Posttranscriptional Regulation of PARG mRNA by HuR Facilitates DNA Repair and Resistance to PARP Inhibitors

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 CRISPR-Cas9-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 3' 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.

Figure 1. HuR expression regulates sensitivity to PARPi in PDA cells

Cell survival of PDA cell lines (A), HuR-knockout CRIPSR cell lines, MIA PaCa-2 and Hs 766T [HuR(+/+) vs HuR(−/−)] (B) and HuR-silenced MiaPaCa-2 and Capan-1 cells (C) treated with increasing doses of olaparib for 7 days. (D) Representative images of MIA.HuR(+/+) vs MIA.HuR(−/−) and HST.HuR(+/+) vs HST.HuR(−/−) cells seeded and cultured in soft agar in the presence of respective IC₅₀ doses of olaparib for 4 weeks. (E) HuR expression in MIA PaCa-2 cells treated with indicated IC₅₀ doses of PARPi for 12hr, and fractionated as indicated. Lamin A/C and α-Tubulin used as controls to determine the integrity of nuclear and cytosolic lysates respectively. Mitomycin C used as positive control for cytoplasmic translocation of HuR. (F) Immunofluorescent images of HuR (green) in MIA PaCa-2 cells treated with PARPi for 12hr. Nuclei were stained with DAPI. Magnification 40X.

Figure 2. HuR regulates PARG mRNA expression

(A) mRNP-IP assay performed with cytoplasmic fraction of MIA PaCa-2 cells treated with IC₅₀ doses of veliparib (12μM) and olaparib (9μM) for 12hr, α-Tubulin used as a loading control for the input and a negative control for the IP samples, Lamin A/C used as a control to detect nuclear contamination in the input. (B) The relative binding of PARG mRNA to HuR, normalized to respective IgG controls, as determined by RT-qPCR using 18S rRNA as a loading control, dCK as positive control and PARP-1 as negative control. (C) HuR- silenced MIA PaCa-2 cells were treated with actinomycin D (5 μg/ml) for the indicated times. PARG, GAPDH and PARP-1 mRNA stability was assayed by RT- qPCR using 18S rRNA as a loading control. (D) RT- qPCR indicating HuR and PARG mRNA expression in HuR-silenced MIA PaCa-2 cells incubated in the presence of olaparib for 24hr. (E) PARG expression in DDR- P MIA PaCa-2 and DDR- D Capan-1 and Hs 766T cells treated with veliparib for indicated time points.

Figure 3. HuR regulates PARG protein expression and function

Luciferase activity in MIA PaCa-2 cells co-expressing a luciferase reporter construct with PARG 3′UTR and (A) HuR overexpression or (B) HuR silencing (C) HuR, PARG and PAR protein expression in total lysates from HuR- and PARG-silenced MIA PaCa-2 cells treated with IC₅₀ doses of indicated PARPi for 24hours, using α-Tubulin as a loading control. (D) ELISA indicating relative PARylation in MIA PaCa-2 cells transfected and treated as above. The indicated fold changes are means of three independent experiments, normalized to control transfected sample under no treatment (NT). (E) DSBs assessed by immunofluorescence staining for γH2AX (green) in MIA PaCa-2 cells transfected and treated as described above. (F) DNA damage foci were quantified and plotted ± SD.

Figure 4. HuR and PARG inhibition enhances PARPi-induced apoptosis and PARP-1 trapping on chromatin and increases PARPi efficacy

(A) Relative number of apoptotic cells quantified and normalized to control- (NT) MIA PaCa-2 and Hs 766T cells. A 3hr treatment with soluble TNF-related apoptosis-inducing ligand (sTRAIL) is used as a positive control. (B) HuR- and PARG- silenced DDR- P MIA PaCa-2 cells treated with IC₅₀ doses of indicated PARPi for 6h were harvested and fractionated to isolate soluble and chromatin- tethered proteins. HuR, PARG, PARP-1 and PAR expression analyzed, with GAPDH (total protein extract) and Histone H3 (nuclear chromatin- tethered fraction) as the loading controls. A representative image of one of three independent experiments is shown. (C) Cell survival in HuR- and PARG- silenced MIA PaCa-2 cells were treated with increasing doses of olaparib and veliparib for 5 days.

Figure 5. PARG overexpression rescues HuR’s regulation of PARPi response

(A) Cell survival of MIA PaCa-2 cells co-transfected with HuR siRNA and PARG overexpression plasmid and treated with olaparib for 7days. (B) PARG rescue and HuR, PARP-1 and PAR expression validated, with GAPDH (total protein extract) and Histone H3 (nuclear chromatin- tethered fraction) as the loading controls. (C) ELISA to quantitate relative PARylation with PARG rescue in HuR silenced MIA PaCa-2 cells. (D) Immunofluorescence of HuR (green) in MIA PaCa-2 cells treated with veliparib for 12hr, with or without a 6hr pre-treatment of small-molecule HuR inhibitor, MS-444. Nuclei stained with DAPI (blue). Magnification 40X. (E) Relative PARylation and immunoblotting of total protein lysates of MIA PaCa-2 cells treated with increasing dosage of MS-444, in the presence of veliparib for 12h. (F) Cell survival of MIA PaCa-2 and Capan-1 cells treated with indicated doses of veliparib, with or without 5μM/L MS-444. (G) Luciferase activity in MIA PaCa-2 cells transfected with luciferase reporter constructs with PARG 3′UTR and incubated in the presence of MS-444 for 24 hr.

Figure 6. HuR silencing in vivo enhances olaparib- mediated suppression of PDA xenograft growth

Mia.shHuR xenografts in athymic, nude mice were randomized into DOX and olaparib treatment groups. (A) Tumor volumes are plotted, with each point representing the mean ± 2SE of each group, *P<0.05. Inset shows differences in number of duplications. (B) Representative image of mice and tumor per group. (C) Tumor duplication time (days) per group (D) HuR, PARG and PARP-1 mRNA expression in extracted tumors, relative to vehicle- treated –DOX group. Each bar represents the mean ± SEM (n = 3 per group). (E) HuR protein expression when tumors were harvested (day 36, n=3). (F) Working model: In response to PARPi stress, cytoplasmic HuR binds to and stabilizes PARG mRNA, thereby increasing PARG expression and modulating PARP1-chromatin dynamics. HuR and PARG inhibition breaks such acute resistance by enhancing chromatin- trapped PARP-1 and accumulation of damaged DNA and apoptosis.