Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130 Cas . Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130 Cas . WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.Lung cancer is the leading cause of cancer death in the United States (1) and is a growing health epidemic worldwide (2). Metastatic lung cancer is virtually incurable. Even when lung cancer is detected early and aggressive intervention is undertaken, its outcome remains disappointing. Patients with completely resected Stage I non-small cell lung cancer (NSCLC) 2 have a 5-year overall survival of 55-70% (3-5). Locoregional and, more commonly, distant recurrence limits the curative potential of surgery.The Eph receptor family represents the largest group of receptor tyrosine kinases (6). The Eph receptors are divided into EphA and EphB based on structural homology and ligandbinding affinity. In all, there are 16 Eph receptors (Eph A1-A10 and B1-B6), but humans lack EphA9 and EphB5 (7). The ligands for the Eph receptors are ephrins (7). The ephrins fall into two subclasses, ephrin-A and -B, based on their mode of membrane attachment and receptor affinity. The physiologic role of Eph receptors is crucial in embryonic developmental processes, such as cell migration, vascular development, tissueborder formation, axonal and synaptic network development, and adult processes such as regulation of neuronal plasticity (7). A peculiarity of the Eph-ephrin system is that signals are transduced by both the receptor (forward signaling) and ligand (reverse signaling) (7).EphA2 is overexpressed in glioblastoma (8), breast (9, 10), colon (9, 11), ovarian (12), pancreatic (13), and prostate (14) cancer. EphA2 is overexpressed (staining intensity 2ϩ/3ϩ) in 70% of NSCLC (15). This receptor promotes cell proliferation (16), motility (16, 17), invasion (8), metastasis (18, 19), and angiogenesis (20) in malignant tumors. Furthermore, somatic mutations in the Eph (EphA3 and -A5) family ha...

Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability in vitro, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.

BackgroundMalignant pleural mesothelioma (MPM) often develops decades following exposure to asbestos. Current best therapy produces a response in only half of patients, and the median survival with this therapy remains under a year. A search for novel targets and therapeutics is underway, and recently identified targets include VEGF, Notch, and EphB4-Ephrin-B2. Each of these targets has dual activity, promoting tumor cell growth as well as tumor angiogenesis.MethodsWe investigated EphB4 expression in 39 human mesothelioma tissues by immunohistochemistry. Xenograft tumors established with human mesothelioma cells were treated with an EphB4 inhibitor (monomeric soluble EphB4 fused to human serum albumin, or sEphB4-HSA). The combinatorial effect of sEphB4-HSA and biologic agent was also studied.ResultsEphB4 was overexpressed in 72% of mesothelioma tissues evaluated, with 85% of epithelioid and 38% of sarcomatoid subtypes demonstrating overexpression. The EphB4 inhibitor sEphB4-HSA was highly active as a single agent to inhibit tumor growth, accompanied by tumor cell apoptosis and inhibition of PI3K and Src signaling. Combination of sEphB4-HSA and the anti-VEGF antibody (Bevacizumab) was superior to each agent alone and led to complete tumor regression.ConclusionEphB4 is a potential therapeutic target in mesothelioma. Clinical investigation of sEphB4-HSA as a single agent and in combination with VEGF inhibitors is warranted.

Esophageal cancer incidence is increasing and has few treatment options. In studying receptor tyrosine kinases associated with esophageal cancers, we have identified EPHB4 to be robustly overexpressed in cell lines and primary tumor tissues. In total, 94 squamous cell carcinoma, 82 adenocarcinoma, 25 dysplasia, 13 Barrett esophagus, and 25 adjacent or unrelated normal esophageal tissues were evaluated by immunohistochemistry. EPHB4 expression was significantly higher in all the different histologic categories than in adjacent normal tissues. In 13 esophageal cancer cell lines, 3 of the 9 SCC cell lines and 2 of the 4 adenocarcinomas expressed very high levels of EPHB4. An increased gene copy number ranging from 4 to 20 copies was identified in a subset of the overexpressing patient samples and cell lines. We have developed a novel 4-nitroquinoline 1-oxide (4-NQO)-induced mouse model of esophageal cancer that recapitulates the EPHB4 expression in humans. A specific small-molecule inhibitor of EPHB4 decreased cell viability in a timeand dose-dependent manner in 3 of the 4 cell lines tested. The small-molecule inhibitor and an EPHB4 siRNA also decreased cell migration (12%-40% closure in treated vs. 60%-80% in untreated), with decreased phosphorylation of various tyrosyl-containing proteins, EphB4, and its downstream target p125FAK. Finally, in a xenograft tumor model, an EPHB4 inhibitor abrogated tumor growth by approximately 60% compared with untreated control. EphB4 is robustly expressed and potentially serves as a novel biomarker for targeted therapy in esophageal cancers. Cancer Res; 73(1); 184-94. Ó2012 AACR.