MYC-Mediated Ribosomal Gene Expression Sensitizes Enzalutamide-resistant Prostate Cancer Cells to EP300/CREBBP Inhibitors

Patients with advanced prostate cancer are frequently treated the antiandrogen enzalutamide. However, resistance eventually develops in virtually all patients, and various mechanisms have been associated this process. The histone acetyltransferases EP300 CREBBP involved regulation of cellular events cancer. This study investigated role EP300/CREBBP inhibitors enzalutamide-resistant led to same inhibition androgen receptor activity -sensitive cells. cells were more sensitive these viability assays. As indicated by RNA-sequencing–based pathway analysis, genes related ribosome MYC significantly altered upon inhibitor treatment. down-regulation ribosomal proteins RPL36 RPL29. High-level amplifications observed castration-resistant samples publicly available Stand Up Cancer data set. An RNA polymerase I–mediated transcription was used evaluate functional implications findings. cell lines In addition, migration rate strongly inhibited Taken together, current show that affect MYC/ribosomal protein axis may promising therapeutic implications. Prostate (PCa) is one most commonly diagnosed types cancer, especially industrialized nations. Therapy localized PCa mostly curative. For recurrent PCa, deprivation therapy via chemical or surgical castration remains standard-of-care treatment.1Barsouk A. Padala S.A. Vakiti Mohammed Saginala K. Thandra K.C. Rawla P. Barsouk Epidemiology, staging management cancer.Med Sci (Basel). 2020; 8: 28Google Scholar Although initially effective, development nearly always inevitable. case, blockade (AR) used, Enzalutamide not only inhibits ligand binding AR, but also reduces nuclear translocation as well DNA AR.2Tran C. Ouk S. Clegg N.J. Chen Y. Watson P.A. Arora V. Wongvipat J. Smith-Jones P.M. Yoo D. Kwon Wasielewska T. 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Open table tab ADT, generated chronic increasing concentrations enzalutamide, described previously.21Puhr Hoefer Eigentler Ploner Schaefer Kroon Leo Heidegger Pluijm Klocker key player survival improved therapy.Clin 24: 927-938Crossref (87) Scholar,22Hoefer Akbor Ofer Parson Critical level generation enzalutamide.Oncotarget. 7: 59781-59794Crossref (38) denoted suffix EnzaR. All cultured RPMI 1640 (PAN-Biotech, Aidenbach, Germany) 10% fetal calf serum (PAN-Biotech), 1% penicillin/streptomycin (Lonza, Basel, Switzerland), GlutaMAX (Thermo Fisher Scientific, Waltham, MA). additionally supplied 8 ?M (DuCaP EnzaR) 5 (LNCaP identity confirmed tandem repeat analysis. knockdown MYC, four different pooled siRNA constructs (ON-TARGETplus Human SMARTPool, THP, Vienna, Austria) control siControl THP) used. transfection, Lipofectamine RNAiMAX Scientific) according manufacturer’s instructions. After 3 days, lysed radioimmunoprecipitation assay buffer, blotted. A total 20 ?g per sample loaded onto 8% Tris-Acetate detection 4 12% Bis-Tris NuPAGE gels electrophoretic separation. Gels then blotted 0.2 ?m Amersham Protran Nitrocellulose membrane (Sigma, St. Louis, MO). Membranes stained using Revert 700 Total Stain (LI-COR Biosciences, Lincoln, NE) quantify protein. incubated Starting Blocking Buffer 1 hour room temperature before overnight incubation primary antibody 4°C. washing Tris-buffered saline 0.1% Tween-20 (hereafter termed TBST), membranes IRDye Goat anti-Rabbit/Mouse IgG Secondary Antibody Biosciences) 45 minutes. Odyssey imaging System scan after TBST Image Studio software (version 5.2, LI-COR amounts. blots probed repeatedly. antibodies dilutions: (7389S, Signaling Technology, Danvers, MA; 1:2000), (ab10485, Abcam, Cambridge, UK; 1:1000), FKBP5/FKBP51(A301-430A, Bethyl, Montgomery, TX; alpha tubulin (sc-5286, Santa Cruz Laboratories, Dallas, (D84C12, Signaling; RPL29 (AP20452c-ev, ABcepta, San Diego, CA; 1:500), (ELA-E-AB-32803 60, Elabscience Biotechnology, Houston, glyceraldehyde-3-phosphate dehydrogenase (ABS16, Millipore, Burlington, 1:5000). Parental seeded 384-well plate (Corning, Corning, NY) triplicate 200 2000 well, respectively, C646 (Sigma), I-CBP112 (Tocris, Bristol, UK), CPI637 (MedChem Express, Monmouth Junction, NJ), CX-5461 Express), solvent dimethyl sulfoxide (Sigma). RealTime-Glo MT (Promega, Madison, WI) manufacturer. Cytation5 (BioTek, Winooski, VT) reader equipped carbon dioxide chamber heated 37°C luminescence over 72 hours. Dose–response curves GraphPad Prism (GraphPad Software Inc., La Jolla, CA) 50% inhibitory compared sum-of-square-F-test. Quantitative real-time PCR performed described.23Puhr Neuwirt Susani Nemeth Kenner Down-regulation suppressor cytokine signaling-3 causes death extrinsic intrinsic apoptosis pathways.Cancer 69: 7375-7384Crossref (75) isolated EXTRACTME Isolation kit (Blirt, Zgierz, Poland) LUNAScript (New England Biolabs, Ipswich, MA) transcribe 250 ng cDNA. quantitative PCR, cDNA mixed LUNA Universal Probe RT-qPCR Master Mix Biolabs) assessed duplicate 7500 Fast Real-Time (Applied Biosystems, Foster City, CA). Geometric mean CT values reference HMBS, HPRT1, TBP normalization. FKBP5, PSA, TMPRSS2 2).Table 2Primer Pairs Used RT-qPCRGenesGeneSequence/Catalog no.ManufacturerHousekeeping genesTBPForward: 5?-CACGAACCACGGCACTGATT-3?Microsynth AGReverse: 5?-TTTTCTTGCTGCCAGTCTGGAC-3?Probe: 5?- TCTTCACTCTTGGCTCCTGTGCACA-3?-TAMRAHPRTForward: GCTTTCCTTGGTCAGGCAGTA-3?Reverse: 5?-GTCTGGCTTATATCCAACACTTCGT-3?Probe: 5?-CAAGGTCGCAAGCTTGCTGGTGAAAAGGA-3?-TAMRAHMBSTaqMan Gene Expression Assay: Hs00609297_m1Thermo ScientificTarget genesFKBP5TaqMan Hs01561006_m1Thermo ScientificTMPRSS2TaqMan Hs01120965_m1Thermo ScientificPSAForward: 5?-GTCTGCGGCGGTGTTCTG-3?Microsynth 5?-TGCCGACCCAGCAAGATC-3?Probe: 5?-CACAGCTGCCCACTGCATCAGGA-3?-TAMRART-qPCR, PCR. RT-qPCR, 96-well plates 2 × 104 respectively. Cells transfected x-tremeGENE HP (Roche, Switzerland) 50 pGL4.53 (Promega) encoding firefly luciferase under constitutive promoter (PGK) normalization pGL4.70 NanoLuc TATA-box preceded elements. 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Growth chemoattractant lower chamber. 48 hours, ?mol/L Calcein AM (Sigma) dissolved Hanks’ Balanced Salt Solution (Lonza) bovine albumin Staining solution added. staining (BioTek) representative images taken JuLI smart fluorescent analyzer (Science Services, Munich, Germany). percentage migrated relative parallel without account reduced CX-5461–treated wells. comparison independent replicates Western blot, reporter assay, Boyden-chamber normalized average signal intensity. One-way two-way variance Dunnett’s Tukey’s comparisons test unless stated otherwise. Significant noted figures ?P < 0.05, **P 0.01, ***P 0.001. setting (henceforth EnzaR, respectively) clearly detectable, no significant differences between (Figure 1A). Therefore, growth effect compounds directed against dose–response proliferation assays tested. domain, BD.32Bowers Yan Orry Holbert Crump N.T. Hazzalin Lisczak Yuan Saldanha Abagyarn Sun Marmorstein Mahadevan L.C. 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