Abstract A32: The small GTPase Rac1 controls the stability of Yes-Associated Protein (YAP) independently of the LATS1/2 kinases

Activating mutations in the K-Ras gene are the earliest and most common genetic alterations detected in human pancreatic cancer (PC) specimens. In genetically engineered mouse models of PC, oncogenic K-Ras drives the formation of PanIN precursor lesions and their progression to invasive PC. Oncogenic K-Ras signals through at least four effector pathways that play distinct roles in malignant transformation: MAPK pathway, PI3K pathway, Ral-GDS, and the Rac1 GTPase (Ras-related C3 botulinum toxin substrate 1). Among these effectors, the least well understood is the Rac1 pathway. Yet, this pathway has now been shown to be critical for Ras-driven PC development and malignant transformation. However, the specific mechanisms by which Rac1 promotes anchorage-independent growth and tumorigenicity are still unclear. In recent studies, we have discovered that the Rac1 pathway regulates the stability of the Yes-Associated Protein (YAP). YAP is a transcriptional coactivator that controls the transcription of genes involved in proliferation and survival. In mouse models of Ras-driven PC, YAP is required for the formation of PanIN precursor lesions and their progression to invasive PC. In our telomerase-immortalized human pancreatic ductal cells, overexpressed YAP alone is sufficient to allow anchorage-independent growth. Our results show that in PC cells treated with Rac1 inhibitors (NSC23766, EHT-1864, AZA1, and Rac1T17N), YAP is rapidly and almost completely degraded within 24 hours. Importantly, this degradation of YAP is blocked by the preincubation of the cells with proteasome inhibitor MG132, thereby implying the involvement of a ubiquitin ligase complex. YAP contains a phosphodegron that controls its ubiqutination and subsequent degradation by the proteasome. Activation of this degron recognized by SCF-βTRCP ubiquitin ligase complexes requires its phosphorylation by the LATS1/2 kinases. To identify the molecular signals that control the degradation of YAP after Rac1 inhibition, Flag-tagged mutants of YAP were tested that were missing either a functional degron (ΔβTRCP mutant) or all of their LATS1/2 phosphorylation sites (5SA mutant). Our results show that while the functionality of the degron is required for YAP degradation after Rac1 inhibition, the phosphorylation of this degron by the LATS1/2 kinases is needed. Not only was the 5SA YAP mutant still degraded after Rac1 inhibition, but the knockdown of the LATS1/2 kinases also did not prevent the degradation of YAP. These findings suggest the existence of a novel SCF ubiquitin ligase complex that regulates YAP stability independently of the LATS1/2 kinases. The screening of a siRNA library has now been undertaken to identify the F-box protein involved. The proposed work is expected to shed light on novel mechanisms underlying the development of PC and of other Ras-driven malignancies. Citation Format: Chitra Palanivel, Bailey M. Gabler, Ying Yan, Surinder K. Batra, Michel M. Ouellette. The small GTPase Rac1 controls the stability of Yes-Associated Protein (YAP) independently of the LATS1/2 kinases [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A32.