Novel Role Of Yap1 In Tumor Angiogenesis And Vascular Mimicry

Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that cancer stem-like cells (CSCs) contribute to the genesis and progression of NSCLC. These cells have been shown to form differentiated tumor cells upon receiving appropriate micro-environmental cues and can give rise to multiple components of the tumor, including the vasculature. We isolated the CSCs from NSCLC cell lines and PDX tumors based on the side population (SP) phenotype. SP cells displayed high self-renewal capacity, were highly drug resistant, and could form metastatic tumors in immunocompromised mice. The present study shows that the transcriptional co-activator YAP1, which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and the ability to form angiogenic tubule-like structures in matrigel, a unique feature of cancer stem-like cells that is also called as vascular mimicry. Inhibition of YAP1 by a small molecule inhibitor Visudyne or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of SP cells. The stem -like SP cells from NSCLC were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2) which are crucial genes during angiogenic tubule regression and growth in addition to YAP1. Further, depletion of YAP1 with siRNAs reduced the expression of VEGF, KDR and AngPT-2 mRNA levels. Overexpression of YAP1 resulted in increase in the promoter activity of both KDR and AngPT-2 in transient transfection experiments. This suggests that YAP1 might play a role in regulating VEGF, KDR and AngPT-2 mediated angiogenic functions in cancer cells. In addition to vascular mimicry, YAP1 appears to play a unique role in regulating the expression of genes involved in angiogenesis. Hypoxia is known to contribute towards angiogenesis as well as cancer progression and metastasis. Preliminary experiments showed that cells grown in hypoxic conditions or treated with hypoxia mimetic compounds like DMOG resulted in increase in YAP1 mRNA and protein expression. However, such an increase was not observed in YAP1 orthologue, TAZ or other canonical Hippo pathway proteins. Hypoxia-inducible factor-1alpha (HIF-1α) is the key transcription factor that regulates the expression of various hypoxia response genes like VEGF. We find that YAP1 directly interacts with HIF-1α as detected by co-immunoprecipitation experiments and proximity ligation assays (PLA). YAP1 also associated with VEGF promoter as seen in ChIP RT-PCRs, and this interaction was elevated under hypoxic environment. Our data suggest a distinct role for YAP1 in regulating hypoxia response, promoting tumor angiogenesis and vascular mimicry. Citation Format: Namrata Bora Singhal, Srikumar Chellappan. Novel role of YAP1 in tumor angiogenesis and vascular mimicry. [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 2496.