Transcriptomics,Genomics

Dataset Information

18

Intraprostatic Androgens and Androgen-Regulated Gene Expression Persist Following Testosterone Suppression


ABSTRACT: Introduction: Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer (PCa). The efficacy of ADT has not been rigorously evaluated by demonstrating suppression of prostatic androgen activity at the target tissue and molecular level. We determined the efficacy and consistency of medical castration in suppressing prostatic androgen levels and androgen-regulated gene-expression. Design: Androgen levels and androgen-regulated gene-expression (by microarray-profiling, qRT-PCR and immunohistochemistry) were measured in prostate samples from a clinical trial of short-term castration (1 month) using the GnRH-antagonist, Acyline, vs. placebo in healthy men. To assess the effects of long-term ADT, gene-expression measurements were evaluated at baseline and after 3, 6 and 9 months of neoadjuvant ADT in prostatectomy samples from men with localized PCa. Results: Medical castration reduced tissue androgens by 75% and reduced the expression of several androgen-regulated genes (NDRG1, FKBP5, TMPRSS2). However, many androgen-responsive genes, including the androgen receptor (AR) and PSA, were not suppressed after short-term castration, nor after 9 months of neoadjuvant ADT. Significant heterogeneity in PSA and AR protein expression was observed in PCa samples at each time-point of ADT. Conclusions: Medical castration based on serum testosterone levels cannot be equated with androgen ablation in the prostate microenvironment. Standard androgen deprivation does not consistently suppress androgen-dependent gene-expression. Suboptimal suppression of tumoral androgen activity may lead to adaptive cellular changes allowing PCa cell survival in a low androgen environment. Optimal clinical efficacy will require testing of novel approaches targeting complete suppression of systemic and intracrine contributions to the prostatic androgen microenvironment. Keywords: prostate, androgen, Acyline, microarray, immunohistochemistry Overall design: Laser Capture Microdissection and RNA Amplification. Prostate samples embedded in OCT were used for laser capture microdissection (LCM). Approximately 2000-3000 epithelial cells per sample were collected from 8µm sections using the Arcturus Veritas™ Laser Capture Microdissection System according to the Arcturus HistoGene LCM Frozen Section Staining Kit Protocol (Mountain View, CA). Total RNA was isolated using the Arcturus Picopure™ Kit, and the samples were treated with DNAse using the Qiagen RNase-Free DNase Set (Qiagen Inc, Valencia, CA). Total RNA was subjected to two rounds of linear amplification using the Ambion MessageAmpII Kit (Ambion Inc, Austin, TX), quantitated in a Gene-Spec III spectrophotometer (Hitachi, Tokyo) and aRNA integrity evaluated using gel electrophoresis. RNA was extracted from microdissected benign epithelial cells from all subjects in the Acyline trial (placebo, castrate , and castrate +T; n=4 in each group), and from microdissected malignant epithelial cells from a randomly selected subset of patients in the neoadjuvant ADT trial (at 0, 3-6, and 6-9 months of treatment; n=3 in each group). cDNA Microarray Hybridization and Analysis. cDNA probe pairs were prepared from samples obtained in the Acyline trial by amino-allyl reverse transcription using 2 µg of amplified RNA from the microdissected samples and 2µg of amplified RNA from a benign prostate reference standard. The reference was created by pooling equal amounts of RNA amplified from the microdissected placebo-treated prostate epithelial samples. Samples from the neoadjuvant trial of ADT were prepared using 2 µg of amplified RNA from the microdissected samples and 30 µg of total RNA from a reference RNA pool comprised of total RNA isolated from LNCaP, DU145, and PC3 prostate cancer cell lines. Probes were labeled with either Cy5 or Cy3 fluors (Amersham Bioscience, Piscataway, NJ) and competitively hybridized to custom cDNA microarrays spotted in duplicate with approximately 6,700 unique cDNA clones from the Prostate Expression Database (PEDB) as previously described (Nelson PS, Pritchard C, Abbott D, and Clegg N. The human (PEDB) and mouse (mPEDB) Prostate Expression Databases. Nucleic Acids Res 2002;30:218-220. AND Pritchard CC, Hsu L, Delrow J, and Nelson PS. Project normal: defining normal variance in mouse gene expression. Proc Natl Acad Sci U S A 2001;98:13266-13271). Fluorescence array images were collected using a GenePix 4000B fluorescent scanner (Axon Instruments, Foster City, CA) and processed as we have previously described (True L, Coleman I, Hawley S, et al. A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc Natl Acad Sci U S A 2006;103:10991-10996). Changes in gene expression were evaluated using the Statistical Analysis of Microarray (SAM) program (http://www-stat.stanford.edu/_tibs/SAM/) to perform a one-sample t-test assessing the effect of Acyline treatment on gene expression (Tusher VG, Tibshirani R, and Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001;98:5116-5121.). An FDR (false discovery rate) of less than 5% was considered significant. Quantile normalization was performed in Bioconductor (Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004;5:R80) to allow single channel comparison of the absolute signal intensities in the experimental channel across different arrays in the same experiment.

INSTRUMENT(S): FHCRC Human Prostate PEDB cDNA Array v8

SUBMITTER: Denise Mauldin   

PROVIDER: GSE8466 | GEO | 2007-10-25

SECONDARY ACCESSION(S): PRJNA101551

REPOSITORIES: GEO

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Publications

Intraprostatic androgens and androgen-regulated gene expression persist after testosterone suppression: therapeutic implications for castration-resistant prostate cancer.

Mostaghel Elahe A EA   Page Stephanie T ST   Lin Daniel W DW   Fazli Ladan L   Coleman Ilsa M IM   True Lawrence D LD   Knudsen Beatrice B   Hess David L DL   Nelson Colleen C CC   Matsumoto Alvin M AM   Bremner William J WJ   Gleave Martin E ME   Nelson Peter S PS  

Cancer research 20070501 10


Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer. The efficacy of ADT has not been rigorously evaluated by demonstrating suppression of prostatic androgen activity at the target tissue and molecular level. We determined the efficacy and consistency of medical castration in suppressing prostatic androgen levels and androgen-regulated gene expression. Androgen levels and androgen-regulated gene expression (by microarray profiling, quantitative reverse t  ...[more]

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