Published OnlineFirst July 7, 2017; DOI: 10.1158/0008-5472.CAN-16-2704
Cancer Research
Therapeutics, Targets, and Chemical Biology
Posttranscriptional Regulation of PARG mRNA by HuR Facilitates DNA Repair and Resistance to PARP Inhibitors Saswati N. Chand1, Mahsa Zarei1, Matthew J. Schiewer2,3, Akshay R. Kamath1, Carmella Romeo1, Shruti Lal1, Joseph A. Cozzitorto1, Avinoam Nevler1, Laura Scolaro1, Eric Londin4,5, Wei Jiang5, Nicole Meisner-Kober6, Michael J. Pishvaian7, Karen E. Knudsen2,3, Charles J. Yeo1, John M. Pascal8, Jordan M. Winter1, and Jonathan R. Brody1
Abstract The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPRCas9–mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 30 untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-
ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR–PARG axis as an opportunity to enhance PARPi-based therapies. Cancer Res; 77(18); 5011–25. 2017 AACR.
Introduction
transmodifying other acceptor proteins (6). PARylated PARP-1 modulates chromatin dynamics, recruits key DNA damage repair factors, and contributes to multiple pathways of DNA strand break repair (7). Poly (ADP-ribose) glycohydrolase (PARG) is a critical DDR-related enzyme that works in concert with PARP-1 to coordinate the efficient repair of DNA lesions. Through exo- and endo-glycolytic activity, PARG removes PAR moieties from PARP-1 and other repair factors, and is critical for restarting replication forks and resolving DDR (8–10). Germline or somatic defects in such DDR and related genes (e.g., BRCA1/2, PALB2, and FA genes) render PDAC cells dependent on PARP-1 for homologous repair–driven repair, thereby making PARPi and platinumbased therapies promising strategies to treat a distinct subset of PDAC tumors (4, 7, 11). Despite the promise of PARPi therapies, most responsive tumors develop drug resistance (12, 13). Previous studies highlight adaptive resistance mechanisms such as genomic alterations and copy-number variations (e.g., BRCA2 reversion mutations; refs. 14, 15). However, genetic events selected for over time are unlikely to solely contribute to the acute plasticity required by cancer cells to rapidly adapt to anticancer agents (16). Beyond mutations, posttranscriptional gene regulation via RNA-binding proteins (RBP) is an adaptable reprogramming mechanism that may drive PARPi resistance. Our group has previously shown that the RBP, HuR [Hu antigen R; embryonic lethal abnormal visionlike 1 (ELAVL1)], promotes a drug-resistant phenotype, through its stress-induced cytoplasmic translocation and stabilization of prosurvival mRNA targets (17–20). Herein, we report for the first time that the antitumor response to several clinically relevant
Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer deaths in the United States (1, 2). PARP inhibitors (PARPi) are the best example of a personalized approach to treating PDAC with mutations in the BRCA2/Fanconi anemia (FA) pathway (3–5). The primary target, PARP-1, senses and initiates DNA damage repair (DDR) through automodification, by covalently adding poly (ADP-ribose) (PAR) onto itself and
1 Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania. 2Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania. 3 Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania. 4Computational Medicine Center, Thomas Jefferson University, Philadelphia, Pennsylvania. 5Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania. 6Novartis Institute for Biomedical Research, Basel, Switzerland. 7Division of Hematology and Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC. 8Department de Montre al, Montre al, of Biochemistry and Molecular Medicine, Universite Qu ebec, Canada.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Jonathan R. Brody, Thomas Jefferson University, Jefferson Medical College, 1015 Walnut Street, Curtis Bldg 618, Philadelphia, PA 19107. Phone: 215-955-2693; Fax: 215-923-6609; E-mail:
[email protected] doi: 10.1158/0008-5472.CAN-16-2704 2017 American Association for Cancer Research.
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Published OnlineFirst July 7, 2017; DOI: 10.1158/0008-5472.CAN-16-2704
Chand et al.
PARPi in PDAC is regulated by the HuR-dependent stabilization of PARG.
Materials and Methods Cell culture PDAC cell lines (MIA PaCa-2, PANC-1, Capan-1, Hs 766T, PL11) were purchased from the ATCC (2012). All cell lines were routinely tested for mycoplasma using the LookOut Mycoplasma PCR Detection Kit (MP0035 SIGMA), and only early passage (
