Abstract B39: Using in vivo phage display to discover novel, nonvascular endothelial growth factor (VEGF)-mediated tumor angiogenic proteins such as hornerin.

Angiogenesis is vital to the growth, progression and ultimately metastasis of tumors. As such, many therapies have attempted to eradicate tumors through anti-angiogenic strategies. Most current and past preclinical and clinically approved therapies focus on the vascular endothelial growth factor (VEGF) pathway. Unfortunately, these VEGF therapies have not shown much success, leading oncologists and scientists to become pessimistic about the usefulness of targeting angiogenesis as an anti-tumor strategy. Therapies against novel and/or non-VEGF proteins could hold the key to effective anti-angiogenic cancer therapies. Therefore, we used an in vivo phage display screen to find new, non-VEGF mediated angiogenic proteins. After selection, the binding partner of one clone was identified as hornerin, a previously unknown player in tumor angiogenesis. The in vivo phage screen was completed in a murine orthotopic pancreatic ductal adenocarcinoma (PDAC) tumor model. Non-VEGF binding phage clones were identified by screening human umbilical vein endothelial cells (HUVEC) treated with tumor conditioned media, VEGF conditioned media or vehicle control. Those that bound tumor conditioned media, but not VEGF conditioned media were selected for binding partner identification via functional proteomics pioneered in our lab. Hornerin, a previously unknown angiogenic protein, was identified via functional proteomics. Subsequent validation by mRNA expression, immunocytochemistry and immunohistochemistry showed that hornerin was indeed not induced by VEGF. Hornerin expression was detected in resected human and murine subcutaneous PDAC tumor vasculature. Interestingly, hornerin expression was also detected in tumor cells of resected human PDAC tumor sections and in tumor cells in vitro, providing a potential new biomarker of pancreatic cancer. A functional role in angiogenesis was confirmed by intratumoral injection of short interfering RNA (siRNA) against endothelial hornerin; treated tumors9 growth was static and their vessels were smaller, less tortuous and had fewer sprouts. Additionally, siRNA knockdown in L36.pl tumor cells caused decreased proliferation in vitro. We have determined that basic fibroblast growth factor (bFGF) upregulates hornerin, and we plan to widen the search include growth factors of highest relative abundance in tumor conditioned media. The discovery of novel, non-VEGF mediated angiogenic proteins provides the potential to develop therapies that synergize with current anti-VEGF therapies. One such protein, hornerin was identified by our in vivo phage screen and shown to be functionally important in both angogenesis and tumor progression. Future studies include identifying the growth factor(s) other than bFGF responsible for endothelial expression, understanding hornerin9s mechanism and role in pathologic angiogenesis, and designing a new therapeutic strategy. Citation Format: Marc E. Seaman, Kimberly A. Kelly. Using in vivo phage display to discover novel, nonvascular endothelial growth factor (VEGF)-mediated tumor angiogenic proteins such as hornerin. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B39.