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      AR intragenic deletions linked to androgen receptor splice variant expression and activity in models of prostate cancer progression

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          Abstract

          Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively-active COOH-terminally truncated AR variants lacking the AR ligand binding domain has emerged as an important mechanism of ADT-resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35 kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts, and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8,579 bp deletion of AR exons 5, 6, and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted re-sequencing of the AR gene in CWR-R1 cells led to the discovery of a 48 kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity, and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa.

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          Most cited references26

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          Molecular determinants of resistance to antiandrogen therapy.

          Using microarray-based profiling of isogenic prostate cancer xenograft models, we found that a modest increase in androgen receptor mRNA was the only change consistently associated with the development of resistance to antiandrogen therapy. This increase in androgen receptor mRNA and protein was both necessary and sufficient to convert prostate cancer growth from a hormone-sensitive to a hormone-refractory stage, and was dependent on a functional ligand-binding domain. Androgen receptor antagonists showed agonistic activity in cells with increased androgen receptor levels; this antagonist-agonist conversion was associated with alterations in the recruitment of coactivators and corepressors to the promoters of androgen receptor target genes. Increased levels of androgen receptor confer resistance to antiandrogens by amplifying signal output from low levels of residual ligand, and by altering the normal response to antagonists. These findings provide insight toward the design of new antiandrogens.
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            Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth.

            Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor (AR). Despite anorchid serum androgen levels, nearly all patients develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative reverse transcription-PCR, we evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men [0.74 ng/g; 95% confidence interval (95% CI), 0.59-0.89] were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/g; 95% CI, 0.03-0.44; P < 0.0001). Compared with primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including up-regulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1, and UGT2B17 and down-regulated expression of SRD5A2 (P < 0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared with eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment.
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              Using RepeatMasker to identify repetitive elements in genomic sequences.

              The RepeatMasker program is used for identifying repetitive elements in nucleotide sequences for further detailed analyses. Users can run RepeatMasker remotely via a Web site, or, for larger input sequences, the program and its dependent programs may be downloaded and run locally on Unix/Linux computers. The protocols in this chapter detail how to use RepeatMasker both remotely and locally to extract repetitive elements data and mask these repetitive elements in nucleotide sequences.
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                Author and article information

                Journal
                8711562
                6325
                Oncogene
                Oncogene
                Oncogene
                0950-9232
                1476-5594
                13 December 2011
                23 January 2012
                8 November 2012
                08 May 2013
                : 31
                : 45
                : 4759-4767
                Affiliations
                [1 ]Masonic Cancer Center, University of Minnesota, Minneapolis, MN
                [2 ]Biostatistics and Bioinformatics Core, Masonic Cancer Center, University of Minnesota, Minneapolis, MN
                [3 ]Cytogenetics Shared Resource, Masonic Cancer Center, University of Minnesota, Minneapolis, MN
                [4 ]Biomedical Genomics Center, University of Minnesota, Minneapolis, MN
                [5 ]Department of Urology, University of Washington Medical Center, Seattle, WA
                [6 ]Puget Sound VA Health Care System, Seattle, WA
                [7 ]Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN
                [8 ]BioNet, University of Minnesota, Minneapolis, MN
                [9 ]Institute of Human Genetics, University of Minnesota, Minneapolis, MN
                Author notes
                Correspondence: Scott M. Dehm, Masonic Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street SE. Phone: 612-625-1504; Fax: 612-626-4915; dehm@ 123456umn.edu
                [*]

                These authors contributed equally to this study.

                Article
                NIHMS343813
                10.1038/onc.2011.637
                3337879
                22266865
                96eb65e8-fa81-4938-b0eb-112067578c48

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Cancer Institute : NCI
                Award ID: R21 CA141011-02 || CA
                Funded by: National Cancer Institute : NCI
                Award ID: R21 CA141011-01 || CA
                Categories
                Article

                Oncology & Radiotherapy
                prostate cancer,androgen receptor variants,castration-resistant,intragenic rearrangement, ar alternative splicing

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