Abstract B45: A feedback gene regulatory mechanism between YAP1 and the RNA-binding protein, Human Antigen R (HuR), in pancreatic cancer cells: Implications for a context-dependent pancreatic cancer cell survival network
Authors: Brown Samantha Z. , Schultz Christopher W. , Nevler Avinoam , Li Tianyun , Jain Aditi , O’Neil Raymond , Jiang Wei , Londin Eric , Dixon Dan A. , Xu Liang , Yeo Charles J. , Brody Jonathan R. 
Published: September 2020

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 preinvasive neoplasms (PanINs) to late-stage ductal adenocarcinoma. Two important facilitators of KRAS function, Human Antigen R (HuR) and Yes-associated protein 1 (YAP1), are both highly overexpressed in PDAC. HuR is an RNA-binding protein that facilitates gene expression through the stabilization and increased translation of target prosurvival mRNAs upon stress. YAP1 is a transcriptional coactivator, which associates with a number of transcription factor families to sense and upregulate targets that lead to tumor growth and cellular crosstalk. While the functions of HuR and YAP1 are well known, the events that lead to their overexpression and regulation are poorly understood.

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.

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 mRNA 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. Impact of YAP1 transcriptional activity was evaluated both by measuring total expression of canonical YAP targets (i.e., CTGF and CYR61) and by using a TEAD reporter construct (i.e., 8xGTIIC). Surprisingly, we found that RNA silencing of YAP1 significantly reduced HuR mRNA and protein expression, as well as established 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 cloning HuR’s promoter region into a luciferase reporter in order to evaluate the impact of YAP1 on HuR transcriptional expression.

Citation Format: Samantha Z. Brown, Christopher W. Schultz, Avinoam Nevler, Tianyun Li, Aditi Jain, Raymond O’Neil, Wei Jiang, Eric Londin, Dan A. Dixon, Liang Xu, Charles J. Yeo, Jonathan R. Brody. A feedback gene regulatory mechanism between YAP1 and the RNA-binding protein, Human Antigen R (HuR), in pancreatic cancer cells: Implications for a context-dependent pancreatic cancer cell survival network [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 B45.