Collection of patient samples
Metastatic tumor samples constituting UW TMAs were collected from patients with metastatic castration-resistant prostate cancer at the UW–Fred Hutchinson Cancer Center (FHCC) after informed consent under Institutional Review Board approval (IRB number 2341). Samples were taken at the time of a rapid autopsy.
Ethics Statement
All animal work was performed in accordance with protocol (number 50870) approved by the Fred Hutchinson Cancer Center Animal Care and Use Committee. Mice were housed under standard temperature and humidity conditions with a 12-h light/12-h dark cycle in the FHCC animal facility. Mice were closely monitored to minimize discomfort, distress, injury, and pain throughout the in vivo experiments.
Cell culture
LNCaP (clone LNCaP FGC; American Type Culture Collection (ATCC), CRL-1740, RRID: CVCL_1379), C4-2B (ATCC, CRL-3315, RRID: CVCL_4784), 22RV1 (ATCC, CRL-2505, RRID: CVCL_1045), DU145 (ATCC, HTB-81, RRID: CVCL_0105) and PC3 (ATCC, CRL−1435, RRID: CVCL_0035) were cultured in RPMI-1640 medium (Gibco, 11875119) supplemented with 10% FBS, 1% GlutaMax (Gibco, 35050061) and 1% penicillin-streptomycin (Gibco, 15140122) and passage with 0.05% trypsin-EDTA (Gibco, 25300120). VCaP (ATCC, CRL-2876, RRID: CVCL_2235) and HEK293T (ATCC, CRL-3216, RRID: CVCL_0063) cells were cultured in high-glucose DMEM medium (Gibco, 11965118) supplemented with 10% FBS and 1% penicillin-streptomycin and passaged with 0.05% solution. trypsin-EDTA. LNCaP-abl cells (RRID: CVCL_4793) were cultured in RPMI-1640 without phenol red (Gibco, 11835055) supplemented with 10% charcoal-stripped FBS (Gibco, 12676029) and 1% penicillin-streptomycin and passaged using TrypLE without phenol red (Gibco, 12604039). LNCaP AD and LP cells and C4-2B AD and LP cells were cultured in neural stem cell medium consisting of advanced DMEM/F12 (Gibco, 12634028) supplemented with 10 ml B-27 supplement (50×) (Gibco, 17504044), 1% GlutaMax, 10 ng µl.−1 bFGF (PeproTech, 100-18B-100UG), 10 ng µl−1 EGF (PeproTech, AF-100-15-100UG) and 1% penicillin-streptomycin. All cell lines were cultured at 37°C with 5% CO2. LP lines were generated as previously described37.
Mouse
Hi-Myc mice (Tg(ARR2/Pbsn-MYC)7Key, RRID: MGI:5486199) were crossed with n-Trct2–/– mouse (C57BL6/J|6J.6N-rs46447118, n-Trct2(-3_90del)) to generate Hi-Myc;n-Trct2+/– mouse with a copy of the Trr-Tct1-1 embarrassed. TRAMP mice (C57BL/6-Tg(TRAMP)8247Ng/J, RRID: IMSR_JAX:003135) were crossed with n-Trct2OE mouse (B6J-Tg(tRNA-Arg-TCT-1-1)529Slac) to generate TRAMP;n-Trct2OE mouse. Male NSG mice (8–12 weeks old) for in vivo metastasis testing were obtained from the Comparative Medicine Translational Research Model Services Core at FHCC. For in vivo studies, no formal sample size calculation was performed. Sample sizes were based on previous studies using similar animal models and experimental settings.
Establishment and culture of mouse prostate organoids
Hi-Myc prostates aged 8 to 12 weeks;n-Trct2+/+ and Hi-Myc;n-Trct2+/– Male mice were collected and dissected in DMEM medium supplemented with 10% FBS, 1% GlutaMax, and 1% penicillin–streptomycin. Prostate lobes were microdissected using a clean blade, followed by incubation with collagenase I (Gibco, 17018029) for 1 h at 37 °C and 0.05% trypsin-EDTA for 5 min on a rocker at 37 °C. The enzymatic reaction was stopped by adding dissection medium containing 1 mg ml−1 DNase (Sigma, 101041590010). Enzyme-dissociated prostates were mechanically dissociated two or three times using a 3 ml syringe with an 18 G needle and filtered through a 40 μm cell strainer. Dissociated prostates were centrifuged at 350g for 5 min and resuspended in 1 ml of dissection medium for cell counting and stained with 3 µl each of CD49F-PE (R&D Systems, FAB13501P, RRID: AB_357017) and EpCAM-APC (BioLegend, 118214, RRID: AB_1134102) antibodies. Isolated prostate cells were enriched for basal stem cell populations (CD49F-positive and EpCAM-positive) by FACS and collected in DMEM medium with 50% FBS, 1% GlutaMax, and 1% penicillin–streptomycin. FACS-sorted prostate basal cells were centrifuged at 350g for 4 min and resuspended in Matrigel for organoid culture (approximately 20,000 cells per well) in 24-well ultralow fixation plates (Corning, 3473). A total of 500 μl of organoid medium (Advanced DMEM/F12 supplemented with B-27 supplement, 10 mM HEPES, 1% GlutaMax, 1% penicillin-streptomycin, 500 ng ml−1 R-spondin, 100 ng ml−1 Caboche, 0.1 g N-acetyl-I-cysteine, 200 nM A83-01, 50 ng ml−1 EGF and 10 µM Y-27632) containing 1 nM DHT were added per well and passed using TrypLE.
Mouse genotyping
Mouse tissues were incubated in tail lysis buffer (100 mM Tris pH 8, 400 mM NaCl, 8 M urea, 20 mM EDTA pH 8, and 1% N-lauroylsarcosine sodium salt) with proteinase K (1.67 mg ml−1) overnight at 55°C. DNA was extracted from lysed mouse tissue samples using phenol-chloroform-isoamyl alcohol (Ambion, AM9732), followed by ethanol precipitation. PCR genotyping was performed using GoTaq Green master mix (Promega, PRM7123) using the primers shown in Supplementary Table 9. PCR products were analyzed by agarose gel electrophoresis.
Cloning and preparation of plasmids
TRR-TCT1-1tRNA2
Arg(CCG) and tRNA3
Arg(UCG) knockdown by shRNA (Tet-pLKO-puro)
THE EYFP the sequence was cloned into Tet-pLKO-puro (Addgene, 21915) by Gibson assembly (NEB, E2611L). The shScramble, shUCU, shCCG, and shUCG oligonucleotide sequences (see Supplementary Table 9 for details) were amplified using 2× High Fidelity Master Mix Q5 (NEB, M0492S) and cloned into Tet-pLKO.1-puro-EYFP using Gibson assembly after enzymatic digestion with AgeI and EcoRI.
tRNA overexpressionArg(UCU) isocoders and TRR-ACG1-1, TRR-CCG2-1 And TRR-CCT4-1 (pure pLKO.1)
For tRNA overexpressionArg(UCU), three tRNA tandem repeats1Arg(UCU), tRNA2Arg(UCU), tRNA3Arg(UCU), tRNA4Arg(UCU) and tRNA5Arg(UCU) and their flanking sequences around genomic loci (±200 bp) (see Supplementary Table 9 for details) were cloned into pLKO.1 puro (Addgene, 8453) after deletion of the U6 promoter region using ClaI and AgeI.
For tRNA overexpressionArg isocoders of different isoacceptor groups, the plasmids were generated by inserting three tRNA tandem repeats1Arg(ACG), tRNA2Arg(CCG) or tRNA4Arg(CCU) and flanking sequences around the genomic loci (±200 bp) (see Supplementary Table 9 for details) up to pLKO.1 puro without the U6 promoter.
AGA reporter assay (pLJM1-YFP)
Plasmid pLJM1-YFP (modified from pLJM1-EGFP from D. Sabatini, Addgene, 19319) was digested with AgeI and EcoRI to replace YFP with Flag-mCherry-12×AGA-DHFR by Gibson assembly. Finally, EBFP2-intron-IRES was inserted into the plasmid after enzymatic digestion with AgeI. See Supplementary Table 9 for gBlock and primer sequences.
After enzymatic digestion of the 12×AGA reporter plasmid with EcoRV and PmeI, 2×, 4×, and 6×AGA reporters were generated by inserting mCherry and DHFR fragments after PCR with the primers listed in Supplementary Table 9. In the 6×AGA reporter, two 6×AGA codon repeats were separated by codons CGU-CGA-CGU-CGA. In the 4×AGA reporter, three repeats of 4×AGA codons were separated by CGU-CGA codons. In the 2 × AGA reporter, six repeats of 2 × AGA codons were separated by CGA codons (Extended Data, Fig. 9a).
SMARCC2 codon switching plasmid (pLVV-CMV-PGK-BSD)
SMARCC2 cDNA sequences with unswitched AGA codons or switched to CGC were ligated to 3×Flag and inserted into pLVV-CMV-PGK-BSD after enzymatic digestion with XhoI and BamHI.
shSMARCC2 (pLKO.1 explosion)
The shScramble and shSMARCC2 oligonucleotide sequences (see Supplementary Table 9 for details) were cloned into pLKO.1-blast (Addgene, 26655) by Gibson assembly after enzymatic digestion with AgeI and EcoRI.
Generation of stable cell lines
The C4-2B TRR-TCT1-1 The gene knockout cell line was generated by CRISPR ribonucleoprotein nucleofection. Briefly, two gene-targeting sgRNAs (TRR-TCT-1-1_gRNA_1: 5′-GACTCCAACAGGTGGCTCCG-3′ and TRR_TCT-1-1_gRNA_2: 5′- AAAAAGCGTTACGACTCCGC-3′) were mixed in a ratio of 1:1 for a final concentration of 50 pmol µl.−1. As negative controls, two AAVS1 sgRNAs (AAVS1_sgRNA_142: 5′-TTCTGGGAGAGGGTAGCGCA-3′ and AAVS1_sgRNA_288: 5′-GAGATGGCTCCAGGAAATGG-3′) were used. sgRNAs were mixed with complete nucleofector solution, SF buffer (Lonza 4D-Nucleofector The 20 μl mixture was incubated for 15 min at room temperature then added to C4-2B cell pellets (3 × 105 cells by nucleofection) in 1.5 ml Eppendorf tubes. The solutions were transferred to individual wells of a 16-well Nucleocuvette strip and nucleofection was performed using the DS-137 program. Nucleofected cells were collected with 150 μl of cell culture medium by gentle pipetting and plated in a 6-well plate containing 2 ml of medium per well. Cells were monitored daily until each well reached approximately 85–90% confluence.
At 72 hours after nucleofection, approximately 50% of the cells in each well were collected for analyzes of CRISPR editing efficiency. gDNA was extracted from sgAAVS1 and sgTRR-TCT-1-1 cells using a Quick-DNA Microprep Plus kit (Zymo Research, D4074). Genomic regions near the sgRNA cut sites were amplified by PCR, and the PCR products were purified using a QIAquick PCR purification kit (Qiagen, 28106) for Sanger sequencing. Sequencing trace files were analyzed on the ICE website (https://ice.synthego.com/#/) to determine the efficiency of CRISPR editing. The knockout score was 100 at TRR-TCT1-1 locus in the knockout cell line.
Lentivirus was produced in HEK293T cells cultured in 150 mm plates. At 90% confluence, cells were transfected with 0.72 pmol of pMD2.G…
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