High-Content Screening Identifies Src Family Kinases as Potential Regulators of AR-V7 Expression and Androgen-Independent Cell Growth.

Abstract

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.

• Authors: Adam T Szafran, Cliff Stephan, Michael Bolt, Maureen G Mancini, Marco Marcelli, Michael A Mancini
• Journal: The Prostate (Prostate) Vol. 77 Issue 1 Pg. 82-93 (01 2017) ISSN: 1097-0045 [Electronic] United States
• PMID: 27699828 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, N.I.H., Extramural)
• Copyright: © 2016 Wiley Periodicals, Inc.

Chemical References

• AR protein, human
• Androgens
• Pyridones
• Pyrimidines
• Receptors, Androgen
• src-Family Kinases
• PD 180970

Topics

• Androgens (metabolism)
• Cell Line, Tumor
• Cell Proliferation (drug effects, physiology)
• Female
• Gene Expression Regulation, Neoplastic
• Genetic Variation (physiology)
• High-Throughput Screening Assays (methods)
• Humans
• MCF-7 Cells
• Male
• Prostatic Neoplasms (metabolism, pathology)
• Pyridones (pharmacology)
• Pyrimidines (pharmacology)
• Receptors, Androgen (biosynthesis, genetics)
• src-Family Kinases (antagonists & inhibitors, physiology)