Regulating growth factor signaling in a controlled and site-specific manner is the main hurdle that current antiangiogenic treatment modalities face. Here we show that the prion-like protein, doppel, is uniquely found in tumoral endothelial cells (TEC) but not in the normal ECs. Tissue sections containing primary human tumor (lung and colon) and normal tissues were used to study doppel protein expression. Strong expression and clear co-localization of doppel protein with CD34, a classical endothelial marker, was observed in both lung and colon cancer tissues compared to normal tissues. To evaluate doppel expression at the molecular level, we isolated TECs from different tumors derived from mouse xenografts. Extensive analysis showed that doppel was highly and ubiquitously expressed in the vasculature of squamous, breast, lung, and colon cancers. To elucidate the role of doppel in TEC during tumor angiogenesis, we generated a gain-of-function EC model to express doppel in ECs. Inducible overexpression of doppel in ECs correlates with high vessel formation in a spheroid-based EC transplantation technique. To integrate doppel expression with the canonical angiogenic circuits, we studied the mechanism by which doppel regulates angiogenesis. We evidently observed that doppel co-localized and formed complexes with vascular endothelial growth factor receptor 2 (VEGFR2). Also, doppel prolonged the surface residency of VEGFR2 and amplified the responsiveness of VEGF to VEGFR2. Genetic deletion of doppel in TEC not only depleted membrane residency but also induced internalization and degradation of VEGFR2. This indicates that doppel blocking can lead to effective control of VEGF-signaling in TECs and selective inhibition of tumor angiogenesis. Based on the functional role of doppel in angiogenesis and tumor growth inhibition, we generated doppel knockout mice on immunogenic C57BL6 mice. Tumor growth was inhibited in both hetero (Dpl+/-) and homo (Dpl-/-)-type mice compared to their wild-type littermate controls when syngeneic cancer cell lines including melanoma (B16f10) and lymphoma (EL4) or allogeneic colon cancer CT26 cell line were inoculated. Moreover, we developed doppel targeting monoclonal antibodies. Anti-doppel antibodies, 5C7 and 4D6, were effective against both murine CT26 and human HCT116 colon cancer cell lines. Our studies thus demonstrate the success of doppel targeting in inhibiting pathological tumor growth and establish doppel-VEGFR2 interactions as a specific molecular and therapeutic target in tumoral angiogenesis.
Citation Format: Taslim Al-Hilal, Yoosoo Yang, In-San Kim, Youngro Byun, Fakhrul Ahsan. Prion-like protein “Doppel” is a selective therapeutic target for tumoral angiogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3277.