Rabbit Anti-Human YAP1 Polyclonal antibody

Mutation or promoter hypermethylation of CDKN2A is found in over 90% of pancreatic ductal adenocarcinomas (PDAC) and leads to loss of function of cell-cycle inhibitors p16 (INK4A) and p14 (ARF) resulting in unchecked proliferation. The CDK4/6 inhibitor, abemaciclib, has nanomolar IC(50)s in PDAC cell lines and decreases growth through inhibition of phospho-Rb (pRb), G1 cell-cycle arrest, apoptosis, and the senescent phenotype detected with beta-galactosidase staining and relevant mRNA elevations. Daily abemaciclib treatments in mouse PDAC xenograft studies were safe and demonstrated a 3.2-fold decrease in tumor volume compared with no treatment (P < 0.0001) accompanying a decrease in both pRb and Ki67. We determined that inhibitors of HuR (ELAVL1), a prosurvival mRNA stability factor that regulates cyclin D1, and an inhibitor of Yes-Associated Protein 1 (YAP1), a pro-oncogenic, transcriptional coactivator important for CDK6 and cyclin D1, were both synergistic with abemaciclib. Accordingly, siRNA oligonucleotides targeted against HuR, YAP1, and their common target cyclin D1, validated the synergy studies. In addition, we have seen increased sensitivity to abemaciclib in a PDAC cell line that harbors a loss of the ELAVL1 gene via CRISP-Cas9 technology. As an in vitro model for resistance, we investigated the effects of long-term abemaciclib exposure. PDAC cells chronically cultured with abemaciclib displayed a reduction in cellular growth rates (GR) and coresistance to gemcitabine and 5-fluorouracil (5-FU), but not to HuR or YAP1 inhibitors as compared with no treatment controls. We believe that our data provide compelling preclinical evidence for an abemaciclib combination-based clinical trial in patients with PDAC.

Background & Aims: In vitro, cell function can be potently regulated by the mechanical properties of cells and of their microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by regulating intracellular pathways, including the transcriptional coactivators YAP/TAZ. Whether mechanical cues are relevant for in vivo regulation of adult organ homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. Methods: We developed Capzb conditional knockout mice and obtained primary fibroblasts to characterize the role of CAPZ in vitro. In vivo functional analyses were carried out by inducing Capzb inactivation in adult hepatocytes, manipulating YAP/Hippo activity by hydrodynamic tail vein injections, and treating mice with the ROCK inhibitor, fasudil. Results: We found that the F-actin capping protein CAPZ restrains actomyosin contractility: Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small cellular geometry; in vivo, it induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. Conclusions: These results indicate a previously unsuspected role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated state and to regulate organ size. More generally, it indicates for the first time that mechanotransduction has a physiological role in maintaining liver homeostasis in mammals. Lay summary: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. Herein, we found that inactivation of the protein CAPZ alters the mechanical properties of cells and liver tissues, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals in the maintenance of adult liver homeostasis. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.