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.

EphB4 is a member of the largest family of receptor tyrosine kinases (RTKs), classically associated with neural development and vascular patterning in embryonic life. The cellular responses to Eph receptor stimulation by their ephrin ligands are important in mediating a wide range of biological activities, including angiogenesis, cell segregation, cell attachment, shape, and motility. EphB4 has been showed to play an oncogenic role in a number of tumors, and we report the first investigation into its role in esophageal cancer, a disease whose survival has not improved and would benefit from the identification of new targets in its treatment. We have addressed this need by exploring the Eph/ephrin receptor tyrosine kinase pathway. We utilized tissues archived at the University of Chicago as well as commercially purchased TMAs to evaluate the expression of EphB4 by IHC. Approximately 93 squamous cell carcinoma, 100 adenocarcinoma and 25 adjacent normal control samples were utilized. In addition, four adenocarcinoma and nine squamous cell carcinma cell lines were used for in vitro studies. Statistical analysis of IHC data showed a consistently higher expression of EphB4 in both squamous and adenocarcinoma compared to adjacent normal tissue. A statistically significant correlation was established between EphB4 expression and higher grades of squamous cell carcinoma, suggesting that overexpression of EphB4 in squamous cell carcinoma is directly linked to clinically aggressive tumors. The EphB4 gene was found amplified in 8/16 squamous cell carcinoma (30% with a gene copy number ≥ 3, 30% with >8 copies and 10% with a copy number of 20. Of the adenocarcinomas, 3/7 samples were amplified (43% with a gene copy number of ≥4, and 14% >8 copies). Three of 9 squamous cell carcinoma cell lines had a gene copy number ≥4 and rest of them had no significant variation. Only one of four adenocarcinoma cell lines had an increased gene copy number. In vitro experiments with EphB4 specific siRNA inhibited the motility and proliferation of cultured cells and the inhibition of EphB4 specifically abrogated the phosphorylation of functional proteins associated with cell motility and proliferation, especially that of phospho-FAK and phospo-AKT, the expression of which were abrogated completely by the inhibition of EphB4. Our data imply that EphB4 plays a crucial role in esophageal cancer and should be further evaluated as a therapeutic target in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1625. doi:10.1158/1538-7445.AM2011-1625