Yoonha Hwang

The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling

Overexpression of the receptor tyrosine kinase EPH receptor A2 (EphA2) is commonly observed in aggressive breast cancer and correlates with a poor prognosis. However, while EphA2 has been reported to enhance tumorigenesis, proliferation, and MAPK activation in several model systems, other studies suggest that EphA2 activation diminishes these processes and inhibits the activity of MAPK upon ligand stimulation. In this study, we eliminated EphA2 expression in 2 transgenic mouse models of mammary carcinoma. EphA2 deficiency impaired tumor initiation and metastatic progression in mice overexpressing ErbB2 (also known as Neu) in the mammary epithelium (MMTV-Neu mice), but not in mice overexpressing the polyomavirus middle T antigen in mammary epithelium (MMTV-PyV-mT mice). Histologic and ex vivo analyses of MMTV-Neu mouse mammary epithelium indicated that EphA2 enhanced tumor proliferation and motility. Biochemical analyses revealed that EphA2 formed a complex with ErbB2 in human and murine breast carcinoma cells, resulting in enhanced activation of Ras-MAPK signaling and RhoA GTPase. Additionally, MMTV-Neu, but not MMTV-PyV-mT, tumors were sensitive to therapeutic inhibition of EphA2. These data suggest that EphA2 cooperates with ErbB2 to promote tumor progression in mice and may provide a novel therapeutic target for ErbB2-dependent tumors in humans. Moreover, EphA2 function in tumor progression appeared to depend on oncogene context, an important consideration for the application of therapies targeting EphA2.

Regulation of EphA2 Receptor Endocytosis by SHIP2 Lipid Phosphatase via Phosphatidylinositol 3-Kinase-dependent Rac1 Activation

Endocytosis of Eph receptors is critical for a number of biological processes, including modulating axon growth cone collapse response and regulating cell surface levels of receptor in epithelial cells. In particular, ephrin-A ligand stimulation of tumor cells induces EphA2 receptor internalization and degradation, a process that has been explored as a means to reduce tumor malignancy. However, the mechanism and regulation of ligand-induced Eph receptor internalization are not well understood. Here we show that SHIP2 (Src homology 2 domain-containing phosphoinositide 5-phosphatase 2) is recruited to activated EphA2 via a heterotypic sterile α motif (SAM)-SAM domain interaction, leading to regulation of EphA2 internalization. Overexpression of SHIP2 inhibits EphA2 receptor endocytosis, whereas suppression of SHIP2 expression by small interfering RNA-mediated gene silencing promotes ligand-induced EphA2 internalization and degradation. SHIP2 regulates EphA2 endocytosis via phosphatidylinositol 3-kinase-dependent Rac1 activation. Phosphatidylinositol 3,4,5-trisphosphate levels are significantly elevated in SHIP2 knockdown cells, phosphatidylinositol 3-kinase inhibitor decreases phosphatidylinositol 3,4,5-trisphosphate levels and suppresses increased EphA2 endocytosis. Ephrin-A1 stimulation activates Rac1 GTPase, and the Rac1-GTP levels are further increased in SHIP2 knockdown cells. A dominant negative Rac1 GTPase effectively inhibited ephrin-A1-induced EphA2 endocytosis. Together, our findings provide evidence that recruitment of SHIP2 to EphA2 attenuates a positive signal to receptor endocytosis mediated by phosphatidylinositol 3-kinase and Rac1 GTPase.

Ephrin-A1 Facilitates Mammary Tumor Metastasis through an Angiogenesis-Dependent Mechanism Mediated by EphA Receptor and Vascular Endothelial Growth Factor in Mice

Ephrin-A1, the prototypic ligand for EphA receptor tyrosine kinases, is overexpressed in vascularized tumors relative to normal tissue. Moreover, ephrin-A1-Fc fusion proteins induce endothelial cell sprouting, migration, and assembly in vitro, and s.c. vascular remodeling in vivo. Based on these data, we hypothesized that native, membrane-bound ephrin-A1 regulates tumor angiogenesis and progression. We tested this hypothesis using a transplantable mouse mammary tumor model. Small interfering RNA-mediated ephrin-A1 knockdown in metastatic mammary tumor cells significantly diminishes lung metastasis without affecting tumor volume, invasion, intravasation, or lung colonization upon i.v. injection in vivo. Ephrin-A1 knockdown reduced tumor-induced endothelial cell migration in vitro and microvascular density in vivo. Conversely, overexpression of ephrin-A1 in nonmetastatic mammary tumor cells elevated microvascular density and vascular recruitment. Overexpression of ephrin-A1 elevated wild-type but not EphA2-deficient endothelial cell migration toward tumor cells, suggesting that activation of EphA2 on endothelial cells is one mechanism by which ephrin-A1 regulates angiogenesis. Furthermore, ephrin-A1 knockdown diminished, whereas overexpression of ephrin-A1 elevated, vascular endothelial growth factor (VEGF) levels in tumor cell-conditioned medium, suggesting that ephrin-A1-mediated modulation of the VEGF pathway is another mechanism by which membrane-tethered ephrin-A1 regulates angiogenic responses from initially distant host endothelium. These data suggest that ephrin-A1 is a proangiogenic signal, regulating VEGF expression and facilitating angiogenesis-dependent metastatic spread.

Vital roles of mTOR complex 2 in Notch-driven thymocyte differentiation and leukemia

Rictor is essential in Notch-driven T-ALL pathogenesis.

Identification and Functional Analysis of Phosphorylated Tyrosine Residues within EphA2 Receptor Tyrosine Kinase

EphA2 is a member of the Eph family of receptor tyrosine kinases. EphA2 mediates cell-cell communication and plays critical roles in a number of physiological and pathologic responses. We have previously shown that EphA2 is a key regulator of tumor angiogenesis and that tyrosine phosphorylation regulates EphA2 signaling. To understand the role of EphA2 phosphorylation, we have mapped phosphorylated tyrosines within the intracellular region of EphA2 by a combination of mass spectrometry analysis and phosphopeptide mapping using two-dimensional chromatography in conjunction with site-directed mutagenesis. The function of these phosphorylated tyrosine residues was assessed by mutational analysis using EphA2-null endothelial cells reconstituted with EphA2 tyrosine-to-phenylalanine or tyrosine-to-glutamic acid substitution mutants. Phosphorylated Tyr587 and Tyr593 bind to Vav2 and Vav3 guanine nucleotide exchange factors, whereas Tyr(P)734 binds to the p85 regulatory subunit of phosphatidylinositol 3-kinase. Mutations that uncouple EphA2 with Vav guanine nucleotide exchange factors or p85 are defective in Rac1 activation and cell migration. Finally, EphA2 mutations in the juxtamembrane region (Y587F, Y593F, Y587E/Y593E), kinase domain (Y734F), or SAM domain (Y929F) inhibited ephrin-A1-induced vascular assembly. In addition, EphA2-null endothelial cells reconstituted with these mutants were unable to incorporate into tumor vasculature, suggesting a critical role of these phosphorylation tyrosine residues in transducing EphA2 signaling in vascular endothelial cells during tumor angiogenesis.

Effects of Cancer-Associated EPHA3 Mutations on Lung Cancer

BACKGROUND Cancer genome sequencing efforts recently identified EPHA3, which encodes the EPHA3 receptor tyrosine kinase, as one of the most frequently mutated genes in lung cancer. Although receptor tyrosine kinase mutations often drive oncogenic conversion and tumorigenesis, the oncogenic potential of the EPHA3 mutations in lung cancer remains unknown. METHODS We used immunoprecipitation, western blotting, and kinase assays to determine the activity and signaling of mutant EPHA3 receptors. A mutation-associated gene signature was generated from one large dataset, mapped to another training dataset with survival information, and tested in a third independent dataset. EPHA3 expression levels were determined by quantitative reverse transcription-polymerase chain reaction in paired normal-tumor clinical specimens and by immunohistochemistry in human lung cancer tissue microarrays. We assessed tumor growth in vivo using A549 and H1299 human lung carcinoma cell xenografts in mice (n = 7-8 mice per group). Tumor cell proliferation was measured by bromodeoxyuridine incorporation and apoptosis by multiple assays. All P values are from two-sided tests. RESULTS At least two cancer-associated EPHA3 somatic mutations functioned as dominant inhibitors of the normal (wild type) EPHA3 protein. An EPHA3 mutation-associated gene signature that was associated with poor patient survival was identified. Moreover, EPHA3 gene copy numbers and/or expression levels were decreased in tumors from large cohorts of patients with lung cancer (eg, the gene was deleted in 157 of 371 [42%] primary lung adenocarcinomas). Reexpression of wild-type EPHA3 in human lung cancer lines increased apoptosis by suppression of AKT activation in vitro and inhibited the growth of tumor xenografts (eg, for H1299 cells, mean tumor volume with wild-type EPHA3 = 437.4 mm(3) vs control = 774.7 mm(3), P < .001). Tumor-suppressive effects of wild-type EPHA3 could be overridden in trans by dominant negative EPHA3 somatic mutations discovered in patients with lung cancer. CONCLUSION Cancer-associated EPHA3 mutations attenuate the tumor-suppressive effects of normal EPHA3 in lung cancer.

Host Deficiency in Vav2/3 Guanine Nucleotide Exchange Factors Impairs Tumor Growth, Survival, and Angiogenesis In vivo

Vav guanine nucleotide exchange factors modulate changes in cytoskeletal organization through activation of Rho, Rac, and Cdc42 small GTPases. Although Vav1 expression is restricted to the immune system, Vav2 and Vav3 are expressed in several tissues, including highly vascularized organs. Here, we provide the first evidence that Vav2 and Vav3 function within the tumor microenvironment to promote tumor growth, survival, and neovascularization. Host Vav2/3 deficiency reduced microvascular density, as well as tumor growth and/or survival, in transplanted B16 melanoma and Lewis lung carcinoma models in vivo. These defects were due in part to Vav2/3 deficiency in endothelial cells. Vav2/3-deficient endothelial cells displayed reduced migration in response to tumor cells in coculture migration assays, and failed to incorporate into tumor vessels and enhance tumor volume in tumor-endothelial cotransplantation experiments. These data suggest that Vav2/3 guanine nucleotide exchange factors play a critical role in host-mediated tumor progression and angiogenesis, particularly in tumor endothelium.(Mol Cancer Res 2009;7(5):615–23)

Regulation of heart valve morphogenesis by Eph receptor ligand, ephrin-A1.

Disease or malformation of heart valves is one of the leading causes of morbidity and mortality in both children and adults. These congenital anomalies can remain undetected until cardiac function is compromised, making it important to understand the underlying nature of these disorders. Here we show that ephrin‐A1, a ligand for class A Eph receptor tyrosine kinases, regulates cardiac valve formation. Exogenous ephrin‐A1‐Fc or overexpression of ephrin‐A1 in the heart inhibits epithelial‐to‐mesenchymal transformation (EMT) in chick atrioventricular cushion explants. In contrast, overexpression of wild‐type EphA3 receptor promotes EMT via a kinase‐dependent mechanism. To analyze ephrin‐A1 in vivo, we generated an ephrin‐A1 knockout mouse through gene targeting. Ephrin‐A1 null animals are viable but exhibit impaired cardiac function. Loss of ephrin‐A1 results in thickened aortic and mitral valves in newborn and adult animals. Analysis of early embryonic hearts revealed increased cellularity in outflow tract endocardial cushions and elevated mesenchymal marker expression, suggesting that excessive numbers of cells undergo EMT. Taken together, these data indicate that ephrin‐A1 regulates cardiac valve development, making ephrin‐A1‐deficient mice a novel model for congenital heart defects. Developmental Dynamics 239:3226–3234, 2010.

Cooperative Signaling between Slit2 and Ephrin-A1 Regulates a Balance between Angiogenesis and Angiostasis

ABSTRACT Slit proteins induce cytoskeletal remodeling through interaction with roundabout (Robo) receptors, regulating migration of neurons and nonneuronal cells, including leukocytes, tumor cells, and endothelium. The role of Slit2 in vascular remodeling, however, remains controversial, with reports of both pro- and antiangiogenic activity. We report here that cooperation between Slit2 and ephrin-A1 regulates a balance between the pro- and antiangiogenic functions of Slit2. While Slit2 promotes angiogenesis in culture and in vivo as a single agent, Slit2 potently inhibits angiogenic remodeling in the presence of ephrin-A1. Slit2 stimulates angiogenesis through mTORC2-dependent activation of Akt and Rac GTPase, the activities of which are inhibited in the presence of ephrin-A1. Activated Rac or Akt partially rescues vascular assembly and motility in costimulated endothelium. Taken together, these data suggest that Slit2 differentially regulates angiogenesis in the context of ephrin-A1, providing a plausible mechanism for the pro- versus antiangiogenic functions of Slit2. Our results suggest that the complex roles of Slit-Robo signaling in angiogenesis involve context-dependent mechanisms.

The Ephrin-A1/EPHA2 Signaling Axis Regulates Glutamine Metabolism in HER2-Positive Breast Cancer

Dysregulation of receptor tyrosine kinases (RTK) contributes to cellular transformation and cancer progression by disrupting key metabolic signaling pathways. The EPHA2 RTK is overexpressed in aggressive forms of breast cancer, including the HER2(+) subtype, and correlates with poor prognosis. However, the role of EPHA2 in tumor metabolism remains unexplored. In this study, we used in vivo and in vitro models of HER2-overexpressing breast cancer to investigate the mechanisms by which EPHA2 ligand-independent signaling promotes tumorigenesis in the absence of its prototypic ligand, ephrin-A1. We demonstrate that ephrin-A1 loss leads to upregulated glutamine metabolism and lipid accumulation that enhanced tumor growth. Global metabolic profiling of ephrin-A1-null, HER2-overexpressing mammary tumors revealed a significant increase in glutaminolysis, a critical metabolic pathway that generates intermediates for lipogenesis. Pharmacologic inhibition of glutaminase activity reduced tumor growth in both ephrin-A1-depleted and EPHA2-overexpressing tumor allografts in vivo Mechanistically, we show that the enhanced proliferation and glutaminolysis in the absence of ephrin-A1 were attributed to increased RhoA-dependent glutaminase activity. EPHA2 depletion or pharmacologic inhibition of Rho, glutaminase, or fatty acid synthase abrogated the increased lipid content and proliferative effects of ephrin-A1 knockdown. Together, these findings highlight a novel, unsuspected connection between the EPHA2/ephrin-A1 signaling axis and tumor metabolism, and suggest potential new therapeutic targets in cancer subtypes exhibiting glutamine dependency. Cancer Res; 76(7); 1825-36. ©2016 AACR.