430 A non-transcriptional role of the oncoprotein YAP in cutaneous squamous cell carcinoma

Keratinocyte cancers (KCs) are the most frequently diagnosed cancers worldwide. Basal cell carcinomas and cutaneous squamous cell carcinomas (cSCC) account for approximately 80% and 20% of cases of KCs, respectively. The paralogous transcriptional regulators YAP and TAZ have emerged as essential drivers of cSCC development and progression, where they execute transcriptional programmes that promote cell proliferation and survival, and epithelial-to-mesenchymal transition. How YAP/TAZ execute their various downstream transcriptional programmes in cSCC has remained an unresolved question. Using a technique called RIME (Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins) we have identified the nuclear interactome of chromatin-associated YAP in a cSCC cell line that is transcriptionally addicted to YAP for cell proliferation and survival. Of the proteins identified, Rif1 – a protein implicated in DNA double-strand break repair and DNA replication – was selected for further validation using co-immunoprecipitation and in-situ proximity ligation assay (PLA) and was confirmed to be a novel YAP interactor. Rif1 knockdown in cSCC cells perturbed cell cycle progression and induced replication stress. RNA sequencing following siRNA-mediated YAP/TAZ knockdown revealed decreased mRNA expression of RIF1. Moreover, using cSCC cell lines expressing YAP-targeting shRNAs, Rif1 protein expression was found to be decreased upon YAP knockdown. These findings suggest that YAP does not only interact with Rif1 on chromatin, but also transcriptionally regulates the expression of Rif1. Upon treatment with the replication stress-inducing chemotherapeutic hydroxyurea, protein levels of YAP and RIF1 and YAP-Rif1 interaction (as assessed by PLA) were found to increase, indicating that YAP and Rif1 might co-operate in response to replication stress. In summary, our work shows that in cSCC cells, YAP interacts with and regulates the expression of Rif1, a key regulator of DNA replication and DNA damage repair, and thus highlights novel and yet unexplored functions of YAP in cancer outside of transcriptional regulation.