Abstract 4388: A novel, reversible YAP1-HuR axis promotes pancreatic tumorigenesis

Abstract

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death in the U.S. In roughly 95% of cases, gain-of-function mutations in KRAS combine with loss of tumor suppressors to progress pre-invasive neoplasms (PanINs) to late-stage ductal adenocarcinoma. Two important mediators of KRASfunction, Human Antigen R (HuR) and Yes-associated protein 1 (YAP1), are both highly overexpressed in PDAC. HuR is a RNA-binding protein that facilitates gene expression through the stabilization and increased translation of target pro-survival mRNAs upon stress. YAP1 is a transcriptional co-activator, which associates with a number of transcription factor families to sense and upregulate targets that lead to tumor growth and cellular cross-talk. While the functions of HuR and YAP1 are well known, the events that lead to their overexpression and regulation are poorly understood. YAP1 was first identified as a HuR target via ribonucleoprotein-immunoprecipitation assays in which HuR-bound mRNAs were run on a whole-transcriptome microarray. YAP1 mRNA was significantly bound to HuR as compared to the IgG isotype control (13.2-fold), and was in line with previously established targets (WEE1, 3.2-fold; PIM1, 13.9-fold). YAP1 mRNA bound to HuR is abolished when treated with a known HuR inhibitor, pyrvinium pamoate, even in the presence of an established HuR stressor (i.e., oxaliplatin). Actinomycin D chase assays demonstrated that YAP1 mRNA stability is significantly dependent on HuR proficiency. We validated that both YAP1 mRNA and protein expression levels are dependent on HuR via real-time quantitative PCR and Western blot analysis. Transcription of downstream YAP1 targets, CTGF and CYR61, was also significantly affected by knockdown of HuR. Surprisingly, we found that RNA silencing of YAP1 significantly reduced HuR mRNA and protein expression, as well as HuR targets, WEE1 and PIM1. Treatment with small-molecule inhibitors, Verteporfin and CA3, which target the interface of YAP1’s transcription-factor binding domain, recapitulated these effects in a dose and time-dependent manner. We are currently using chromatin immunoprecipitation sequencing to identify HuR’s promoter/enhancer domains for YAP1-mediated transcription. Our previous work has shown that overexpression of cytoplasmic HuR correlates strongly with tumor staging. Low-levels of HuR correspond to early PanINs with staining steadily increasing in late-stage PanIN lesions and gross overexpression in invasive adenocarcinoma. Conversely, YAP1 overexpression seems to be most critical for initial development and expansion of the tumor cells, while it converts to a maintenance role once PDAC is fully developed. Ongoing studies will address whether the temporal regulation of these proteins could explain their overexpression patterns in pancreatic pathologic stages as they relate to cooperating with KRAS activity. Citation Format: Samantha Z. Brown, Christopher W. Schultz, Tianyun Li, Aditi Jain, Raymond O'Neill, Wei Jiang, Charles P. Yeo, Jonathan R. Brody. A novel, reversible YAP1-HuR axis promotes pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4388.