative proteomic and RNomic analyses, which gives a better characterization of EVs. The protein composition and its change upon RalA/B KD could have been used to try to confirm (or not) the MVB origin of the EVs controlled by RalA or RalB, but it is not crucial for the message. Alternatively, to demonstrate that the CD146-bearing EVs that carry the prometastatic function are bona fide exosomes, the authors could have shown its localization in the cells, upon or not RalA/B depletion, and show if a drastic change in ratio of localization in MVBs vs the PM occurs, but this would be an additional study, not necessary for the current paper.

    1. The authors insist that RalA/B control exosome secretion, and discuss the bases and limitations of their demonstration properly. The summarizing schemes (fig2f and fig5i) of their model show the release of RalA/B-depent pro-metastatic exosomes, and RalA/B-independent exosomes which are not pro-metastatic. However, EVs that are released in the absence of RalA/B could instead be formed at the plasma membrane, and correspond to ectosomes. Nothing in this study demonstrates the origin of RalA/B-independent EVs, thus PM-derived EVs should be represented in the scheme.
  2. Version published to 10.1101/2020.07.10.196691v1 on bioRxiv
  3. Version published to 10.1101/2020.07.10.196691v1 on bioRxiv