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 4

miR-30c gain of function in the endothelium inhibits tumor growth and reduces fibrin abundance, whereas miR-30c loss of function has the opposite effect.

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miR-30c gain of function in the endothelium inhibits tumor growth and re...
(A) qPCR analysis in E0771 mammary tumor cells and ECs treated with 10 ng/ml TGF-β for 48 hours. Samples were assayed in triplicate (n = 3). (B) Western blot analysis of PAI-1 in E0771 mammary tumor cells or ECs treated with 10 ng/ml TGF-β for 48 hours. PS was used to show equal loading. (C) Tumor volume measurements of mice bearing orthotopic E0771 mammary tumors treated intravenously with 1.5 mg/kg 7C1 scrambled versus 1.5 mg/kg 7C1 mimic or 1.5 mg/kg 7C1 scrambled versus 7C1 antagomiR. Data were combined from 2 independent experiments (n = 10–11 mice/group). (D) Tumor weights in mice treated as in C. Data were combined from 2 independent experiments (n = 10–11 mice/group). (E) Tumor volume measurements of PAI-1–KO mice bearing orthotopic E0771 mammary tumors treated with 7C1 scrambled, 7C1 mimic, or 7C1 antagomiR (n = 3–4 mice/group). (F) qPCR analysis of Serpine1 expression in FACS-sorted ZSGreen+ ECs and ZSGreen– non-ECs from orthotopic E0771 mammary tumors from mice treated with 7C1 scrambled, 7C1 mimic, or 7C1 antagomiR as in C (n = 3 mice/group; n = 2 replicates). (G) qPCR analysis of miR-30c using FACS-enriched ZSGreen+ ECs and ZSGreen– non-ECs as in F. (H) Plasma PAI-1 levels in mice treated as indicated (n = 5 mice/group). (I) Plasma PAI-1 levels in mice treated as indicated (n = 5 mice/group). (J) Immunofluorescence images of E0771 mammary tumors from mice treated with 7C1 scrambled, 7C1 mimic, or 7C1 antagomiR, as above. Nuclei were counterstained with DAPI. Scale bar: 100 μm. (K) Quantification of vascular area and fibrin deposition area in E0771 mammary tumors from mice treated with 7C1 scrambled, 7C1 mimic, or 7C1 antagomiR (n = 5 tumors/group). *P < 0.05, by Student’s t test (A, D, F–I, and K). Data represent the mean ± SEM.