Guanglei Zhuang

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.

Elevation of Receptor Tyrosine Kinase EphA2 Mediates Resistance to Trastuzumab Therapy

One arising challenge in the treatment of breast cancer is the development of therapeutic resistance to trastuzumab, an antibody targeting the human epidermal growth factor receptor-2 (HER2), which is frequently amplified in breast cancers. In this study, we provide evidence that elevated level of the receptor tyrosine kinase Eph receptor A2 (EphA2) is an important contributor to trastuzumab resistance. In a screen of a large cohort of human breast cancers, we found that EphA2 overexpression correlated with a decrease in disease-free and overall survival of HER2-overexpressing patients. Trastuzumab-resistant cell lines overexpressed EphA2, whereas inhibiting EphA2 restored sensitivity to trastuzumab treatment in vivo. Notably, trastuzumab treatment could promote EphA2 phosphorylation by activating Src kinase, leading in turn to an amplification of phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signaling in resistant cells. Our findings offer mechanistic insights into the basis for trastuzumab resistance and rationalize strategies to target EphA2 as a tactic to reverse trastuzumab resistance.

Impaired tumor microenvironment in EphA2-deficient mice inhibits tumor angiogenesis and metastatic progression

EphA2 belongs to a unique family of receptor tyrosine kinases that play critical roles in development and disease. Since EphA2 is required for ephrin‐A1 ligand‐induced vascular remodeling and is overexpressed in a variety of vascularized human adenocarcinomas, we assessed tumor angiogenesis and metastatic progression in EphA2‐deficient host animals. 4T1 metastatic mammary adenocarcinoma cells transplanted subcutaneously and orthotopically into EphA2‐deficient female mice displayed decreased tumor volume, tumor cell survival, microvascular density, and lung metastasis relative to tumor‐bearing littermate controls. To determine if the phenotype in EphA2‐deficient mice was endothelial cell intrinsic, we also analyzed endothelial cells isolated from EphA2‐deficient animals for their ability to incorporate into tumor vessels in vivo, as well as to migrate in response to tumor‐derived signals in vitro. EphA2‐deficient endothelial cells displayed impaired survival and failed to incorporate into tumor microvessels in vivo, and displayed impaired tumor‐mediated migration in vitro relative to controls. These data suggest that host EphA2 receptor tyrosine kinase function is required in the tumor microenvironment for tumor angiogenesis and metastatic progression.

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.

Essential Role of Vav Family Guanine Nucleotide Exchange Factors in EphA Receptor-Mediated Angiogenesis

ABSTRACT Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2−/− Vav3−/− mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.

Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism

EPHA2 receptor tyrosine kinase is overexpressed in several human cancer types and promotes malignancy. However, the mechanisms by which EPHA2 promotes tumor progression are not completely understood. Here we report that overexpression of a wild-type EPHA2, but not a signaling-defective cytoplasmic truncation mutant (ΔC), in human mammary epithelial cells weakens E-cadherin-mediated cell-cell adhesion. Interestingly, the total level of cadherins and the composition of the adherens junction complexes were not affected, nor was the tyrosine phosphorylation of the cadherin complex components changed. By contrast, RhoA GTPase activity was significantly affected by modulating the EPHA2 activity in MCF-10A cells. Treatment with a ROCK kinase inhibitor rescued cell-cell adhesion defects in EPHA2-overexpressing cells, whereas expression of constitutively activated Rho disrupted adherens junctions in ΔC-expressing cells. EPHA2-dependent Rho activation and destabilization of adherens junctions appeared to be regulated via a signaling pathway involving Src kinase, low molecular weight phosphotyrosine phosphatase (LMW-PTP) and p190 RhoGAP. EPHA2 interacted with both Src and LMW-PTP, and the interactions increased in EPHA2-overexpressing cells. In addition, LMW-PTP phosphatase activity was elevated, and this elevation was accompanied by a decrease in tyrosine phosphorylation of p190 RhoGAP and destabilization of cell-cell adhesion. Expression of either a dominant negative LMW-PTP mutant, C12S, or a wild-type p190 RhoGAP rescued adhesion defects in EPHA2-overexpressing cells. Together, these data suggest that EPHA2 promotes tumor malignancy through a mechanism involving RhoA-dependent destabilization of adherens junctions.

Eph Receptors and Ephrins in Cancer: Common Themes and Controversies

Ephrins and Eph receptors are key regulators of physiologic and pathologic processes in development and disease. Expression of Eph receptors is often elevated in many types of malignant tumors, yet their precise role in cancer is not well-understood. The purpose of the first “Eph/Ephrins and

Effects of Cancer-Associated EPHA3 Mutations on Lung Cancer

BACKGROUNDnCancer 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.nnnMETHODSnWe 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.nnnRESULTSnAt 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.nnnCONCLUSIONnCancer-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)