Satish Pasula

Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling.

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.

Dynamin 2 regulation of integrin endocytosis, but not VEGF signaling, is crucial for developmental angiogenesis

Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of β1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of β1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo.

Temporal and spatial regulation of epsin abundance and VEGFR3 signaling are required for lymphatic valve formation and function

Proteins involved in endocytosis promote the internalization and degradation of VEGFR3, ensuring valve formation in lymphatic vessels. Ensuring a One-Way Flow of Lymph Lymphatic vessels return lymph, which consists of fluid and mostly immune cells that go into the tissues from the blood, back to the circulation. Valves in lymphatic vessels ensure that lymph flows in one direction into veins and prevent the accumulation of lymph in tissues, a condition called edema. The receptor VEGFR3 is required for lymphatic vessel development. Liu et al. noted that the regions of developing lymphatic vessels that became valves had high VEGFR3 amounts but low amounts of epsin 1 and 2, proteins that are involved in endocytosis. They determined that epsin 1 and 2 suppressed VEGFR3 signaling in collecting lymphatic trunks by triggering endocytosis and degradation of VEGFR3. Furthermore, mice lacking epsin 1 and 2 in the endothelial cells that line lymphatic vessels had defective lymphatic valves and impaired drainage. Lymphatic valve formation was restored in these mice by deletion of a single allele of Vegfr3 or treatment with a VEGFR3 inhibitor. Lymphatic valves prevent the backflow of the lymph fluid and ensure proper lymphatic drainage throughout the body. Local accumulation of lymphatic fluid in tissues, a condition called lymphedema, is common in individuals with malformed lymphatic valves. The vascular endothelial growth factor receptor 3 (VEGFR3) is required for the development of lymphatic vascular system. The abundance of VEGFR3 in collecting lymphatic trunks is high before valve formation and, except at valve regions, decreases after valve formation. We found that in mesenteric lymphatics, the abundance of epsin 1 and 2, which are ubiquitin-binding adaptor proteins involved in endocytosis, was low at early stages of development. After lymphatic valve formation, the initiation of steady shear flow was associated with an increase in the abundance of epsin 1 and 2 in collecting lymphatic trunks, but not in valve regions. Epsin 1 and 2 bound to VEGFR3 and mediated the internalization and degradation of VEGFR3, resulting in termination of VEGFR3 signaling. Mice with lymphatic endothelial cell–specific deficiency of epsin 1 and 2 had dilated lymphatic capillaries, abnormally high VEGFR3 abundance in collecting lymphatics, immature lymphatic valves, and defective lymph drainage. Deletion of a single Vegfr3 allele or pharmacological suppression of VEGFR3 signaling restored normal lymphatic valve development and lymph drainage in epsin-deficient mice. Our findings establish a critical role for epsins in the temporal and spatial regulation of VEGFR3 abundance and signaling in collecting lymphatic trunks during lymphatic valve formation.

Genetic Reduction of Vascular Endothelial Growth Factor Receptor 2 Rescues Aberrant Angiogenesis Caused by Epsin Deficiency

Objective—We previously showed that endothelial epsin deficiency caused elevated vascular endothelial growth factor receptor 2 (VEGFR2) and enhanced VEGF signaling, resulting in aberrant tumor angiogenesis and reduced tumor growth in adult mice. However, direct evidence demonstrating that endothelial epsins regulate angiogenesis specifically through VEGFR2 downregulation is still lacking. In addition, whether the lack of epsins causes abnormal angiogenesis during embryonic development remains unclear. Approach and Results—A novel strain of endothelial epsin-deleted mice that are heterozygous for VEGFR2 (Epn1fl/fl; Epn2−/−; Flkfl/+; iCDH5 Cre mice) was created. Analysis of embryos at different developmental stages showed that deletion of epsins caused defective embryonic angiogenesis and retarded embryo development. In vitro angiogenesis assays using isolated primary endothelial cells (ECs) from Epn1fl/fl; Epn2−/−; iCDH5 Cre (EC-iDKO) and Epn1fl/fl; Epn2−/−; Flkfl/+; iCDH5 Cre (EC-iDKO-Flkfl/+) mice demonstrated that VEGFR2 reduction in epsin-depleted cells was sufficient to restore normal VEGF signaling, EC proliferation, EC migration, and EC network formation. These findings were complemented by in vivo wound healing, inflammatory angiogenesis, and tumor angiogenesis assays in which reduction of VEGFR2 was sufficient to rescue abnormal angiogenesis in endothelial epsin-deleted mice. Conclusions—Our results provide the first genetic demonstration that epsins function specifically to downregulate VEGFR2 by mediating activated VEGFR2 internalization and degradation and that genetic reduction of VEGFR2 level protects against excessive angiogenesis caused by epsin loss. Our findings indicate that epsins may be a potential therapeutic target in conditions in which tightly regulated angiogenesis is crucial, such as in diabetic wound healing and tumors.

AIP1 Suppresses Atherosclerosis by Limiting Hyperlipidemia-Induced Inflammation and Vascular Endothelial Dysfunction

Objective—Apoptosis signal–regulating kinase 1–interacting protein-1 (AIP1) is a signaling adaptor molecule implicated in stress and apoptotic signaling induced by proinflammatory mediators. However, its function in atherosclerosis has not been established. In the present study, we use AIP1-null (AIP1−/−) mice to examine its effect on atherosclerotic lesions in an apolipoprotein E-null (ApoE−/−) mouse model of atherosclerosis. Approach and Results—ApoE−/− control mice developed atherosclerosis in the aortic roots and descending aortas on Western-type diet for 10 weeks, whereas the atherosclerotic lesions are significantly augmented in ApoE−/−AIP1−/− double knockout (DKO) mice. DKO mice show increases in plasma inflammatory cytokines with no significant alterations in body weight, total cholesterol levels, or lipoprotein profiles. Aortas in DKO mice show increased inflammation and endothelial cell (EC) dysfunction with nuclear factor-&kgr;B activity, correlating with increased accumulation of macrophages in the lesion area. Importantly, macrophages from DKO donors are not sufficient to augment inflammatory responses and atherogenesis when transferred to ApoE-KO recipients. Mechanistic studies suggest that AIP1 is highly expressed in aortic EC, but not in macrophages, and AIP1 deletion in EC significantly enhance oxidized low-density lipoprotein–induced nuclear factor-&kgr;B signaling, gene expression of inflammatory molecules, and monocyte adhesion, suggesting that vascular EC are responsible for the increased inflammatory responses observed in DKO mice. Conclusion—Our data demonstrate that loss of AIP1 in aortic EC primarily contributes to the exacerbated lesion expansion in the ApoE−/−AIP1−/− mice, revealing an important role of AIP1 in limiting inflammation, EC dysfunction, and atherosclerosis.

CD82 Restrains Pathological Angiogenesis by Altering Lipid Raft Clustering and CD44 Trafficking in Endothelial Cells

Background— Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. Methods and Results— Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. Conclusions— Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.

Epsin Family of Endocytic Adaptor Proteins as Oncogenic Regulators of Cancer Progression.

Tumor angiogenesis, tumor cell proliferation, and tumor cell migration result from an accumulation of oncogenic mutations that alter protein expression and the regulation of various signaling cascades. Epsins, a small family of clathrin-mediated endocytic adaptor proteins, are reportedly upregulated in a variety of cancers. Importantly, loss of epsins protects against tumorigenesis, thus supporting an oncogenic role for epsins in cancer. Although a clear relationship between epsins and cancer has evolved, the importance of this relationship with regards to cancer progression and anti-cancer therapies remains unclear. In this review, we summarize epsins’ role as endocytic adaptors that modulate VEGF and Notch signaling through the regulated internalization of VEGFR2 and trans-endocytosis of Notch receptors. As both VEGF and Notch signaling have significant implications in angiogenesis, we focus on the newly identified role for epsins in tumor angiogenesis. In addition to epsins’ canonical role in receptor-mediated endocytosis, and the resulting downstream signaling regulation, we discuss the non-canonical role of epsins as regulators of small GTPases and the implications this has on tumor cell proliferation and invasion. Given epsins’ identified roles in tumor angiogenesis, tumor cell proliferation, and tumor cell invasion, we predict that the investigative links between epsins and cancer will provide new insights into the importance of endocytic adaptors and their potential use as future therapeutic targets.

Epsin is required for Dishevelled stability and Wnt signalling activation in colon cancer development.

Uncontrolled canonical Wnt signaling supports colon epithelial tumor expansion and malignant transformation. Understanding the regulatory mechanisms involved is crucial for elucidating the pathogenesis of and will provide new therapeutic targets for colon cancer. Epsins are ubiquitin-binding adaptor proteins upregulated in several human cancers; however, epsins’ involvement in colon cancer is unknown. Here we show that loss of intestinal epithelial epsins protects against colon cancer by significantly reducing the stability of the crucial Wnt signaling effector, dishevelled (Dvl2), and impairing Wnt signaling. Consistently, epsins and Dvl2 are correspondingly upregulated in colon cancer. Mechanistically, epsin binds Dvl2 via its epsin N-terminal homology domain and ubiquitin-interacting motifs and prohibits Dvl2 polyubiquitination and degradation. Our findings reveal an unconventional role for epsins in stabilizing Dvl2 and potentiating Wnt signaling in colon cancer cells to ensure robust colon cancer progression. Epsins’ pro-carcinogenic role suggests they are potential therapeutic targets to combat colon cancer.

Selective targeting of a novel Epsin-VEGFR2 interaction promotes VEGF-mediated angiogenesis

RATIONALE We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. OBJECTIVE The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo. METHODS AND RESULTS Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. CONCLUSIONS Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Endocytic Adaptor Protein Epsin Is Elevated in Prostate Cancer and Required for Cancer Progression

Epsins have an important role in mediating clathrin-mediated endocytosis of ubiquitinated cell surface receptors. The potential role for epsins in tumorigenesis and cancer metastasis by regulating intracellular signaling pathways has largely not been explored. Epsins are reportedly upregulated in several types of cancer including human skin, lung, and canine mammary cancers. However, whether their expression is elevated in prostate cancer is unknown. In this study, we investigated the potential role of epsins in prostate tumorigenesis using the wild type or epsin-deficient human prostate cancer cells, LNCaP, in a human xenograft model, and the spontaneous TRAMP mouse model in wild type or epsin-deficient background. Here, we reported that the expression of epsins 1 and 2 is upregulated in both human and mouse prostate cancer cells and cancerous tissues. Consistent with upregulation of epsins in prostate tumors, we discovered that depletion of epsins impaired tumor growth in both the human LNCaP xenograft and the TRAMP mouse prostate. Furthermore, epsin depletion significantly prolonged survival in the TRAMP mouse model. In summary, our findings suggest that epsins may act as oncogenic proteins to promote prostate tumorigenesis and that depletion or inhibition of epsins may provide a novel therapeutic target for future prostate cancer therapies.