The role of the Hippo pathway in etiology and progression of pancreato-biliary tumors and clear cell renal cell carcinoma

Abstract

In the quest for the discovery of novel targets for cancer therapy, aberrantly reactivated embryonic signaling pathways have proven to be a rich mine and recent years have seen the introduction of the hedgehog-inhibitor cyclopamine into clinical practice. Dysfunctional signaling via the growth inhibitory embryonic signaling pathway clustered around the Hippo kinase and aberrant expression of its main target Yes-associated protein (YAP) is likewise emerging to be involved in maintenance and progression of various human cancers.<br /> The purpose of this dissertation is to identify the incidence, functional relevance and mechanistic significance of aberrant Hippo signaling in etiology and progression of different solid tumor entities that vary in their biology as well as tissue of origin, but share an unfavorable clinical prognosis and the dire need for novel therapeutic approaches. Specifically cancers of the pancreato-biliary tract, more precisely pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCC), as well as clear cell renal cell carcinoma (ccRCC) have been analyzed in the course of this dissertation. For these tumor entities initial evidence of aberrant Hippo signaling has been published.<br /> In a first step, aberrant expression of the transcriptional co-activator YAP, which constitutes the principal target of the growth inhibitory Hippo-pathway, was confirmed in human tumor tissue samples by immunohistochemistry. Nuclear YAP expression correlated with nodal stadium in PDAC and was also more frequent in ccRCC patients with tumor-positive lymph nodes. A common feature in all three tumor entities were solitary, fibroblast-like cells residing inside the tumor adjacent stroma that were highlighted by robust nuclear YAP-staining, suggesting that YAP might be a mediator of tumor-stroma crosstalk. <br /> In a second step, the role of aberrant YAP activity in tumor cell lines was examined by shRNA-mediated knockdown of the protein in selected, highly YAP-expressing cell lines. Phenotypic consequences of the knockdown included inhibition of cell proliferation as measured by the MTS-assay as well as impairment of colony formation in vitro for PDAC, CCC and ccRCC cell lines and decreased tumorigenicity in vivo for ccRCC cells in tumor xenograft experiments. However, shRNA-mediated YAP knockdown impaired in vitro cancer cell migration only in ccRCC cell lines.<br /> In a third and final step, the transcriptomic output of YAP in the three different tumor entities was compared by gene expression analysis of YAP-knockdown vs. mock transfected cancer cell lines. Although the phenotype of YAP-knockdown cells was functionally similar in PDAC, CCC and ccRCC cell lines, the transcriptional output of the Hippo-YAP axis as determined by transcriptomic profiling appeared to be fundamentally different and seemed to be highly tissue context-dependent. In fact, certain pathways were activated by YAP-knockdown in one cell line while they were inhibited in another (e.g. MAPK in PK9 v. MZ1774 cells). As no single, clearcut mechanism of YAP-mediated tumorigenicity could be observed, mechanistic considerations were made for each of the three tumor entities individually and feature CTGF and Wnt-signaling for pancreatic cancer as well as c-Myc and endothelins in ccRCC. The observation of solitary cells that exhibit robust nuclear YAP-expression was common in all three tumor entities analyzed, suggesting a role of Hippo-YAP signaling in tumor-stroma crosstalk which is certainly a highly interesting starting point for future research.