Identification of Androgen Receptor Modulators in a Prostate Cancer Cell Line Microarray Compendium

High-throughput transcriptomic (HTTr) technologies are increasingly being used to screen environmental chemicals in vitro to identify molecular targets and provide mechanistic context for regulatory testing. Here, we describe the development and validation of a novel gene expression biomarker to identify androgen receptor (AR)-modulating chemicals using a pattern matching method. Androgen receptor biomarker genes were identified by their consistent expression after exposure to 4 AR agonists and 4 AR antagonists and included only those genes that were regulated by AR. The 51 gene biomarker was evaluated as a predictive tool using the fold-change, rank-based Running Fisher algorithm. Using 158 comparisons from cells treated with 95 chemicals, the biomarker gave balanced accuracies for prediction of AR activation or AR suppression of 97% or 98%, respectively. The biomarker correctly classified 16 out of the 17 AR reference antagonists including those that are "weak" and "very weak". Predictions based on microarray profiles from AR-positive LAPC-4 cells treated with 28 chemicals in antagonist mode were compared with those from an AR pathway model which used 11 in vitro HT assays. The balanced accuracy for suppression was 93%. Using our approach, we identified conditions in which AR was modulated in a large collection of microarray profiles from prostate cancer cell lines including (1) constitutively active mutants or knockdown of AR, (2) decreases in availability of androgens by castration or removal from media, and (3) exposure to chemical modulators that work through indirect mechanisms including suppression of AR expression. These results demonstrate that the AR gene expression biomarker could be a useful tool in HTTr to identify AR modulators.

[1]  V. Noé,et al.  Urolithin A causes p21 up-regulation in prostate cancer cells , 2016, European Journal of Nutrition.

[2]  Joanne M Yeakley,et al.  A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling , 2017, PloS one.

[3]  I. Mills,et al.  The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis , 2011, The EMBO journal.

[4]  K. Lam,et al.  ROR-γ drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer , 2016, Nature Medicine.

[5]  Parantu K. Shah,et al.  Targeting Poly(ADP-Ribose) Polymerase and the c-Myb–Regulated DNA Damage Response Pathway in Castration-Resistant Prostate Cancer , 2014, Science Signaling.

[6]  Scott Auerbach,et al.  Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium , 2015, Nuclear receptor signaling.

[7]  Shu-Dong Zhang,et al.  Application of connectivity mapping in predictive toxicology based on gene-expression similarity. , 2010, Toxicology.

[8]  R. Judson,et al.  Identifying environmental chemicals as agonists of the androgen receptor by using a quantitative high-throughput screening platform. , 2017, Toxicology.

[9]  D. Kazmin,et al.  Pharmacological uncoupling of androgen receptor-mediated prostate cancer cell proliferation and prostate-specific antigen secretion. , 2003, Cancer research.

[10]  F. Claessens,et al.  Comparing the rules of engagement of androgen and glucocorticoid receptors , 2017, Cellular and Molecular Life Sciences.

[11]  David M. Reif,et al.  Test driving ToxCast: endocrine profiling for 1858 chemicals included in phase II. , 2014, Current opinion in pharmacology.

[12]  T. Golub,et al.  Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators. , 2006, Cancer cell.

[13]  Karl G. Kohlgraf,et al.  Mechanisms of Cell Death Induced by Histone Deacetylase Inhibitors in Androgen Receptor–Positive Prostate Cancer Cells , 2006, Molecular Cancer Research.

[14]  Richard A Becker,et al.  Developing scientific confidence in HTS-derived prediction models: lessons learned from an endocrine case study. , 2014, Regulatory toxicology and pharmacology : RTP.

[15]  Robert J Kavlock,et al.  Phenotypic screening of the ToxCast chemical library to classify toxic and therapeutic mechanisms , 2014, Nature Biotechnology.

[16]  K. A. Klein,et al.  Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice , 1997, Nature Medicine.

[17]  David M. Reif,et al.  Analysis of the Effects of Cell Stress and Cytotoxicity onIn Vitro Assay Activity Across a Diverse Chemical and Assay Space , 2016, Toxicological sciences : an official journal of the Society of Toxicology.

[18]  Ruili Huang,et al.  Predictive endocrine testing in the 21st century using in vitro assays of estrogen receptor signaling responses. , 2014, Environmental science & technology.

[19]  Jianfei Qi,et al.  Refinement of the androgen response element based on ChIP-Seq in androgen-insensitive and androgen-responsive prostate cancer cell lines , 2016, Scientific Reports.

[20]  N. Guseva,et al.  Inhibition of p53 expression modifies the specificity of chromatin binding by the androgen receptor , 2012, Oncotarget.

[21]  J. Palvimo,et al.  Androgen receptor amplification is reflected in the transcriptional responses of Vertebral-Cancer of the Prostate cells , 2011, Molecular and Cellular Endocrinology.

[22]  Nisha S. Sipes,et al.  In vitro and modelling approaches to risk assessment from the U.S. Environmental Protection Agency ToxCast programme. , 2014, Basic & clinical pharmacology & toxicology.

[23]  Paul A Clemons,et al.  The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease , 2006, Science.

[24]  R. Vessella,et al.  Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer. , 2009, Cancer research.

[25]  Zhou Zhu,et al.  Dose-dependent effects of small-molecule antagonists on the genomic landscape of androgen receptor binding , 2012, BMC Genomics.

[26]  L. Borsu,et al.  Histone deacetylases are required for androgen receptor function in hormone-sensitive and castrate-resistant prostate cancer. , 2009, Cancer research.

[27]  V. Arora,et al.  Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer , 2015, Nature Reviews Cancer.

[28]  Ruili Huang,et al.  Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor. , 2015, Toxicological sciences : an official journal of the Society of Toxicology.

[29]  L. Giudice,et al.  Endocrine-disrupting chemicals: an Endocrine Society scientific statement. , 2009, Endocrine reviews.

[30]  Joshua M. Korn,et al.  An F876L mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 (enzalutamide). , 2013, Cancer discovery.

[31]  Ruili Huang,et al.  Development and Validation of a Computational Model for Androgen Receptor Activity , 2016, Chemical research in toxicology.

[32]  J. Isaacs,et al.  The Prostate 69 : 1724 ^ 1729 ( 2009 ) TissueCultureMedia SupplementedWith 10 % Fetal Calf SerumContains aCastrate Levelof Testosterone , 2009 .

[33]  Hideo Araki,et al.  Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome. , 2003, Cancer research.

[34]  J. Céraline,et al.  Constitutively Active Androgen Receptor Variants Upregulate Expression of Mesenchymal Markers in Prostate Cancer Cells , 2013, PloS one.

[35]  J. Corton,et al.  Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα) in a Mouse Liver Gene Expression Compendium , 2015, PloS one.

[36]  Lauren M Aleksunes,et al.  Screening a mouse liver gene expression compendium identifies modulators of the aryl hydrocarbon receptor (AhR). , 2015, Toxicology.

[37]  Yu-Mei Tan,et al.  Adverse Outcome Pathways—Organizing Toxicological Information to Improve Decision Making , 2016, The Journal of Pharmacology and Experimental Therapeutics.

[38]  G. Jenster,et al.  The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens , 1992, Journal of Steroid Biochemistry and Molecular Biology.

[39]  Edwin Cheung,et al.  A transcriptional repressor co‐regulatory network governing androgen response in prostate cancers , 2012, The EMBO journal.

[40]  P. Dervan,et al.  Suppression of androgen receptor-mediated gene expression by a sequence-specific DNA-binding polyamide , 2007, Proceedings of the National Academy of Sciences.

[41]  L. Butler,et al.  Co-targeting AR and HSP90 suppresses prostate cancer cell growth and prevents resistance mechanisms. , 2015, Endocrine-related cancer.

[42]  Raymond R Tice,et al.  Moving Toward Integrating Gene Expression Profiling Into High-Throughput Testing: A Gene Expression Biomarker Accurately Predicts Estrogen Receptor α Modulation in a Microarray Compendium. , 2016, Toxicological sciences : an official journal of the Society of Toxicology.

[43]  Hongjuan Cui,et al.  Hormone depletion-insensitivity of prostate cancer cells is supported by the AR without binding to classical response elements. , 2011, Molecular endocrinology.

[44]  Daehee Hwang,et al.  Integrated Expression Profiling and ChIP-seq Analyses of the Growth Inhibition Response Program of the Androgen Receptor , 2009, PloS one.

[45]  M. Ronaghi,et al.  Ontology-Based Meta-Analysis of Global Collections of High-Throughput Public Data , 2010, PloS one.

[46]  M. Waters,et al.  Characterizing and predicting carcinogenicity and mode of action using conventional and toxicogenomics methods. , 2010, Mutation research.

[47]  Erick R. Scott,et al.  Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay , 2014, Nucleic acids research.