Castiglioni, Ilaria (2014) Development of drug screening assays for identification of new molecules against pancreatic ductal adenocarcinoma. PhD thesis, University of Trento.
|PDF - Doctoral Thesis|
The yes-associated protein, YAP, is a transcriptional co-activator, able to regulate the expression level of a wide range of target genes. Despite deregulation of YAP has been associated with cancer etiology, no compounds are known to specifically modulate its functions. Here we identified an inhibitor of YAP functionality, called GF 10923X, and we proposed it as a potential lead molecule to be used against disease where YAP is found deregulated, pancreatic ductal adenocarcinoma (PDAC) included. GF 109203X is an already known kinase inhibitor, preferentially targeting PKCα. We developed a high throughput approach by which we identified this compound able to reduce YAP-induced proliferation and clonogenicity of PDAC cellsin vitro, despite it leads to increased YAP nuclear levels. The Hippo pathway is the main inhibitor of YAP. One component of this signaling cascade,Lats1/2, phosphorylates YAP at Serine 127, thereby promoting its cytosolic retention and degradation.In line with YAP nuclear retention after the treatment, we observed that GF 109203X caused an increase of both acetylations and phosphorylations on YAP protein, with the exception of P-S127, suggesting a Hippo pathway-independent mechanism of action of the identified compound. TEAD is the major transcriptional factor partner in the functional activity of YAP and CTGF is considered one of the main representative target genes whose expression is regulated by this couple of transcriptional regulators.Chromatin immunoprecipitation experiments allowed us to demonstrate that GF 109203X interferes with YAP binding to CTGF promoter, without affecting the presence of TEAD at the same region. This inhibition is responsible of CTGF downregulation during GF 109203X administration and it can explain the phenotypic effects we observed. These effects, associated with the observed toxicity in pancreatic cancer cells but not in immortalized HPNE cell lines, make GF 109203X a potential lead compound to be used in drug development for PDAC treatment.
|Item Type:||Doctoral Thesis (PhD)|
|Doctoral School:||Biomolecular Sciences|
|Subjects:||Area 05 - Scienze biologiche|
|Funders:||[FP7-2007-2013] No. HEALTH No. F2-2011-256986 (A.P., G.F. A.N:)|
|Repository Staff approval on:||29 Apr 2014 11:52|
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