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Similarities and Distinctions in Actions of Surface-Directed and Classic Androgen Receptor Antagonists.

Suh JH, Chattopadhyay A, Sieglaff DH, Storer Samaniego C, Cox MB, Webb P - PLoS ONE (2015)

Bottom Line: These effects are related to arrest of an AR/chaperone complex in the cytoplasm.Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide.Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes.

View Article: PubMed Central - PubMed

Affiliation: Genomic Medicine Program, Houston Methodist Research Institute, 66670 Bertner Avenue, R8-114, Houston, Texas, 77030, United States of America.

ABSTRACT
The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.

No MeSH data available.


Related in: MedlinePlus

MJC13 blocks AR interaction with coactivators.(A) Co-immunoprecipitations from extracts of LNCaP cells treated +/- DHT (1nM), +/- flutamide (1μM), or +/- MJC13 (30μM) for 12 h. AR antibody was used for immunoprecipitation and antibody used for western analysis is indicated at the left hand side. Panels below represent western blots of input proteins or GAPDH loading control. (B) Co-immunoprecipitation assay from extracts of HEK293T cells cotransfected with plasmids of Gal4-AR LBD fusion and SRC-2 or β-catenin. AR LBD was immunoprecipitated with an anti-Gal4 antibody in lysates from cells treated +/- DHT, +/- Flutamide or +/- MJC13 as above. Antibody used for western analysis is indicated at the left hand side. Panels below represent western blots of input proteins or GAPDH loading control.
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pone.0137103.g008: MJC13 blocks AR interaction with coactivators.(A) Co-immunoprecipitations from extracts of LNCaP cells treated +/- DHT (1nM), +/- flutamide (1μM), or +/- MJC13 (30μM) for 12 h. AR antibody was used for immunoprecipitation and antibody used for western analysis is indicated at the left hand side. Panels below represent western blots of input proteins or GAPDH loading control. (B) Co-immunoprecipitation assay from extracts of HEK293T cells cotransfected with plasmids of Gal4-AR LBD fusion and SRC-2 or β-catenin. AR LBD was immunoprecipitated with an anti-Gal4 antibody in lysates from cells treated +/- DHT, +/- Flutamide or +/- MJC13 as above. Antibody used for western analysis is indicated at the left hand side. Panels below represent western blots of input proteins or GAPDH loading control.

Mentions: We next compared abilities of flutamide and MJC13 to inhibit AR/coactivator interactions. We performed co-immunoprecipitations from LNCaP cells with anti-AR antibody in the presence and absence of DHT and antagonists. SRC2 associated with AR in the presence of DHT (Fig 8A). While flutamide and MJC13 did not induce AR/SRC2 association when used alone, both compounds blocked DHT-dependent AR/SRC2 interaction [34]. Unlike SRC2, β-catenin coprecipitated with AR in the presence and absence of DHT (Fig 8A). This binding event was unaffected by antagonists in the absence of DHT, but MJC13 specifically inhibited AR/β-catenin binding in the presence of DHT, consistent with predictions of a distinct binding mode for this ligand [27]. Similar results were obtained in co-immunoprecipitations from HEK293T cells transfected with GAL4-AR LBD and coactivators. Again, flutamide and MJC13 blocked DHT-dependent AR/SRC2 association and MJC13 selectively inhibited hormone-independent AR LBD/β-catenin interactions (Fig 8B).


Similarities and Distinctions in Actions of Surface-Directed and Classic Androgen Receptor Antagonists.

Suh JH, Chattopadhyay A, Sieglaff DH, Storer Samaniego C, Cox MB, Webb P - PLoS ONE (2015)

MJC13 blocks AR interaction with coactivators.(A) Co-immunoprecipitations from extracts of LNCaP cells treated +/- DHT (1nM), +/- flutamide (1μM), or +/- MJC13 (30μM) for 12 h. AR antibody was used for immunoprecipitation and antibody used for western analysis is indicated at the left hand side. Panels below represent western blots of input proteins or GAPDH loading control. (B) Co-immunoprecipitation assay from extracts of HEK293T cells cotransfected with plasmids of Gal4-AR LBD fusion and SRC-2 or β-catenin. AR LBD was immunoprecipitated with an anti-Gal4 antibody in lysates from cells treated +/- DHT, +/- Flutamide or +/- MJC13 as above. Antibody used for western analysis is indicated at the left hand side. Panels below represent western blots of input proteins or GAPDH loading control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4557941&req=5

pone.0137103.g008: MJC13 blocks AR interaction with coactivators.(A) Co-immunoprecipitations from extracts of LNCaP cells treated +/- DHT (1nM), +/- flutamide (1μM), or +/- MJC13 (30μM) for 12 h. AR antibody was used for immunoprecipitation and antibody used for western analysis is indicated at the left hand side. Panels below represent western blots of input proteins or GAPDH loading control. (B) Co-immunoprecipitation assay from extracts of HEK293T cells cotransfected with plasmids of Gal4-AR LBD fusion and SRC-2 or β-catenin. AR LBD was immunoprecipitated with an anti-Gal4 antibody in lysates from cells treated +/- DHT, +/- Flutamide or +/- MJC13 as above. Antibody used for western analysis is indicated at the left hand side. Panels below represent western blots of input proteins or GAPDH loading control.
Mentions: We next compared abilities of flutamide and MJC13 to inhibit AR/coactivator interactions. We performed co-immunoprecipitations from LNCaP cells with anti-AR antibody in the presence and absence of DHT and antagonists. SRC2 associated with AR in the presence of DHT (Fig 8A). While flutamide and MJC13 did not induce AR/SRC2 association when used alone, both compounds blocked DHT-dependent AR/SRC2 interaction [34]. Unlike SRC2, β-catenin coprecipitated with AR in the presence and absence of DHT (Fig 8A). This binding event was unaffected by antagonists in the absence of DHT, but MJC13 specifically inhibited AR/β-catenin binding in the presence of DHT, consistent with predictions of a distinct binding mode for this ligand [27]. Similar results were obtained in co-immunoprecipitations from HEK293T cells transfected with GAL4-AR LBD and coactivators. Again, flutamide and MJC13 blocked DHT-dependent AR/SRC2 association and MJC13 selectively inhibited hormone-independent AR LBD/β-catenin interactions (Fig 8B).

Bottom Line: These effects are related to arrest of an AR/chaperone complex in the cytoplasm.Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide.Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes.

View Article: PubMed Central - PubMed

Affiliation: Genomic Medicine Program, Houston Methodist Research Institute, 66670 Bertner Avenue, R8-114, Houston, Texas, 77030, United States of America.

ABSTRACT
The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.

No MeSH data available.


Related in: MedlinePlus