The deubiquitinating enzyme USP5 promotes pancreatic cancer via modulating cell cycle regulators

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors. With an overall five-year survival rate remaining below 6%, there is an explicit need to search for new molecular targets for therapeutic interventions. We undertook a barcode labelled short-hairpin (shRNA) library screen in pancreatic cancer cells in order to identify novel genes promoting cancer survival and progression. Among the candidate genes identified in this screen was the deubiquitinase USP5, which subsequent gene expression analyses demonstrated to be significantly upregulated in primary human pancreatic cancer tissues. Using different knockdown approaches, we show that expression of USP5 is essential for the proliferation and survival of pancreatic cancer cells, tested under different 2D and 3D cell culture conditions as well as in in vivo experiments. These growth inhibition effects upon knockdown of USP5 are mediated primarily by the attenuation of G1/S phase transition in the cells, which is accompanied by accumulation of DNA damage, upregulation of p27, and increased apoptosis rates. Since USP5 is overexpressed in cancer tissues, it can thus potentially serve as a new target for therapeutic interventions, especially given the fact that deubiquitinases are currently emerging as new class of attractive drug targets in cancer.


Barcode labelled shRNA based dropout screen Cell culture and Screening conditions
The pancreatic cancer cell line BxPC-3 was obtained from ATCC (Rockville, MD, USA). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Darmstadt) supplemented with 10% foetal calf serum (Life Technologies, Darmstadt) and 1% penicillin-streptomycin (Life Technologies, Darmstadt).
We targeted 10,000 genes for shRNA-mediated knockdown using modules 1 and 2 of the Decipher library (Cellecta, Mountain View, CA, USA) in two screens under identical conditions. The majority of genes were targeted by two or more individual shRNA expression constructs (average: five), which are identifiable by barcode sequences. The target cell line BxPC-3 was infected with lentiviral particles containing either module 1 or 2 at a multiplicity of infection of 0.3. Positively infected cells were selected by addition of 0.5 μg/ml puromycin 24 hours after infection. Selection took place for 48 hours, after which puromycin was removed and cells were allowed to recover for another 48 hours. A sample of 20 million cells was taken for the t 0 reference time point, shock-frozen and kept at −80°C. Cells were kept under selective pressure of 0.5 μg/ml puromycin, which was added after cells had attached to the substrate. After an additional 144 hours, cells were harvested, shock-frozen and kept at −80°C.

Barcode amplification
To determine the abundance of cells at t 0 and t end , barcode sequences were amplified from genomic DNA and subjected to high-throughput sequencing. Amplification of barcode sequences was achieved by two rounds of PCR according to the following protocol. Cell pellets from samples obtained at the t 0 and t end time points were thawed and subjected to ultrasonication at 50% intensity for 10 seconds on ice to shear genomic DNA into fragments of 1-10 kb. Genomic DNA was isolated from cell pellets using DNeasy Blood & Tissue Kit (69581, Qiagen, Hilden, Germany). For the first round of PCR, 50 μg of fragmented genomic DNA were used in eight 100 μl reactions containing: 1 μl Titantium Taq polymerase and corresponding PCR buffer (639210, Clontech of Takara Bio, Saint-Germain-en-Laye, France), 250 μM dNTP mix (M3015.4100, Genaxxon, Ulm, Germany), 3 μM HTS forward primer: 5′-TTCTCTGGC AAGCAAAAGACGGCATA-3′ and 3 μM reverse primer: 5′-TGCCATTTGTCTCGAGGTCGAGAA-3′ in nucleasefree water (AM9938, Life Technologies, Darmstadt, Germany). Desalted purity primers were obtained from Sigma Aldrich (Munich, Germany). Reaction mixtures were prepared on ice and subjected to a first round PCR starting with activation of the polymerase at 94°C for 3 minutes, followed by 16 cycles of denaturation at 94°C for 30 seconds, annealing at 65°C for 10 seconds, and elongation at 72°C for 20 seconds. Final extension was done at 68°C for 2 minutes. Second round PCR was done using 2 μl of the first-round reaction as template in three 100 μl reactions per condition. This time, primers GEX forward 5′-CAAGCAGAAGACGGCATACGAGA-3′ and GEX reverse 5′-AATGATACGGCGACCACCGAGA-3′ were used, while all other reagents remained the same. Thermal cycler settings were also unchanged, with the exception of a reduced elongation time of 10 seconds and a reduction of the cycle number to ten. Amplified barcode sequences were purified using PCR purification and gel extraction kits (28104 and 28704, Qiagen). Barcodes of module 1 and 2 were combined for each time point and quantified by next-generation sequencing (Genome Analyzer IIx, Illumina, San Diego, CA, USA). Each module contained a two-base-pair identifier attached to the barcode sequence for later discrimination.

Sequencing data analysis
Sequences were analysed to determine the abundance of each barcode using the software 'Barcode Deconvoluter' provided by Cellecta. The software counts the number of 18-nucleotide barcode sequences with a tolerance of two mismatches and assigns each barcode to the corresponding shRNA construct. Furthermore, each module was identified by two module-specific base pairs attached to the barcode sequence. Further analysis was done in Microsoft Excel. In order to compare reads of different modules and time points, each group of modules and time points was normalized to the average number of reads. Barcodes below 200 reads for t 0 were filtered out for reasons of quality assurance. Finally, drop-out ratios were calculated by dividing normalised reads of t end by normalised reads of t 0 (expressed as % of t0).

Production of lentiviral particles
Lentiviral particles were produced using a second generation system described and engineered by Didier Trono's group (Zufferey et al., 1997). HEK-293T cells were seeded in DMEM without antibiotics at a density of 65,000 cells per cm 2 in a six-well format for single constructs or 175 cm 2 flasks for production of viral pools. Helper plasmids psPAX2 and pMD2.G were cotransfected with the construct carrier pRSI9 the following day using Lipofectamine (18324010, Life Technologies) and Plus Reagent (11514015, Life Technologies) according to the manufacturer's instructions. The next day, the medium was exchanged for high-serum DMEM (30% FCS). Supernatant containing lentiviral particles was harvested the following day, filtered through a 0.45 μm PES membrane (295-4545, Thermo Scientific) and stored in aliquots at −80°C.