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Cancer-cell-secreted extracellular vesicles suppress insulin secretion through miR-122 to impair systemic glucose homeostasis and contribute to tumour growth

Nature Cell Biology volume 24, pages 954–967 (2022)Cite this article

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Abstract

Epidemiological studies demonstrate an association between breast cancer (BC) and systemic dysregulation of glucose metabolism. However, how BC influences glucose homeostasis remains unknown. We show that BC-derived extracellular vesicles (EVs) suppress pancreatic insulin secretion to impair glucose homeostasis. EV-encapsulated miR-122 targets PKM in β-cells to suppress glycolysis and ATP-dependent insulin exocytosis. Mice receiving high-miR-122 EVs or bearing BC tumours exhibit suppressed insulin secretion, enhanced endogenous glucose production, impaired glucose tolerance and fasting hyperglycaemia. These effects contribute to tumour growth and are abolished by inhibiting EV secretion or miR-122, restoring PKM in β-cells or supplementing insulin. Compared with non-cancer controls, patients with BC have higher levels of circulating EV-encapsulated miR-122 and fasting glucose concentrations but lower fasting insulin; miR-122 levels are positively associated with glucose and negatively associated with insulin. Therefore, EV-mediated impairment of whole-body glycaemic control may contribute to tumour progression and incidence of type 2 diabetes in some patients with BC.

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Fig. 1: BC-derived EVs suppress insulin signalling in liver and enhance hepatic glucose production.
Fig. 2: miR-122 in BC-derived EVs suppresses insulin secretion.
Fig. 3: Tumours require EV secretion and miR-122 to alter insulin secretion and glucose homeostasis.
Fig. 4: A PDX model also exhibits impairments of insulin secretion and glucose homeostasis.
Fig. 5: miR-122 acts as a main effector in BC-associated dysregulation of insulin and glucose homeostasis.
Fig. 6: Restoration of PKM2 expression in β-cells abolishes the effects of BC tumours on insulin and glucose metabolism.
Fig. 7: Supplementing insulin or lowering glucose levels intervenes BC tumour growth.
Fig. 8: miR-122 in circulating EVs is associated with fasting blood glucose and insulin in patients with BC.

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Data availability

RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE152391, GSE173408, and GSE173276. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH)/National Cancer Institute (NCI) grants R01CA218140 (S.E.W.), R01CA206911 (S.E.W.) and R01CA179977 (D.D.S.). J.D.B. is supported by a grant from the Hartwell Foundation. Research reported in this publication included work performed in the core facilities supported by the NIH/NCI under grant number P30CA23100 (UCSD Moores Cancer Center) and in the City of Hope Integrative Genomics Core supported by NIH/NCI under grant number P30CA33572. The authors would like to thank the UCSD/CMM EM facility for EM sample preparation. The EM facility is supported by NIH equipment grant 1S10OD023527. We thank X. Xiao for kindly providing plasmid pEMBOL-D(+)-Ins-GFP.

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Author notes

  1. These authors jointly supervised this work: Dorothy D. Sears, Jerrold M. Olefsky, Shizhen Emily Wang.

Authors and Affiliations

  1. Department of Pathology, University of California, San Diego, La Jolla, CA, USA

    Minghui Cao, Wei Yan, Xianhui Ruan, Li Jiang, Yuhao Wan, Jessica Wang, Emily Wang, Christine Caron, Donald P. Pizzo, Xuxiang Liu, Andrew R. Chin, Miranda Y. Fong, Oluwole Fadare, Jack D. Bui & Shizhen Emily Wang

  2. Department of Medicine, University of California, San Diego, La Jolla, CA, USA

    Roi Isaac, Richard B. Schwab, Wei Ying, Dorothy D. Sears & Jerrold M. Olefsky

  3. Regulus Therapeutics Inc., San Diego, CA, USA

    Steven Neben & Denis Drygin

  4. Department of Molecular and Cellular Biology, City of Hope, Duarte, CA, USA

    Xiwei Wu

  5. Department of Chemistry, University of California, Riverside, Riverside, CA, USA

    Ziting Gao, Kaizhu Guo & Wenwan Zhong

  6. Department of Medical Oncology & Therapeutics Research, City of Hope, Duarte, CA, USA

    Yuan Yuan, Susan E. Yost & Joanne Mortimer

  7. College of Health Solutions, Arizona State University, Phoenix, AZ, USA

    Dorothy D. Sears

  8. Department of Family Medicine, University of California, San Diego, La Jolla, CA, USA

    Dorothy D. Sears

  9. Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA

    Dorothy D. Sears & Shizhen Emily Wang

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Contributions

S.E.W. and M.C. conceived ideas, and J.M.O., D.D.S. and W. Ying contributed to project planning. M.C. and S.E.W. designed and performed most of the experiments. R.I., W. Yan, X.R., L.J., Y.W., J.W., E.W., X.L., A.R.C. and M.Y.F. assisted with mouse experiments, cell line construction and EV preparation. S.N. and D.D. provided miR-122 ONI and control oligonucleotides and helped design experiments involving oligonucleotide treatment. D.P.P. assisted with tissue processing and histological analyses. X.W. assisted with RNA-seq. C.C. and J.D.B. assisted with flow cytometry. Z.G., K.G. and W.Z. assisted with NTA analysis of EVs. D.D.S., O.F., R.B.S., Y.Y., S.E.Y. and J.M. assisted with clinical sample assembly and assessment. S.E.W. and M.C. wrote the manuscript.

Corresponding author

Correspondence to Shizhen Emily Wang.

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S.N. and D.D. are employees of Regulus Therapeutics, Inc. The other authors declare no competing interests.

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Nature Cell Biology thanks David Hodson, Luis Fajas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Additional assessments of the EV injection model in Fig. 1.

a, Food consumption was calculated by total food consumed in 48 h after the 8th EV/PBS injection (n = 3 mice per group). b, Body weight was monitored (n = 8 mice for MDA-MB-231 EV and n = 7 mice for other groups). c, Liver tissues collected at ZT10 from mice that had received 5 weeks of EV/PBS injections were subjected to RNA-seq and GSEA, showing enrichment of genes related to IRS1/2 signalling (n = 6 mice per group). d, Heatmap showing the relative levels of selected genes based on the RNA-seq data. e, Triglyceride levels measured in the liver (n = 4 mice for PBS and MCF-10A/vec EV group and n = 5 for other groups). f-h, Mouse serum glucagon (f), GLP-1 (g), and ghrelin (h) levels were determined by ELISA kits (n = 6 mice for PBS and MCF-10A/vec EV group and n = 8 for other groups). Data are shown as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for a, e-h. Two-way ANOVA followed by Tukey’s multiple comparison test was used for b. ns: not significant. *P < 0.05. Numerical source data and statistics are available online.

Source data

Extended Data Fig. 2 Characterization of BC-cell-secreted EVs.

a, NTA of MDA-MB-231 EVs (n = 3 replicates). b, Immunoblots of indicated proteins in MDA-MB-231 whole cell lysate and EV fractions from OptiPrep gradient ultracentrifugation showing EV markers and a Golgi marker (GM130, as a negative control for EV-specific proteins). c, RT-qPCR-determined miR-122 (left) and miR-16 (right) levels in OptiPrep gradient fractions of MDA-MB-231 EVs (n = 3 replicates; normalized to an ath-miR159a spike-in control). d, miR-122 levels in EVs treated with Proteinase K (PK, 10 μg/mL) followed by RNase If (RNase, 40 U) or with PBS (as control) in the presence or absence of 1% Triton X-100 (TX-100) (n = 3 replicates). RT-qPCR data were normalized to an ath-miR159a spike-in control added after all treatments. e, RT-qPCR showing relative miR-122 levels (normalized to spike-in control) in EVs isolated from the sera of mice in Fig. 1 (n = 6 mice per group). Data are shown as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for d and e. ns: not significant. *P < 0.05, **P < 0.01, ***P < 0.001. Numerical source data, statistics and unprocessed blots are available online.

Source data

Extended Data Fig. 3 Functional assessments of EVs and PKM2 in MIN6 β-cells.

a, Uptake of CFSE-labelled EVs by MIN6 cells (CFSE, green; DAPI, blue). Bar=50 μm. b, Immunoblots of MIN6 cells after EV/PBS treatment for 48 h. When indicated, MIN6 cells were pre-transfected with a PKM2 cDNA expression plasmid or empty vector. c, Pyruvate kinase activity in MIN6 cells (n = 3 replicates). d, Relative ATP/ADP ratio in MIN6 cells (n = 4 replicates) after low glucose (3.3 mM) or high glucose (16.7 mM) stimulation. Data were normalized to the first bar. e, Changes of the intracellular Ca2+ levels measured by Fura-2/AM in MIN6 cells after low or high glucose stimulation (n = 6 replicates). f, Insulin secretion by EV/PBS-treated MIN6 cells (n = 3 replicates) under 3.3 mM glucose, 16.7 mM glucose, or 20 mM monomethyl succinate (MMS). g, Immunoblots of MIN6 cells transfected with siRNA against Pkm2 (scrambled siRNA served as a negative control). h,i, Relative ATP/ADP ratio (h; n = 6 replicates) and insulin secretion (i; n = 4 replicates) in siRNA-transfected MIN6 cells after low glucose (3.3 mM) or high glucose (16.7 mM) stimulation. For all bar and line graphs, values are shown as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for c and right panel of f. Two-way ANOVA with repeated measures followed by Tukey’s multiple comparison test was used for e. Two-way ANOVA followed by Tukey’s multiple comparison test was used for d and left panel of f, or by Bonferroni’s multiple comparison test used for h and i. *P < 0.05, **P < 0.01, ***P < 0.001 compared to PBS or as indicated; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to MCF-10A/vec EV. In e, the upper set of signs indicate MCF-10A/miR-122 EV vs. PBS or MCF-10A/vec EV, and the lower set indicate MDA-MB-231 EV vs. PBS or MCF-10A/vec EV. Numerical source data, statistics and unprocessed blots are available online.

Source data

Extended Data Fig. 4 Characterization of MDA-MB-231-derived cells.

a, Immunoblots showing protein levels in indicated MDA-MB-231-derived cells and their EVs. EVs secreted from equal number of cells were loaded. b, NTA of EVs from indicated cells (n = 3 replicates). The inserted graph shows the numbers of EVs secreted in 24 hours per 107 cells (n = 3 replicates). c, Sanger sequencing showing genetic knockout of hsa-mir-122 gene in MDA-MB-231/miR-122 KO cells. Dashes indicate the region deleted by the CRISPR-Cas9 genome editing system. d, RT-qPCR-determined miR-122 and miR-16 levels (normalized to U6 snRNA) in indicated MDA-MB-231-derived cells (n = 6 replicates). e, Cell proliferation indicated by cell numbers counted every 24 hours (n = 4 replicates). f, Cell proliferation indicated by MTS assay. Optical density (O.D.) at 490 nm was shown (n = 8 replicates). g-j, Volcano plots showing differentially expressed miRNAs in indicated cells and EVs based on small RNA-seq data (n = 2 replicates). The position of miR-122 was noted in each plot. Data in bar and line graphs are shown as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparison test was used for e and f. *P < 0.05, **P < 0.01. Numerical source data, statistics and unprocessed blots are available online.

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Extended Data Fig. 5 Additional assessments of the MDA-MB-231-derived tumour model in Fig. 3a-n.

a, Body weight over time (n = 6 mice per group). b,c, Serum glucagon (b) and ghrelin (c) levels (n = 6 mice per group). d, Weight of gonadal white adipose tissue (WAT) (n = 7 mice per group). Values were normalized to whole body weight. e, RT-qPCR showing the relative mRNA levels (normalized to Gapdh mRNA) in gonadal WAT (n = 7 mice per group). f, Immunoblots of indicated proteins in the gastrocnemius muscle tissues from indicated groups. g, Representative images showing 2-NBDG uptake in the gastrocnemius from indicated groups (left; Bar=50μm) and overall 2-NBDG signal intensity quantified by ImageJ (right; n = 5 mice per group). Data in bar and line graphs are shown as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparison post was used for a. One-way ANOVA followed by Tukey’s multiple comparison test was used for b-e and g. *P < 0.05, **P < 0.01, ***P < 0.001. Numerical source data, statistics and unprocessed blots are available online.

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Extended Data Fig. 6 Cell proliferation and migration under different culture conditions.

a, Cell number counting every 24 hours under different glucose concentrations (n = 4 biologically independent plates of cells). Medium containing indicated glucose concentration was replenished every 24 hours. b, Wound closure migration assay under indicated glucose concentration. Bar=200μm. c, Cell number counting every 24 hours under different insulin concentrations (n = 4 biologically independent plates of cells). Medium containing indicated insulin concentration was replenished every 24 hours. Data are shown as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparison test was used for a-c. *P < 0.05, **P < 0.01, ***P < 0.001. Numerical source data and statistics are available online.

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Extended Data Fig. 7 Assessments of the pancreatic islets of mice in Fig. 7g-j.

a, Immunofluorescence of pancreas sections (left) showing PDX1 expression (green) in β-cells (insulin+; red). Bar=50μm. The overall frequency of PDX1+ among insulin+ cells was calculated and plotted in the right panel (n = 4 mice per group except n = 5 mice for MDA-MB-231/control + SGLT2i group). b, Immunofluorescence of pancreas sections (left) showing apoptosis events (indicated by cleaved caspase-3 staining; green) in β-cells (insulin+; red). Bar=50μm. The overall frequency of cleaved caspase-3+ among insulin+ cells was calculated and plotted in the right panel (n = 4 mice per group except n = 5 mice for MDA-MB-231/control + SGLT2i group). c, Representative H&E staining of pancreas sections (left) showing the regular morphology of mouse islets. Bar=50μm. Area of at least 8 islets from each mouse was calculated and plotted in the right panel (n = 4 mice per group except n = 5 mice for MDA-MB-231/control + SGLT2i group). Data are shown as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for a-c. ns: not significant. Numerical source data and statistics are available online.

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Extended Data Fig. 8 Characterization of CD24+ EVs enriched from human serum samples.

a, Immunoblots of indicated proteins in CD24+ EVs enriched from equal volume of human serum samples, including 3 non−cancer controls and 3 cases of BC. b, NTA and a representative EM image of the CD24+ EVs enriched from the serum of a BC patient. Bar=200 nm. c, Calculated ratio of miR-122 abundance in CD24+ vs. total (CD9/CD63/CD81+) circulating EVs. d, RT-qPCR showing relative miR-122 levels in CD24+ EVs (from 3 cases of BC patients) following indicated treatment. Data are shown as mean ± SEM. One-way ANOVA followed by Holm-Sidak’s multiple comparison test was used for c. *P < 0.05 and ***P < 0.001. Numerical source data, statistics and unprocessed blots are available online.

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Extended Data Fig. 9 Gating strategies used for islet cell flow cytometry.

Gating strategies used to analyse uptake of CFSE-labelled EVs by insulin+ pancreatic islet β-cells presented in Fig. 2c. a-d, Islets were treated with PBS or EVs as indicated.

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Tables 1–3

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Cao, M., Isaac, R., Yan, W. et al. Cancer-cell-secreted extracellular vesicles suppress insulin secretion through miR-122 to impair systemic glucose homeostasis and contribute to tumour growth. Nat Cell Biol 24, 954–967 (2022). https://doi.org/10.1038/s41556-022-00919-7

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