Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling
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
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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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 6

Lack of endothelial epsins 1 and 2 impairs VEGF-induced VEGFR2 endocytosis (A) WT or DKO MECs were incubated with biotinylated VEGF-A/streptavidin–Alexa Fluor 488 at 4°C for 30 minutes, shifted to 37°C for 0 to 20 minutes, and processed for immunofluorescence.

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Lack of endothelial epsins 1 and 2 impairs VEGF-induced VEGFR2 endocytos...
Colocalization of biotinylated VEGF-A/streptavidin–Alexa Fluor 488–labeled VEGFR2 with Alexa Fluor 594–labeled epsin 1 at 2 minutes, EEA1 at 10 minutes, and LAMP1 at 20 minutes seen by confocal microscopy. Arrows indicate colocalization of the 2 proteins. Quantification of colocalized proteins is on right. (B) Cell surface of WT or DKO MECs was labeled with cleavable biotin at 4°C for 30 minutes, incubated with VEGF-A (50 ng/ml) at 37°C for the time points as indicated, followed by cleavage of surface biotin; internalized biotinylated VEGFR2 was determined by streptavidin bead pull-down and Western blotted with anti-VEGFR2. Quantification of internalized VEGFR2 is on right. n > 5 per group in all panels. Scale bar: 10 μm (A).