Abstract
Tumour-derived microvesicles (TMVs) comprise a class of extracellular vesicles released from tumour cells that are now understood to facilitate communication between the tumour and the surrounding microenvironment. Despite their significance, the regulatory mechanisms governing the trafficking of bioactive cargos to TMVs at the cell surface remain poorly defined. Here we describe a molecular pathway for the delivery of microRNA (miRNA) cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran–GTP following nuclear export and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6–GTP and GRP1. As such, ARF6 activation increases the pre-miRNA cargo contained within TMVs through a process that requires the casein kinase 2-mediated phosphorylation of RanGAP1. Furthermore, TMVs were found to contain pre-miRNA processing machinery including Dicer and Argonaute-2, which allow for cell-free pre-miRNA processing within shed vesicles. These findings offer cellular targets to block the loading and processing of pre-miRNAs within TMVs.
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Data availability
Deep-sequencing (miRNA–seq) data that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession code GSE130316. Previously published protein–protein interaction data that was analysed for this manuscript is available through the Intact database (www.ebi.ac.uk/intact) accession no. EBI-105937047, pubid:17353931 (ref. 45; Figs. 2b and 5e). Data used for the PRISM predictions was accessed through http://cosbi.ku.edu.tr/prism/ using structures stored in the RCSB Protein Data Bank (https://www.rcsb.org/; Figs. 2a, 5d and Supplementary Fig. 3a). The Cancer Genome Atlas data analysed in this manuscript was accessed via the Xena Functional Genomics Explorer52 (www.xenabrowser.net; Supplementary Figs. 4e,f and 5). All other data generated for and analysed in this manuscript is available by contacting the corresponding author. The source data for Figs. 1–6 and Supplementary Figs. 2–4,6 have been provided as Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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Acknowledgements
This work was supported in part by grants from the National Cancer Institute (grant no. R01CA115316) and the Catherine Peachey Foundation to C.D.-S.
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J.W.C. provided conceptual input, designed and performed experiments (shown in Figs. 1a-d; 2a,d,e,f,h; 3c,e; 4; 5; 6; and Supplementary Figs. S1a,e-g; S2a, c-g; S3b, d-i, k-n; S4; S5; S6a-c, e-i, k), analysed the data, proposed the model, assembled the figures and wrote the manuscript. Y.Z. provided conceptual input, designed and performed experiments (shown in Figs. 1e-l; 2a-e,i; 3a-e; 4a, 5; 6f; and Supplementary Figs. S1b,c; S2a-c; S3a,b, j; S6d, j), analysed the data and contributed to writing of the manuscript. C.S. designed and performed experiments (shown in Supplementary Figs. S1b; S6j) and assisted with experiments. C.D.-S. provided conceptual input, contributed to experimental design, analysed the data, wrote the manuscript and was responsible for the overall project administration.
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Supplementary Figure 1 TMVs are distinct from other populations of extracellular vesicles.
a. 20 µg of total protein isolated from LOX cells, TMVs, or exosomes as outlined in methods, were separated by SDS-PAGE and probed, as indicated, by western blotting. Representative blots from N=3 biologically independent experiments shown. b. 2x106 LOX cells were plated and treated with vehicle control (water) or 50 nM okadaic acid for 24 hours to induce apoptosis. Apoptotic bodies and TMVs were fractionated according to methods, and the total secretome separated by SDS-PAGE and probed by western blotting. 5% of total cell lysate used as input control. Representative blots from N=4 biologically independent experiments shown. c. Bioanalyzer analysis for Small RNA TMV cargo from invasive breast cancer (MDA-MB-231) and prostate (PC-3) cells reveals the presence of miRNA cargo within shed TMVs. Representative images from N=3 biologically independent samples shown. Unprocessed blot images shown in Supplemental Image 7.
Supplementary Figure 2 ARF6 activation increases TMV shedding and miRNA content in shed TMVs.
a. Nanoparticle tracking analysis (NTA) and total particle concentration of TMVs released from equal numbers of LOX or LOXARF6-Q67L cells. Data represents mea±SEM for each diameter (NTA) or mean±SD (total particle concentration) for N=5 biologically independent samples. p-value determined by unpaired, two-tailed t-test. b. Heat-map analysis of the 50 most abundant TMV miRNAs from LOX or LOXARF6-Q67L cell lines reveals a consistent increase in TMV miRNA content with ARF6 activation. c. qRT–PCR analysis reveals an increase in miRNA cargo content in TMVs released by tumour cells of melanoma (LOX), ovarian (OvCar3), and breast (MDA-MB-231) origins. Data presented as mean±SD for N=3 biologically independent experiments. p-values determined by unpaired, two-tailed t-test between control and treatment conditions for each independent miRNA amplification reaction. p-values ≤0.05 were considered significant. d. qRT–PCR analysis of LOX cellular miRNA levels with expression of ARF6-Q67L. For each condition, data presented as mean±SD of N=3 biologically independent experiments. p-values determined by unpaired, two-tailed t-test between control and treatment conditions for each independent miRNA amplification reaction. p-values ≤0.05 were considered significant. e. ARF6 activity following expression of the fast-cycling ARF6-T157N mutant was measured using an MT2 ARF6–GTP specific pulldown as outlined in methods. Representative western blots and quantification for N=3 biologically independent samples shown. Data presented as mean±SD. p-value determined by unpaired, two-tailed t-test. f. NTA and total particle concentration of TMVs released from equal numbers of LOX or LOXARF6-T157N cells. Data represents mean±SEM for each diameter (NTA) or mean±SD (total particle concentration) for N=5 biologically independent samples. p-value determined by unpaired, two-tailed t-test. g. qRT–PCR analysis confirms an increase in pre-miRNA and miRNA cargo content in TMVs released by cells expressing ARF6-T157N. For each condition, data presented as mean±SD of N=3 biologically independent experiments. p-values determined by unpaired, two-tailed t-test for each independent miRNA amplification reaction. P-values ≤0.05 were considered significant. Unprocessed blot images shown in Supplemental Image 7. Statistical Source in Supplementary Table 1.
Supplementary Figure 3 Exportin-5 interacts with ARF6, affecting TMV cargo.
a. PRISM predicted interaction between Exportin-5 and ARF6–GDP. b. Myc immunoprecipitation from cells expressing ARF6-WT-HA and myc-Exportin-5. Representative blots (N=3 biologically independent experiments) shown. c. Myc-tag co-immunoprecipitation from cells expressing ARF6-T27N-HA or ARF6-WT-HA; and myc-Exportin-5. Representative blots (N=3 biologically independent experiments) shown. Endogenous Exportin-5 and ARF6 imaging in LOX cell TMVs (d) and perinuclear region (e). Panels d and e represent orthogonal view of Fig. 3h. Representative images (N=3 biologically independent experiments) shown. f. Immunofluorescence of endogenous Exportin-5 and ARF6 in isolated TMVs. Representative images shown (N=4 biologically independent samples). g. Western blot of Exportin-5 in TMVs from invasive tumour lines. Representative blots (N=4 biologically independent experiments) shown. h. Dicer and Argonaute-2 western blotting in control or LOXARF6-Q67L TMVs. Representative blots (N=3 biologically independent samples) shown. i. qRT–PCR of TMV miRNA cargo from control or TBB treated cells. Data presented as mean±SD of N=3 biologically independent experiments. p-values determined by unpaired, two-tailed t-test between control and treatment reactions for each miRNA. j. Western blot of Exportin-5 in 20 μg total protein from control or TBB treated LOX cells. Representative blots (N=3 biologically independent samples) shown. k. Western blot of equal amounts of nuclear and cytosolic fractions from control or TBB treated LOX cells. Representative blots (N=3 biologically independent samples) shown. Data presented as mean±SD. p-values determined by unpaired, two-tailed t-test between each control and treatment condition. l. Western blot of Exportin-5 in 20 μg total protein from control or TBB treated LOXARF6-Q67L cells. Representative blots (N=3 biologically independent samples) shown. m. Western blot of equal amounts of nuclear and cytosolic fractions from control or TBB treated LOXARF6-Q67L cells. Representative blots (N=3 biologically independent samples) shown. Data presented as mean±SD. p-values determined by unpaired, two-tailed t-test. n. Western blot of Ran and RanGAP1 in 20 µg total protein from LOX cells or TMVs. Representative blots (N=3 biologically independent experiments) shown. For all panels, P-values ≤0.05 were considered significant. Unprocessed blot images shown in Supplemental Image 7. Statistical Source in Supplementary Table 1.
Supplementary Figure 4 Cytohesin activity alters Exportin-5 localization and expression has varying effects on patient outcomes.
a. Control or SecinH3 treated LOX cells were fractionated as detailed in methods. Equal amounts of nuclear and cytosolic fractions were resolved by SDS-PAGE and probed as indicated by western blotting. Representative blots (N=3 biologically independent samples) shown. Data presented as mean±SD. p-values determined by unpaired, two-tailed t-test. b. Western blotting using 10 μg of total protein from cells treated with vehicle control or SecinH3 show no reduction in total levels of Exportin-5. Representative blots (N=3 biologically independent samples) shown. c. Similar to western blotting, qRT–PCR showed no change in Exportin-5 mRNA levels upon inhibition of the cytohesins by treatment with SecinH3. Data presented as mean±SD for N=3 biologically independent experiments. No significant relationship was found by one-way ANOVA with Tukey’s correction for multiple comparisons. d. LOX cells treated with SecinH3 alone or SecinH3 and Chloroquine were fractionated as outlined in methods. Cytosolic Exportin-5 levels were measured by western blotting of equal amounts of nuclear and cytosolic fractions. Representative blots (N=3 biologically independent samples) shown. Data presented as mean±SD. p-values determined by unpaired, two-tailed t-test. e. High levels of GRP1 expression correlate with poor overall survival. f. Cytohesin 1 and cytohesin 2 show an inverse correlation between expression levels and overall survival in pan-cancer analysis. Kaplan-Meier data reported with log-rank test statistic (χ2) and the p-value (χ2-distribution). In all panels, P-values ≤0.05 were considered significant. Unprocessed blot images shown in Supplemental Image 7. Statistical Source in Supplementary Table 1.
Supplementary Figure 5 GRP1 expression correlates with poor patient outcomes in multiple tumour types.
GRP1 expression data contained within the publicly available datasets comprising The Cancer Genome Atlas was analysed for correlation between GRP1 expression and patient survival. High levels of GRP1 (levels above the median values noted for each cancer type) expression correlate with poor outcomes in a. melanoma, b. ovarian, c. breast, d. lung squamous cell, and e. bladder cancers. Data reported with log-rank test statistic (χ2) and the p-value (χ2-distribution). For all panels, P-values ≤0.05 were considered significant.
Supplementary Figure 6 GRP1 is necessary for Exportin-5 and miRNA trafficking to TMVs.
a. Co-immunoprecipitation of GRP1. Representative blots (N=3 biologically independent experiments) shown. b. myc-Exportin-5 was precipitated from control or GRP1-shRNA transduced LOX cells. Blots are representative of N=3 biologically independent experiments. c. NTA and total particle concentration of TMVs released from equal numbers of LOX or LOXGRP1sh cells. Data represents mean±SEM for each diameter (NTA) or mean±SD (total particle concentration) for N=5 biologically independent samples. p-value determined by unpaired, two-tailed t-test. d. Western blotting of Exportin-5, Dicer, and Argonaute-2 in TMVs from LOXGRP1sh cells. Representative blots (N=3 biologically independent experiments). e. 15 μg of total protein from control or GRP1-shRNA cells; and control or GRP1-shRNA TMVs were separated by SDS-PAGE and protein cargo analysed by western blotting. Representative blots (N=3 biologically independent experiments) shown. f, g. Mature miRNA cargo is lost from shed TMVs upon the introduction of either of 2 independent shRNA sequences targeting GRP1. For each condition, data presented as mean±SD of N=3 biologically independent experiments. p-values determined by unpaired, two-tailed t-test between control and shRNA reactions for each independent miRNA amplification reaction. Cellular pre-miRNA (h) and mature miRNA (i) cargo is increased with depletion of GRP1. For each condition, data presented as mean±SD of N=3 biologically independent experiments. p-values determined by unpaired, two-tailed t-test between control and shRNA reactions for each independent miRNA amplification reaction. j. LOX cells were independently transduced with shRNA targeting the 3 cytohesin family members known to act on ARF6. Equal numbers of TMVs from control or shRNA cells were lysed and Exportin-5 cargo examined by western blotting. Representative blots (N=3 biologically independent experiments) shown. For all panels, P-values ≤0.05 were considered significant. k. Interaction between Exportin-5 and ARF6 is facilitated by the ARF GEF GRP1. Trafficking complex formation allows Exportin-5 and pre-miRNA cargo to be transferred from Ran–GTP to ARF6–GTP for outward trafficking and inclusion into nascent TMVs at peripheral sites of TMV biogenesis. Unprocessed blot images shown in Supplemental Image 7. Statistical Source in Supplementary Table 1.
Supplementary Figure 7 Unprocessed images of all gels and blots.
Unprocessed images of gels and blots presented in main and supplementary figures.
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Supplementary Table 1
Statistics source data.
Supplementary Table 2
Table of antibody information.
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Clancy, J.W., Zhang, Y., Sheehan, C. et al. An ARF6–Exportin-5 axis delivers pre-miRNA cargo to tumour microvesicles. Nat Cell Biol 21, 856–866 (2019). https://doi.org/10.1038/s41556-019-0345-y
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DOI: https://doi.org/10.1038/s41556-019-0345-y
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