Unknown,Transcriptomics,Genomics,Proteomics

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Androgen receptor splice variants mediate enzalutamide resistance in castration-resistant prostate cancer cell lines

ABSTRACT: The goal of this experiment was to compare the gene expression programs mediated by androgen/AR vs. constitutively active, truncated AR variants in castration-resistant CWR-R1 prostate cancer cells. Because constitutive activity of truncated AR variants can mask androgen/AR target genes, the androgen/AR transcriptional program was assessed by silencing the trucnated AR 1/2/3/CE3 variant with siRNA targeting AR exon CE3 and treating cells with vehicle (ethanol) or 1nM DHT. Similarly, because full-length AR activity can mask truncated AR variant target genes, the AR variant transcriptional program was assessed under castrate conditions by selectively silencing full-length AR with siRNA targeting AR exon 7, and comparing this profile with CWR-R1 cells transfected wtih siRNA targeting AR exon 1, which silences all AR expression (full-length and truncated AR variants). CWR-R1 cells were maintained under castrate conditions in long term culture in order to enrich for the population of cells harboring a 48kb intragenic deletion in AR intron 1. These late-passage CWR-R1 cells were electroporated with siRNAs targeting AR exon 1, AR exon 7, or AR exon CE3. Electroporated cells were seeded in RPMI 1640 medium containing antibiotics and 5% charcoal-stripped, steroid-depleted medium and allowed to recover for 48h. After this 48h recovery, electroporated cells were switched to serum-free RPMI 1640 with 1nM DHT or 0.1% ethanol (vehicle control) for an additional 24h prior to extraction of total RNA. Three independent biological replicates were performed.

ORGANISM(S): Homo sapiens

SUBMITTER: Scott Dehm

PROVIDER: E-GEOD-41784 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 in the N-terminal transactivation domain. In this study, the role of this phosphorylation site was investigated by characterizing the phosphorylation site mutant in the context of full length and truncated AR lacking the ligand-binding domain. The Y267F mutant showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. Expression of AR responsive genes in cells expressing truncated AR wt and AR-Y267F mutant under androgen deprived conditions was assessed by microarray gene expression analysis. The AR pathway score was increased in cells expressing truncated AR wt and decreased in cells expressing truncated AR-Y267F. These results support the concept that phosphorylation of Tyr-267 is required for AR nuclear translocation and recruitment and DNA binding and transcription of AR responsive genes. Gene expression profiling was performed using RNA samples from LNCaP cells expressing truncated AR-wt and truncated AR-Y267F growing in androgen deprived conditions. The RNA sample from vector control cells were used as reference sample. Two biological replicates were used per each cell line.

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Androgen receptor splice variants mediate enzalutamide resistance in castration-resistant prostate cancer cell lines

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