Inhibiting cytoplasmic accumulation of HuR synergizes genotoxic agents in urothelial carcinoma of the bladder

HuR, an RNA-binding protein, post-transcriptionally regulates nearly 4% of encoding proteins implicated in cell survival. Here we show that HuR is required for the efficacy of chemotherapies in urothelial carcinoma of the bladder. We identify pyrvinium pamoate, an FDA-approved anthelminthic drug, as a novel HuR inhibitor that dose-dependently inhibited cytoplasmic accumulation of HuR. Combining pyrvinium pamoate with chemotherapeutic agents (e.g. cisplatin, doxorubicin, vincristine and oxaliplatin) not only led to enhanced cytotoxicity in bladder cancer cells but also synergistically suppressed the growth of patient-derived bladder tumor xenografts in mice (P < 0.001). Mechanistically, pyrvinium pamoate promoted nuclear import of HuR by activating the AMP-activated kinase/importin α1 cascade and blocked HuR nucleo-cytoplasmic translocation by inhibiting the checkpoint kinase1/cyclin-dependent kinase 1 pathway. Notably, pyrvinium pamoate-additive treatment increased DNA double-strand breaks as indicated by elevated γH2AX expression, suggesting an involvement of DNA damage response. We further found that pyrvinium pamoate dramatically downregulated several key DNA repair genes in genotoxically-stressed cells, including DNA ligase IV and BRCA2, leading to unbearable genomic instability and cell death. Collectively, our findings are the first to characterize a clinical HuR inhibitor and provide a novel therapeutically tractable strategy by targeting cytoplasmic translocation of HuR for treatment of urothelial carcinoma of the bladder.


HuR knockout cell lines
The SpCas9 targeting vector lentiCRISPR v2 was obtained from Addgene (Cambridge, MA). HuR sgRNA (ACCACATGGCCGAAGACTGC) was designed using the Optimized CRISPR Design Tool (Zhang lab, MIT). Lentiviruses were produced by co-transfection of lentiviral backbone constructs (lentiCRISPR v2) and packaging vectors (pMD2.G and pSPAX2; Addgene) into 293T cells. Supernatant was collected after 48 h post-transfection and consequently added to bladder cancer cells in a 6-well plate. Infected cells were selected with puromycin (Sigma). Single cells with HuR frame-shift mutations were selected.

Primary bladder tumor xenograft mouse models
The mice used in the present study were purchased from National Rodent Laboratory Animal Resources (Shanghai, China). Animals were caged in groups of 5 in a laminar airflow cabinet under specific pathogen-free conditions, fed with sterilized food and water and kept on a 12-hour light/dark cycle. All treatments were administered according to the guidelines of Institution Animal Care and Use Committee and all the protocols were approved by East China Normal University.
For establishment of UCB patient-derived xenograft mouse model, fresh tumor samples from 23 bladder cancer patients were obtained from the Shanghai Changhai Hospital (Shanghai, China). Prior written informed consent was obtained from all patients, and the study protocol was approved by the local hospital ethics committee. In brief, surgically removed tumor tissues were cut into fragments (approximately 2 mm 3 ) and implanted subcutaneously into the flanks of male nude mice. When tumor volume reached approximately 1000-1500 mm 3 , primary xenografts (designated as P0) at exponential growth phase were removed by serial passage to other immunodeficient mice (designated as P1 to P6). Histological morphology of two primary UCB tumor xenograft models was characterized by hematoxylin and eosin staining, and recurrent UCB mutations were sequenced as well. In this study, UCBPDX0615 model (P4; pT1 stage; TP53, FGFR3, PIK3A mutation) and UCBPDX0826 model (P3; pT2b stage; TP53, FGFR3, HRAS mutation) were applied to drug treatments.
Primary tumor-bearing mice with an average tumor volume reaching approximately 200 mm 3 were randomly divided into the following groups (n = 8-12 each group): vehicle control (phosphate-buffered saline; i.p.; daily); pyrvinium pamoate (0.8 mg/kg; i.p.; daily); cisplatin (4 mg/kg; i.p.; weekly); or a combination of pyrvinium pamoate and cisplatin at the same dosages as single treatments. All groups were continuously treated for 4 weeks, and the body weight was measured every other day. Tumor size was evaluated by caliper measurements, and the approximate volume of the tumor mass was calculated using the following formula: V = (L × [W] 2 ) × 0.52; where L is the longest diameter of the tumor; and W is the shortest diameter of the tumor. At the end of the experiment, solid tumors were removed, weighed and processed for immunohistochemistry and immunofluorescence.

Immunoprecipitation assay and Western blotting assay
For immunoprecipitation assay, 5637 cells were co-transfected with flag-HuR and myc-importin α1. Meanwhile, cells co-transfected with pcDNA3.0-5ʹflag and myc-importin α1 vectors served as negative controls. Two days after transfection, cells were exposed to indicated treatments followed by lysed with EBC buffer (20 mmol/L Tris-HCl, 125 mmol/L NaCl, 2 mmol/L EDTA, and 0.5% NP-40) supplemented with protease inhibitors. Cell lysates were consequently incubated with protein A-conjugated sepharose beads and the anti-flag M2 agarose beads at 4°C with rotation. Beads were boiled with SDS sample buffer and subjected to Western blotting assays.
For Western blotting assays, the whole-cell extracts were prepared in RIPA buffer. Approximately 40-50 μg of cellular protein from each treatment was applied to 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Millipore, Billerica, MA). Membranes were incubated overnight with primary antibodies followed by incubation with fluorescent secondary antibodies. After several washes, the signals were detected by the Li-Cor Odyssey Infrared system (LI-COR Biosciences, Lincoln, NE).

Immunofluorescence and Immunohistochemistry
Treated cells were plated onto gelatin-coated coverslips and fixed with 3.7% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked with 1% bovine serum albumin for 1 h. Cells were then subjected to specific primary and secondary antibodies. Phalloidin and 4, 6-diamidino-2phenylindole (Thermo Scientific, Grand Island, NY) were further used to stain cytoskeleton and nuclei. The images were acquired using a laser confocal microscope (Zeiss Axiovert 200M LSM510).
The expression of HuR and γH2AX in treated tumors was detected by immunohistochemistry and immunofluorescence, respectively. Primary tumors were fixed, embedded in paraffin and were sectioned (4-μm). Randomly selected slides were stained with HuR and γH2AX antibodies following to standard protocols. Images were obtained with Leica microscope (Leica, DM4000b).