title: High-Content Screening Identifies Src Family Kinases as Potential Regulators of AR-V7 Expression and Androgen-Independent Cell Growth.
abstract: AR-V7 is an androgen receptor (AR) splice variant that lacks the ligand-binding domain and is isolated from prostate cancer cell lines. Increased expression of AR-V7 is associated with the transition from hormone-sensitive prostate cancer to more advanced castration-resistant prostate cancer (CRPC). Due to the loss of the ligand-binding domain, AR-V7 is not responsive to traditional AR-targeted therapies, and the mechanisms that regulate AR-V7 are still incompletely understood. Therefore, we aimed to explore existing classes of small molecules that may regulate AR-V7 expression and intracellular localization and their potential therapeutic role in CRPC.
  
  
  
  We used AR high-content analysis (AR-HCA) to characterize the effects of a focused library of well-characterized clinical compounds on AR-V7 expression at the single-cell level in PC3 prostate cancer cells stably expressing green fluorescent protein (GFP)-AR-V7 (GFP-AR-V7:PC3). In parallel, an orthogonal AR-HCA screen of a small interfering (si)RNA library targeting 635 protein kinases was performed in GFP-AR-V7:PC3. The effect of the Src-Abl inhibitor PD 180970 was further characterized using cell-proliferation assays, quantitative PCR, and western blot analysis in multiple hormone-sensitive and CRPC cell lines.
  
  
  
  Compounds that tended to target Akt, Abl, and Src family kinases (SFKs) decreased overall AR-V7 expression, nuclear translocation, absolute nuclear level, and/or altered nuclear distribution. We identified 20 protein kinases that, when knocked down, either decreased nuclear GFP-AR-V7 levels or altered AR-V7 nuclear distribution, a set that included the SFKs Src and Fyn. The Src-Abl dual kinase inhibitor PD180970 decreased expression of AR-V7 by greater than 46% and decreased ligand-independent transcription of AR target genes in the 22RV1 human prostate carcinoma cell line. Further, PD180970 inhibited androgen-independent cell proliferation in endogenous-AR-V7-expressing prostate cancer cell lines and also overcame bicalutamide resistance observed in the 22RV1 cell line.
  
  
  
  SFKs, especially Src and Fyn, may be important upstream regulators of AR-V7 expression and represent promising targets in a subset of CRPCs expressing high levels of AR-V7. Prostate 77:82-93, 2017. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas.
authors.forename: Adam T
authors.initials: AT
authors.lastname: Szafran
authors.affiliation: Institute for Bioscience and Technology, Texas A&M University Health Science Center, Houston, Texas.
authors.forename: Cliff
authors.initials: C
authors.lastname: Stephan
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas.
authors.forename: Michael
authors.initials: M
authors.lastname: Bolt
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas.
authors.forename: Maureen G
authors.initials: MG
authors.lastname: Mancini
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas.|Department of Medicine, Baylor College of Medicine, Houston, Texas.|Michael E. DeBakey VA Medical Center, Baylor College of Medicine, Houston, Texas.|Diana Helis Henry Medical Research Foundation, New Orleans, Louisiana.
authors.forename: Marco
authors.initials: M
authors.lastname: Marcelli
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas.|Diana Helis Henry Medical Research Foundation, New Orleans, Louisiana.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Mancini
mesh_terms: D000728:Androgens / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D014644:Genetic Variation / Q000502:physiology*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D061986:MCF-7 Cells; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D011728:Pyridones / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011944:Receptors, Androgen / Q000096:biosynthesis* / Q000235:genetics; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: C410798:AR protein, human; D000728:Androgens; D011728:Pyridones; D011743:Pyrimidines; D011944:Receptors, Androgen; D019061:src-Family Kinases; C410732:PD 180970
keywords: castration-resistant prostate cancer; high content analysis; human kinome siRNA screen; small molecule screen