Abstract
The Yes-associated protein, YAP, is a transcriptional co-activator, mediating the Epithelial to Mesenchymal Transition program in pancreatic ductal adenocarcinoma (PDAC). With the aim to identify compounds that can specifically modulate YAP functionality in PDAC cell lines, we performed a small scale, drug-based screening experiment using YAP cell localization as the read-out. We identified erlotinib as an inducer of YAP cytoplasmic localization, an inhibitor of the TEA luciferase reporter system and the expression of the bona fide YAP target gene, Connective Tissue Growth Factor CTGF. On the other hand, BIS I, an inhibitor of PKCδ and GSK3β, caused YAP accumulation into the nucleus. Activation of β-catenin reporter and interfering experiments show that inhibition of the PKCδ/GSK3β pathway triggers YAP nuclear accumulation inducing YAP/TEAD transcriptional response. Inhibition of GSK3β by BIS I reduced the expression levels of SMADs protein and reduced YAP contribution to EMT. Notably, BIS I reduced proliferation, migration and clonogenicity of PDAC cells in vitro, phenocopying YAP genetic down-regulation. As shown by chromatin immunoprecipitation experiments and YAP over-expressing rescue experiments, BIS I reverted YAP-dependent EMT program by modulating the expression of the YAP target genes E-cadherin, vimentin, CTGF and of the newly identified target, CD133. In conclusion, we identified two different molecules, erlotinib and BIS I, modulating YAP functionality although via different mechanisms of action, with the second one specifically inhibiting the YAP-dependent EMT program in PDAC cell lines.
Highlights
The Yes-associated protein, YAP, is a transcriptional co-activator containing a proline-rich region responsible for the interaction with SH3 domains of c-Yes and many other proteins [1]
In pancreatic ductal adenocarcinoma (PDAC) cell lines cultured at high-density, YAP is partially redistributed in the cytoplasm, it has a transcriptional effect controlling the expression of known target genes, it regulates proliferation and the ability of PDAC cells to grow in anchorage-independent conditions
We show that BIS I inhibits Epithelial to Mesenchymal Transition (EMT) in PDAC cell lines triggering the expression of epithelium markers by down-regulating SMAD2/3 and blunting YAP co-transcriptional activity
Summary
The Yes-associated protein, YAP, is a transcriptional co-activator containing a proline-rich region responsible for the interaction with SH3 domains of c-Yes and many other proteins [1]. Multiple post-translational modifications (PTMs) regulate the functions of YAP. The Hippo signaling pathway, initially defined as a tissue growth and organ size regulator in Drosophila, is a kinase cascade able to negatively regulate YAP localization and activity, by phosphorylating YAP at Serine127. Phosphorylation of YAP by the Hippo pathway leads to its accumulation in the cytoplasm and, by interaction with 14–3–3 proteins, YAP is degraded by a ubiquitinationdependent proteasomal process. The Hippo pathway negatively regulates YAP functionality and presence in the nucleus by modulating its cell distribution and its protein expression levels too. The Hippo pathway-induced phosphorylation of YAP rules its functionality according to cell density.
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