HuR Plays a Role in Double-Strand Break Repair in Pancreatic Cancer Cells and Regulates Functional BRCA1-Associated-Ring-Domain-1(BARD1) Isoforms.

Abstract

Simple SummaryRNA binding proteins (RBPs) post-transcriptionally regulate and stabilize a variety of target mRNA transcripts. Human Antigen R (HuR) binds to several pro-survival mRNA transcripts and promotes tumor cell survival and chemotherapeutic resistance. Some of these targets are within the DNA damage repair pathway that help cells repair damaged DNA and allow them to proliferate. Homologous recombination repair is the preferred method of DNA repair in the cells. Here, we aim to understand if HuR regulates homologous recombination repair in pancreatic ductal adenocarcinoma (PDAC). Our analysis, using Ribonucleo-protein Immunoprecipitation (RNP-IP) coupled with microarray and RNA-sequencing studies, revealed that HuR binds BARD1 (BRCA-1 Associated Ring Domain 1) and regulates expression of BARD1 mRNA full length transcripts and alternative isoforms. Silencing BARD1 mRNA potentiated the DNA damage effects of clinically relevant drugs, olaparib and oxaliplatin, in PDAC cells. Together, this work underscores a transient regulatory DNA repair pathway (i.e., HuR—BARD1 mRNA), which is likely a PDAC therapeutic resistance mechanism.Human Antigen R (HuR/ELAVL1) is known to regulate stability of mRNAs involved in pancreatic ductal adenocarcinoma (PDAC) cell survival. Although several HuR targets are established, it is likely that many remain currently unknown. Here, we identified BARD1 mRNA as a novel target of HuR. Silencing HuR caused a >70% decrease in homologous recombination repair (HRR) efficiency as measured by the double-strand break repair (pDR-GFP reporter) assay. HuR-bound mRNAs extracted from RNP-immunoprecipitation and probed on a microarray, revealed a subset of HRR genes as putative HuR targets, including the BRCA1-Associated-Ring-Domain-1 (BARD1) (p < 0.005). BARD1 genetic alterations are infrequent in PDAC, and its context-dependent upregulation is poorly understood. Genetic silencing (siRNA and CRISPR knock-out) and pharmacological targeting of HuR inhibited both full length (FL) BARD1 and its functional isoforms (α, δ, Φ). Silencing BARD1 sensitized cells to olaparib and oxaliplatin; caused G2-M cell cycle arrest; and increased DNA-damage while decreasing HRR efficiency in cells. Exogenous overexpression of BARD1 in HuR-deficient cells partially rescued the HRR dysfunction, independent of an HuR pro-oncogenic function. Collectively, our findings demonstrate for the first time that BARD1 is a bona fide HuR target, which serves as an important regulatory point of the transient DNA-repair response in PDAC cells.