Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling
Satish Pasula, … , Wang Min, Hong Chen
Satish Pasula, … , Wang Min, Hong Chen
Published November 26, 2012
Citation Information: J Clin Invest. 2012;122(12):4424-4438. https://doi.org/10.1172/JCI64537.
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Research Article

Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling

Abstract

Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

Authors

Satish Pasula, Xiaofeng Cai, Yunzhou Dong, Mirko Messa, John McManus, Baojun Chang, Xiaolei Liu, Hua Zhu, Robert Silasi Mansat, Seon-Joo Yoon, Scott Hahn, Jacob Keeling, Debra Saunders, Genevieve Ko, John Knight, Gail Newton, Francis Luscinskas, Xiaohong Sun, Rheal Towner, Florea Lupu, Lijun Xia, Ottavio Cremona, Pietro De Camilli, Wang Min, Hong Chen

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Figure 7

Loss of endothelial epsins 1 and 2 inhibits endocytosis of VEGFR2 but not transferrin receptor, leading to surface accumulation of VEGFR2.

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Loss of endothelial epsins 1 and 2 inhibits endocytosis of VEGFR2 but no...
(A) WT or DKO MECs were incubated with 100 ng/ml of biotinylated VEGF-A/streptavidin–Alexa Fluor 488 and 10 μg/ml transferrin Alexa Fluor 594 conjugate at 4°C for 30 minutes, shifted to 37°C for 0 to 10 minutes, and processed for immunofluorescence. (B) Cell surface of WT or DKO MECs was labeled with cleavable biotin at 4°C for 30 minutes, incubated with VEGF (50 ng/ml) and holo-transferrin (10 μg/ml) for 0 to 5 minutes, followed by cleavage of surface biotin, and internalized biotinylated VEGFR2 and transferrin receptor were determined by streptavidin bead pull-down and Western blotting with the indicated antibodies. (C) WT or DKO MECs were incubated with VEGF-A (50 ng/ml) for the times indicated, and cell-surface VEGFR2 was labeled by cell-surface biotinylation, and analyzed by streptavidin bead pull-down followed by Western blotting with antibodies indicated. Turnover rate of cell-surface VEGFR2 was plotted based on the expression of cell-surface VEGFR2 after VEGF-A treatment normalized against nontreated samples. n > 5 per group in all panels. Scale bar: 10 μm (A).