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 5

Heterogeneous tumor–associated ECs show a spectrum of miR-30c and Serpine1 expression that defines their in vitro sprouting abilities.

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Heterogeneous tumor–associated ECs show a spectrum of miR-30c and Serpin...
(A) qPCR analysis of FACS-enriched TECs from orthotopic E0771 mammary tumors, orthotopic 344SQ lung tumors, and human lung tumor specimens. Samples were assayed in triplicate (n = 3 tumors). (B) PCA of NanoString array using TECs treated with 10 ng/ml TGF-β for 48 hours. A heatmap of miR-30 family members is also shown. (C) qPCR analysis of individual isolates of TECs from the indicated murine tumor model. Samples were assayed in triplicate (n = 3). (D) qPCR analysis in C3-TAg TECs or KRASG12D TECs treated with 10 ng/ml TGF-β for 48 hours. Samples were assayed in triplicate. Samples are arranged from low (far left) to high (far right) miR-30c expression on the graph (n = 3). (E) Western blot analysis of PAI-1 using conditioned media from miR-30clo PAI-1hi and miR-30chi PAI-1lo ECs treated with TGF-β (0, 1, 5, and 10 ng/ml for 48 hours). PS was used to show equal loading. (F) Images of miR-30clo PAI-1hi and miR-30chi PAI-1lo ECs in a fibrin-sprouting assay. Arrowheads indicate aberrant sprout formation in miR-30chi PAI-1lo ECs. The scale at bottom right indicates sprout depth in the 3D fibrin matrix. Scale bar: 40 μm (x) and 54 μm (y). (G) Number of sprouts and length of sprouts per bead in miR-30clo PAI-1hi and miR-30chi PAI-1lo ECs. Sprouts were counted at the indicated time points (n = 30 beads per time point). *P < 0.05, by ANOVA. (H) Schematic of TEC subtypes showing angiogenic (miR-30clo PAI-1hi) versus dysmorphic (miR-30chi PAI-1lo) TECs identified in this study. Data represent the mean ± SEM.