Novel Pharmacological Targets for NF2-Mediated Yap/Taz Signaling
Annual summary 1 Sep 2011-31 Aug 2012
CHILDREN'S HOSPITAL CORP BOSTON MA
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Mutations within the Nf2 gene, encoding the protein product Merlin, are the primary cause of NF2, however the precise mechanism by which Merlin promotes cellular proliferation and tumor formation is unclear. Recent studies have shown that Nf2-mediated cellular proliferation is a consequence of aberrant signaling of the Hippo pathway. Activation of the Hippo pathway via a series of phosphorylation events leads to the inactivation and phosphorylation of Yap, the downstream transcriptional coactivator of the TEAD family of transcription factors. In the absence of Merlin, Yap translocates to the nucleus where it binds to TEADs to promote pro-proliferative and anti-apoptotic programs. Yap and Merlin interact indirectly, however the direct binding partners linking Yap and Merlin have not been discovered to date. Thus, the identification of putative binding partners and novel downstream targets of Nf2-mediated Hippo pathway activation is of interest. The primary objective of this proposal was to identify novel downstream targets of Nf2-mediated cell proliferation in the context of Hippo signaling. Since Yap is a downstream target of Merlin, I plan to identify therapeutic targets that lie between the Merlin and Yap signaling cascade that could result in the inhibition Merlin-mediated cell proliferation using a reporter-based high-throughput genome-wide RNAi screen in a human schannoma cell line, HEI-193. Since the TEAD family of transcription factors are essential in mediating Yap-dependent gene expression we developed DNA constructs that carry multimerized copies of a TEAD-DNA binding site TBS upstream of a promoter driving the expression of luciferase or mCherry reporter. The dynamic range of the reporter indicates that we are able to reliably reproduce a 15-fold induction of the reporter by transfection of constitutively active Yap1 YapS127A or siRNA knockdown of NF2.
- Medicine and Medical Research