Unbiased Functional Proteomics to Identify Novel Mediators of Tumor Vessel Development

Most current and past preclinical and clinically approved anti-tumor vessel therapies focus on the vascular endothelial growth factor (VEGF) pathway. Although targeting VEGF has proven effective in certain cancers, overall, the clinical efficacy has been modest at best. Where efficacious, tumors initially respond then recur becoming resistant to VEGF inhibitors in the process. Therapies against non-VEGF mediated proteins that might augment VEGF inhibition could hold the key to improving patient survival. Therefore, we identified VEGF-independent factors through a combination of in vivo phage display in mice with orthotopically implanted pancreatic ductal adenocarcinoma (PDAC) tumors and functional proteomics methods. Hornerin, an S100 family member, was identified and subsequently validated via immunofluorescence and immunoblotting as a protein important in tumor endothelial cells in vitro and in vivo. In human PDAC vessels, hornerin was highly expressed, indicating potential clinical relevance. Knockdown of tumor vessel hornerin by intratumoral injections of siRNA reduced tumor volume and important vessel parameters as measured by software we developed and published called Rapid Analysis of Vessel Elements (RAVE). Functionally, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) revealed reduced vascularity and perfusion in hornerin knockdown tumors. Further, anti-hornerin therapy in combination with a VEGF receptor inhibitor AV-951 resulted in additive decreases in tumor volume, providing evidence that targeting independent pathways may result in enhanced therapeutic efficacy.

Mechanistically, we showed that hornerin expression contributes to protection from oxidative stress through NF-κB induced BCL2 localization to the mitochondria. This protective effect was eliminated after incubation with an NF-κB inhibitor. The identification of hornerin, a VEGF-independent protein important in tumor vessel development and endothelial survival, provides a potential therapeutic target to be used alone or in combination with existing VEGF inhibitor therapy.