Unknown,Transcriptomics,Genomics,Proteomics

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The androgen receptor coordinates biosynthesis and proliferation in prostate cancer

ABSTRACT: The androgen receptor (AR) is the major therapeutic target in prostate cancer, although the important targets of the AR have remained obscure. Here we report a detailed genomic profile of AR signalling and find that the AR directly regulates glycolysis, anabolic metabolism and cell cycle regulators in prostate cancer. This coordinated transcriptional program promotes cancer cell proliferation and enhances the macromolecular synthesis needed to produce daughter cells. Clinical gene expression profiles and mechanistic studies highlight the importance of CAMKK2, an AR target which regulates both cell proliferation and metabolism. Thus our genomics study identifies a direct link between AR signalling and aerobic glycolysis (the Warburg effect), providing a new perspective on the oncogenic function of the AR in prostate cancer. 8 Samples: Chromatin IP using AR and PolII in stimulated and unstimulated LNCaP and VCaP cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Suraj Menon

PROVIDER: E-GEOD-28126 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aberrant activation of androgen receptor (AR)-dependent transcriptional programs is a hallmark of human prostate cancers. At the molecular level, ligand-mediated AR activation is coordinated through spatial and temporal protein-protein interactions (PPIs) involving AR-interacting proteins, which we designate the “AR-interactome”. Despite many years of research, the ligand-sensitive protein complexes involved in ligand-mediated AR activation in prostate-tumor cells has not been clearly defined. Here, we describe the development, characterization, and utilization of a novel human LNCaP prostate-tumor cell line, N-AR, which stably expresses wild-type AR containing the streptavidin-binding peptide epitope tagged at its N-terminus (SBP-AR). A bioanalytical workflow involving streptavidin-chromatography and label-free quantitative mass spectrometry was used to identify SBP-AR and associated ligand-sensitive proteins/protein complexes functionally linked to AR activation in the cytosol of N-AR cells. Functional studies verified that ligand-sensitive streptavidin-copurified proteins encoded modulators of AR-mediated transcription, suggesting that these novel proteins were putative SBP-AR-interacting proteins in N-AR cells. This was supported by biochemical associations between recombinant SBP-AR and the ligand-sensitive COPI retrograde trafficking complex in vitro. Extensive biochemical and molecular experiments showed that the COPI-retrograde complex regulates ligand-mediated AR transcriptional activation through the mobilization of Golgi-localized ARA160 coactivator into the nuclear compartment of prostate-tumor cells. Collectively, this study provides a bioanalytical strategy to validate the AR-interactome and define novel AR-interacting proteins involved in ligand-mediated AR activation in prostate-tumor cells. Moreover, we describe a cellular system to study how compartment-specific AR-interacting proteins influence AR activation and contribute to aberrant AR-dependent transcription that underlies the majority of human prostate cancers.

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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The androgen receptor coordinates biosynthesis and proliferation in prostate cancer

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