Abstract 1618: Erythropoietin meets a new receptor
Authors: Mora Edna M. , Pecot Chad V. , Nick Alpa M. , Mangala Lingegowda S. , Han Hee Dong , Stein Martin A. , Brock Stephan , Vivas-Mejias Pablo , Sung Bokyung , Nishimura Masato , Matsuo Koji , Kannan Sankaranarayanan , Zweidler-McKay Patrick A. , Armaiz-Pena Guillermo N. , Shahzad Mian M.K. , Stone Rebecca L. , Liu Jinsong , Hu Wei , Aggarwal Bharat B. , Lopez-Berestein Gabriel , Jackson David , Sood Anil K. 
Published: June 2012

Abstract
Introduction: Recent clinical trials suggest that use of erythropoietin to treat chemotherapy-induced anemia results in enhanced tumor progression and impaired survival in certain cancer patients. Rationalizing these observations with Epo's pleiotropic functionality and the lack of compelling evidence for Epo receptor (EpoR) involvement, we hypothesized the existence of a novel Epo receptor. Capturing this premise in terms of a holistic in silico strategy, we analyzed the secreted human proteome for receptors possessing structural, regulatory and functional features consistent with Epo binding and subsequent tumorigenic signaling. Our results provided EphB4 as the most likely candidate molecule.
Methods: To determine the possible association between EphB4 and Epo-mediated tumor growth, we examined several ovarian (A2780, HeyA8-MDR, SKOV3ip1) and breast (MDA-231) cancer cell lines. In addition, we selected A2780 ovarian cancer cells to develop stable clones in which either EpoR or EphB4 was silenced using shRNA (A2780 shEpoR, A2780 shEphB4). Competitive and kinetic binding studies were performed using 125I- Epo. Proliferation, migration, and invasion assays were performed as described previously. To assess the role of EpoR and EphB4 in Epo-induced tumor growth, we silenced EpoR and/or EphB4 with specific siRNAs loaded into DOPC nanoliposomes. Statistical significance was established at p&lt; 0.05.
Results: Binding studies with 125I- Epo revealed Epo binding to EphB4 in a low-affinity fashion. Exposure of shEpoR cells to soluble EpoR showed non-competitive inhibition; however, exposure to soluble EphB4 competitively inhibited 125I- Epo binding. These results demonstrate the specificity of Epo binding to the EphB4 receptor. At pharmacologically relevant doses, Epo treatment of shEpoR cells led to activation of the Stat-3 pathway. In addition, in vitro functional assays revealed significant effects of Epo on proliferation (p=0.003), migration (p=0.006), and invasion (p=0.02) of shEpoR cells, but not shEphB4 cells. In orthotopic ovarian (Hey-A8 MDR, SKOV3ip1, A2780) and breast (MDA-231) cancer models, treatment with Epo resulted in increased tumor growth compared to untreated animals (1.1 vs. 1.6 g, p=0.05; 0.7 vs. 1.75 g, p=0.03; 0.6 vs. 1.5 g, p=0.02; .012 vs 0.4 g, p=0.006), respectively. In vivo, (A2780 and MDA-231 models), EpoR siRNA did not affect Epo-stimulated tumor growth. In contrast, EphB4 siRNA-DOPC completely blocked Epo-stimulated tumor growth.
Conclusions: Collectively, these results point to EphB4 as an alternative Epo receptor that mediates Epo-induced tumor growth by activating Stat-3. These findings offer exciting theranostic avenues for management of anemic cancer patients.
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 1618. doi:10.1158/1538-7445.AM2011-1618