This fundamental paradigm shift is currently being more and more commonly adapted and is finding its way into basic understanding of CSF-1R inhibitor cancer research as well as into everyday routine clinical applications in the field of medical oncology [17] and [18]. To identify signaling pathways potentially affected by altered signaling through the Hippo/warts axis, signaling pathway impact analysis was performed as previously described elsewhere [14]. Of note, the top three pathways found to be affected were the p53, MAP kinase, as well as cell cycle progression pathways, all of which have long been well established

as centrally involved in carcinogenesis and maintenance of a malignant phenotype across several tumor entities (Table 4). These findings thus further support our hypothesis that Hippo signaling might be a crucial driver of carcinogenesis and represents a promising potential therapeutic target in ccRCC. Among the most prominently downregulated genes were two members of the endothelin family, EDN1 and EDN2, VE-821 as well as c-Myc. Cross-validation of mRNA expression of these genes in MZ1774, A498, and ACHN YAP knockdown cells confirmed

significant c-Myc and EDN1 down-regulation in MZ1774 and A498 on YAP knockdown (MZ1774: fold changes = 0.34 ± 0.006, P < .0001 for c-Myc and 0.41 ± 0.009, P < .0001 for EDN1; A498: fold changes = 0.79 ± 0.026, P = .0085 for c-Myc and 0.41 ± 0.019, P

= .0001 for EDN1, respectively; see Figure 5B). EDN2 expression was significantly reduced in all three cell lines examined (fold changes = 0.06 ± 0.003, P < .0001 for MZ1774, 0.62 ± 0.025, P = .001 for A498, and 0.17 ± 0.0067, P < .0001 for ACHN, respectively). Of note, immunohistochemistry and real-time RT-qPCR confirmed consistent knockdown of YAP1 as well as down-regulation of EDN2, both at the mRNA and protein levels, respectively, in murine xenografts of human ccRCCs as well (Figure 6, C and D). To investigate a direct relationship between YAP and its putative target genes in ccRCC, we performed ChIP-qPCR on selected ID-8 regions containing TEAD-binding motifs within the promoter region of those genes (Figure 5C). A well-characterized YAP/TEAD1-binding site in the promoter region of the bona fide YAP target gene CTGF was selected as a positive control. We found YAP and TEAD to be simultaneously present on the promoter regions of the MYC, EDN1, as well as EDN2 genes in MZ1774 ( Figure 5D). We next analyzed the expression of the thus identified proposed downstream effector of YAP, EDN2, in primary tissue samples of human ccRCC tumors using immunohistochemistry. YAP expression was found to significantly correlate with positivity for EDN2 (P = .0067; Table 5).