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High‐Content Screening Identifies Src Family Kinases as Potential Regulators of AR‐V7 Expression and Androgen‐Independent Cell Growth
Author(s) -
Szafran Adam T.,
Stephan Cliff,
Bolt Michael,
Mancini Maureen G.,
Marcelli Marco,
Mancini Michael A.
Publication year - 2017
Publication title -
the prostate
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.295
H-Index - 123
eISSN - 1097-0045
pISSN - 0270-4137
DOI - 10.1002/pros.23251
Subject(s) - androgen receptor , cancer research , prostate cancer , kinase , biology , nuclear receptor , protein kinase b , chemistry , cancer , microbiology and biotechnology , signal transduction , transcription factor , biochemistry , gene , genetics
BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.