The ubiquitin ligase TRIM25 targets ERG for degradation in prostate cancer

Ets related gene (ERG) is a transcription factor that is overexpressed in 40% of prostate tumors due to a gene fusion between ERG and TMPRSS2. Because ERG functions as a driver of prostate carcinogenesis, understanding the mechanisms that influence its turnover may provide new molecular handles to target the protein. Previously, we found that ERG undergoes ubiquitination and then is deubiquitinated by USP9X in prostate cancer cells to prevent its proteasomal degradation. Here, we identify Tripartite motif-containing protein 25 (TRIM25) as the E3 ubiquitin ligase that ubiquitinates the protein prior to its degradation. TRIM25 binds full-length ERG, and it also binds the N-terminally truncated variants of ERG that are expressed in tumors with TMPRSS2-ERG fusions. We demonstrate that TRIM25 polyubiquitinates ERG in vitro and that inactivation of TRIM25 resulted in reduced polyubiquitination and stabilization of ERG. TRIM25 mRNA and protein expression was increased in ERG rearrangement-positive prostate cancer specimens, and we provide evidence that ERG upregulates TRIM25 expression. Thus, overexpression of ERG in prostate cancer may cause an increase in TRIM25 activity, which is mitigated by the expression of the deubiquitinase USP9X, which is required to stabilize ERG.


Cell culture and transfection. VCaP cells, HeLa cells, 22Rv1 cells and HEK293T cells were
obtained from ATCC and cultured in DMEM with 10% FBS. Provenance of all cell lines was validated prior to submission by microsatellite fingerprinting in the McDermott Sequencing Core (UT Southwestern). Plasmids were transfected with Effectene Transfection Reagent (Qiagen); for siRNA transfection Lipofectamine RNAiMax (Invitrogen) was used for HeLa and HEK293T cells, HiPerFect Transfection Reagent (Qiagen) and Stemfect RNA Transfection Reagent (Stemgent) were used for VCaP cells. To generate cells that stably express ERG, 22Rv1 cells were transfected with pIRESneo3-ERG-V5 plasmids or pIRESneo3 (empty vector) and cells stably expressing the IRES-neo transcripts were selected with geneticin (600 µg/ml).
Antibodies and reagents. The following antibodies were used for immunoprecipitation: ERG (Epitomics,5115)

ERG immunoprecipitation and Mass spectrometry. ERG-V5 was transiently expressed in
HEK293T cells by transfection with the pCMV-ERG-V5 expression vector. 48 hours after transfection, cells were lysed (lysis buffer: 25mM HEPES [pH7.5], 400 mM NaCl, 0.5% IGEPAL CA-630, 1mM DTT, 5% glycerol and protease inhibitors). The soluble fraction of the lysate was diluted to adjust the NaCl concentration and IGEPAL CA-630 concentration to 100 mM and 0.125%, respectively, and then pre-cleared with GammaBind G Sepharose (GE Healthcare). Pre-cleared lysates were incubated with a mouse anti-V5 antibody (Genscript, A01724) at 4°C for 3 hours, followed by immunoprecipitation with GammaBind G Sepharose beads at 4°C for 1 hour. Then beads were washed four times with 100 mM NaCl and 0.1% IGEPAL CA-630. Proteins were eluted with V5 peptide (500 ng/ml) at 4°C for 30 minutes. The eluted fraction was mixed with 2X Laemmli buffer and boiled for subsequent SDS-PAGE. For mass spectrometry, PAGE gel slices with bands of interest were digested with trypsin and desalted with C18 ZipTips (Millipore) according to the manufacturer's instructions. An integrated system that includes an Agilent 1100 series Nanoflow LC system (Agilent) and a LTQ 2D trap mass spectrometer (Thermo Electron) equipped with a nanoelectrospray ionization (NSI) source was used to perform high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) analysis: Tryptic peptides were separated by a capillary HPLC column packed in-house with Luna C18 resin (Phenomenex). The eluted peptides were directly electrosprayed into the LTQ ion trap mass spectrometer, with a data-dependent mode. Mascot (version 2.3, Matrix Science) database was used to map peptides to proteins.
Migration assay. For the migration assay, VCaP cells were transfected with TRIM25 siRNA (#1) or ERG siRNA (#1) or non-targeting siRNA for 48 hours. Cells were then reseeded on the outer surface of an 8.0-mm 24-well plate Transwell ® insert (Corning Costar) with serum-free medium and complete medium in the lower chamber. After 24 hours of incubation at 37 °C with 5% CO 2 , cells remaining on the outer surface of the Transwell insert were gently removed with a cotton swab. Migrated cells (adherent to the inner surface of the insert) were stained with 1.0% crystal violet and air-dried. To quantify the migration, inserts were incubated with 200 μl of 10% acetic acid (v/v), the OD at 560 nm of the resulting solution was measured with a spectrophotometer (BMG Labtech).
After induction for 5 hours with 0.4 mM IPTG at 32°C, bacteria were pelleted by centrifugation.
The beads were washed four times with lysis buffer. Then, GST-fusion proteins were eluted with 10 mM glutathione and stored at -80°C. For the GST pulldown, VCaP cells were lysed in high salt buffer (20 mM Tris [pH 8.0], 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, 400 mM NaCl, 0.5% IGEPAL CA-630, Protease Inhibitor Cocktail [Sigma]). Extracts were diluted to adjust the NaCl concentration to 100 mM and IGEPAL CA-630 to 0.125%, and subjected to pre-clearing with GST beads. Pre-cleared supernatants were incubated with 30 µl GST or GST-fusion protein beads for 2 hours at 4°C. The beads were then washed with washing buffer (20 mM Tris [pH 5 8.0], 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, 100 mM NaCl, 0.1% IGEPAL CA-630) four times, boiled in 2X Laemmli buffer for SDS-PAGE. Eluted proteins were separated on a 4-20% SDS-PAGE gel and visualized by Coomassie blue staining or detected by immunoblotting.
Pre-cleared lysates were incubated with V5, FLAG or HA antibodies at 4°C for 3 hours, followed by immunoprecipitation to the sepharose beads at 4°C for 1 hour. Beads were washed four times with 100 mM NaCl and 0.1% IGEPAL CA-630, boiled in Laemmli buffer for Western blot analysis.

Construction of the TRIM25 CRISPR/Cas9 vector.
To generate TRIM25-knockout HeLa cells, the TRIM25 gene was edited using the GeneArt ® CRISPR Nuclease (OFP Reporter) Vector Kit (A21174, Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, TRIM25 sgRNA-specific oligonucleotides for (synthesized by Sigma, sequences are listed in Table S2) were annealed to form a double-stranded oligonucleotide with compatible ends for cloning and ligated into the GeneArt ® CRISPR Nuclease (OFP Reporter) Vector. The constructs were introduced into NEB ® 5-alpha Competent E. coli (NEB) by chemical transformation, and TRIM25 CRISPR nuclease constructs were confirmed by sequencing.
Generation of TRIM25-knockout HeLa cells. TRIM25 CRISPR nuclease constructs were transfected into HeLa cells by using Effectene transfection reagent (Qiagen) according to the manufacturer's instructions. After 48h transfection, cells were trypsinized and single OFP positive cell was directly sorted into 96 well plate by MoFlo ® Cell Sorter (Beckman Coulter) for single cell cloning. Single cell clones were expanded, and then TRIM25-knockout HeLa cells were identified by immunoblot analysis with a TRIM25 antibody.

Analysis of ERG protein stability in VCaP cells. VCaP cells transfected with siRNA against
TRIM25 or a non-targeting control were treated with cycloheximide, or DMSO for 2, 4, 6, or 8 hours starting 72 hours after siRNA transfection. Immunoblot analysis of TRIM25, ERG, and GAPDH was performed, and quantified by densitometry using ImageJ. Band intensities of TRIM25 and ERG were normalized using GAPDH intensity as reference. For quantification three replicate experiments were performed.

Analysis of ERG ubiquitination in TRIM25-knockout HeLa cells. TRIM25-knockout
Hela cells grown in DMEM with 10% FBS were co-transfected with ERG-V5 and HA-ubiquitin expression plasmids. Immunoprecipitation with a V5 antibody was performed as described above. For Western blot analysis antibodies against TRIM25, HA, V5, and GAPDH were used.

Immunohistochemical analysis and scoring
Tumor cells with ERG staining and/or TRIM25 staining were evaluated manually for each tissue core by a reviewer who was blinded to the clinical data. ERG and TRIM25 protein expression levles were registered semi-quantitatively as follows: Staining intensity (0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining) and the proportion of stained cells (0, no staining; 1, 1-25% staining; 2, 26-50%; 3, 51-75%; and 4, if more than 75% of the tumor cells were positive. Analysis of RNA-seq data. RNA-seq data for prostate adenocarcinoma primary tumor samples was retrieved from TCGA database (N=498). The tags per million (TPM) values for ERG and TRIM25 genes were converted to FPKM values and samples that have ERG expression greater than 25 were selected (N=193). A concordance plot was generated using ERG and TRIM25 expression from the selected 193 ERG-positive tumors.
Analysis of ERG occupancy at the TRIM25 gene promoter. ERG ChIP-seq reads generated for VCaP cells was retrieved from NCBI GEO (GSE14092). SRA files were converted to fastq files and mapped to hg19 using bowtie (v.2.2.5). Bigwig files were generated from uniquely mapped reads using Homer (v. 4.6). For chromatin immunoprecipitation, the chromatin of 10 million VCaP cells were crosslinked with 20 ml of pre-warmed DMEM (37ºC) containing 1% formaldehyde. After 10 minutes of incubation at room temperature 2.2 ml 1.25 M glycine was added, and after 5 minutes of incubation at room temperature the cells were washed twice with ice-cold PBS, scraped off the dish and collected in 5 ml ice-cold PBSpelleted by centrifugation at 700g for 10 minutes. The cell pellet was then lysed in 1 ml ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors, Roche) and sonicated at 4ºC with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes).