Endothelial miR-30c suppresses tumor growth via inhibition of TGF-β–induced Serpine1
James V. McCann, … , Nigel Mackman, Andrew C. Dudley
James V. McCann, … , Nigel Mackman, Andrew C. Dudley
Published March 11, 2019
Citation Information: J Clin Invest. 2019;129(4):1654-1670. https://doi.org/10.1172/JCI123106.
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Research Article Oncology Vascular biology

Endothelial miR-30c suppresses tumor growth via inhibition of TGF-β–induced Serpine1

Abstract

In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKO mice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-β–mediated suppression of miR-30c. Bypassing TGF-β signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1–dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-β, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.

Authors

James V. McCann, Lin Xiao, Dae Joong Kim, Omar F. Khan, Piotr S. Kowalski, Daniel G. Anderson, Chad V. Pecot, Salma H. Azam, Joel S. Parker, Yihsuan S. Tsai, Alisa S. Wolberg, Stephen D. Turner, Kohei Tatsumi, Nigel Mackman, Andrew C. Dudley

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Figure 2

Tgfbr2iECKO ECs show impaired fibrin-mediated angiogenesis in vitro and fail to upregulate Serpine1 and downregulate miR-30 miRNAs.

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Tgfbr2iECKO ECs show impaired fibrin-mediated angiogenesis in vitro and...
(A) Validation of EC isolation from ZSGreen reporter mice. Phase and ZSGreen images of isolated ECs. Scale bar: 10 μm. (B) qPCR analysis of EC and fibroblast genes from control and Tgfbr2iECKO mice. Samples were assayed in triplicate. (C) Western blot showing phosphorylated SMAD2 (p-SMAD2) in ECs challenged with 10 ng/ml TGF-β to confirm disabled Tgfbr2 signaling. (D) In vivo fibrin plugs from control versus Tgfbr2iECKO ECs. Lectin594 was injected prior to euthanasia. Scale bar: 100 μm. Phalloidin-stained ECs in the fibrin bead–sprouting assay at 72 hours. Arrows indicate examples of individual sprouts that were quantified. The scale at bottom right indicates sprout depth in the 3D fibrin matrix. Scale bars: 40 μm (x) and 54 μm (y). (E) Quantification of the fibrin-sprouting assay over time. The number and length of sprouts were measured for each indicated time point (n = 30 beads per time point). (F) qPCR analysis of TGF-β–induced and fibrinolysis-regulating genes in control versus Tgfbr2iECKO ECs. Samples were assayed in triplicate. (G) miR-30 family miRNA expression in ECs treated with 10 ng/ml TGF-β for 48 hours. Samples were assayed in triplicate. (H) Ectopic miR-30c mimic transfection and qPCR analysis of miR-30c levels along with fibrinolysis-regulating genes. Samples were assayed in triplicate. *P < 0.05, by ANOVA (E) and Student’s t test (F–H) Data represent the mean ± SEM.