A Novel Phd2/Vhl-Mediated Regulation Of Yap1 And Its Role In Vascular Mimicry And Tumor Angiogenesis

Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death worldwide. In this study, we show that transcriptional co-activator YAP1, the oncogenic component of the Hippo pathway, may contribute to the progression of NSCLC by supporting tumor angiogenesis and vascular mimicry of cancer stem-like cells (CSCs). NSCLC CSCs were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2); depletion of YAP1 inhibited the promoter activity as well as the mRNA expression of VEGF, KDR and AngPT-2, with a corresponding reduction in vascular mimicry as well as tumor growth in a mouse xenograft studies. These results suggest a possible mechanism by which YAP1 regulates tumor angiogenesis. A role for YAP1 in tumor angiogenesis was further supported by the finding that NSCLC cells grown in hypoxic conditions showed higher levels of YAP1. There was little to no changes in the canonical Hippo pathway proteins like LATS1/2, MST1/2, SAV and MOB as well as in the expression of TAZ, an orthologue of YAP1. Elevated YAP1 was found to associate with HIF1α under hypoxic conditions and enhance its transcriptional activity; YAP1 could increase HIF1α-mediated induction of the VEGF promoter confirmed by chromatin immunoprecipitations and transient transfection assays. Elevated levels of YAP1 and HIF1α interaction was detected in lung tumor tissues compared to normal lung tissue, as detected by proximity ligation assay (PLA), suggesting that the higher association of YAP1 with HIF1α and resulting transcriptional activity might have contributed to tumor growth. An examination of the underlying mechanism by which YAP1 levels are elevated under hypoxic conditions revealed a novel regulation of YAP1 protein by prolyl hydroxylase PHD2 and E3 ubiquitin ligase VHL, which are mainly known to regulate HIF1α under normoxia. PHD2 was found to hydroxylate proline residue(s) of YAP1 between aa 284 to aa 289 as seen by mutational studies. YAP1 was found to directly associate with PHD2 as well as with VHL. Depletion of PHD2 or treatment with DMOG, an inhibitor of prolyl hydroxylases, reduced YAP1 association with VHL ligase. This further elevated YAP1 levels in the nucleus. Interestingly, disruption of the YAP1-PHD2 interaction using a domain specific peptide enhanced the angiogenic tubule formation by endothelial cells. Our data therefore identifies a novel non-canonical pathway of regulation of YAP1 that supports angiogenesis and tumor growth. Citation Format: Namrata Bora Singhal, Biswarup Saha, Srikumar Chellappan. A novel PHD2/VHL- mediated regulation of YAP1 and its role in vascular mimicry and tumor angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2052.