Identification of novel androgen-responsive genes by sequencing of LongSAGE libraries

<p>Abstract</p> <p>Background</p> <p>The development and maintenance of the prostate is dependent on androgens and the androgen receptor. The androgen pathway continues to be important in prostate cancer. Here, we evaluated the transcriptome of prostate cancer cells in response to androgen using long serial analysis of gene expression (LongSAGE) libraries.</p> <p>Results</p> <p>There were 131 tags (87 genes) that displayed statistically significant (p ≤ 0.001) differences in expression in response to androgen. Many of the genes identified by LongSAGE (35/87) have not been previously reported to change expression in the direction or sense observed. In regulatory regions of the promoter and/or enhancer regions of some of these genes there are confirmed or potential androgen response elements (AREs). The expression trends of 24 novel genes were validated using quantitative real time-polymerase chain reaction (qRT-PCR). These genes were: <it>ARL6IP5, BLVRB, C19orf48, C1orf122, C6orf66, CAMK2N1, CCNI, DERA, ERRFI1, GLUL, GOLPH3, HM13, HSP90B1, MANEA, NANS, NIPSNAP3A, SLC41A1, SOD1, SVIP, TAOK3, TCP1, TMEM66, USP33</it>, and <it>VTA1</it>. The physiological relevance of these expression trends was evaluated <it>in vivo </it>using the LNCaP Hollow Fibre model. Novel androgen-responsive genes identified here participate in protein synthesis and trafficking, response to oxidative stress, transcription, proliferation, apoptosis, and differentiation.</p> <p>Conclusion</p> <p>These processes may represent the molecular mechanisms of androgen-dependency of the prostate. Genes that participate in these pathways may be targets for therapies or biomarkers of prostate cancer.</p

Targeting androgen receptor activation function-1 with EPI to overcome resistance mechanisms in castration-resistant prostate cancer

Acknowledgments The authors thank Kate Watt (University of Aberdeen, Aberdeen, Scotland) for technical support. The authors are also grateful to Country Meadows Senior Men's Golf Charity Classic for financial support of this research. Financial support: This research was supported by grants to MDS from the NCI (2R01CA105304), the Canadian Institutes of Health Research (MOP79308) and the US Army Medical Research and Materiel Command Prostate Cancer Research Program (E81XWH-11-1-0551). Research by IJM’s group was supported by the Chief Scientist’s Office of the Scottish Government (ETM-258 and -382). We are grateful to Country Meadows Senior Men’s Golf Charity Classic for financial support of this research.Peer reviewedPostprin

FUS/TLS Is a Co-Activator of Androgen Receptor in Prostate Cancer Cells

Androgen receptor (AR) is a member of the nuclear receptor family of transcription factors. Upon binding to androgens, AR becomes transcriptionally active to regulate the expression of target genes that harbor androgen response elements (AREs) in their promoters and/or enhancers. AR is essential for the growth and survival of prostate cancer cells and is therefore a target for current and next-generation therapeutic modalities against prostate cancer. Pathophysiologically relevant protein-protein interaction networks involving AR are, however, poorly understood. In this study, we identified the protein FUsed/Translocated in LipoSarcoma (FUS/TLS) as an AR-interacting protein by co-immunoprecipitation of endogenous proteins in LNCaP human prostate cancer cells. The hormonal response of FUS expression in LNCaP cells was shown to resemble that of other AR co-activators. FUS displayed a strong intrinsic transactivation capacity in prostate cancer cells when tethered to basal promoters using the GAL4 system. Chromatin immunoprecipitation experiments showed that FUS was recruited to ARE III of the enhancer region of the PSA gene. Data from ectopic overexpression and “knock-down” approaches demonstrated that AR transcriptional activity was enhanced by FUS. Depletion of FUS reduced androgen-dependent proliferation of LNCaP cells. Thus, FUS is a novel co-activator of AR in prostate cancer cells

Analysis of the prostate cancer cell line LNCaP transcriptome using a sequencing-by-synthesis approach

BACKGROUND: High throughput sequencing-by-synthesis is an emerging technology that allows the rapid production of millions of bases of data. Although the sequence reads are short, they can readily be used for re-sequencing. By re-sequencing the mRNA products of a cell, one may rapidly discover polymorphisms and splice variants particular to that cell. RESULTS: We present the utility of massively parallel sequencing by synthesis for profiling the transcriptome of a human prostate cancer cell-line, LNCaP, that has been treated with the synthetic androgen, R1881. Through the generation of approximately 20 megabases (MB) of EST data, we detect transcription from over 10,000 gene loci, 25 previously undescribed alternative splicing events involving known exons, and over 1,500 high quality single nucleotide discrepancies with the reference human sequence. Further, we map nearly 10,000 ESTs to positions on the genome where no transcription is currently predicted to occur. We also characterize various obstacles with using sequencing by synthesis for transcriptome analysis and propose solutions to these problems. CONCLUSION: The use of high-throughput sequencing-by-synthesis methods for transcript profiling allows the specific and sensitive detection of many of a cell's transcripts, and also allows the discovery of high quality base discrepancies, and alternative splice variants. Thus, this technology may provide an effective means of understanding various disease states, discovering novel targets for disease treatment, and discovery of novel transcripts

Rational optimization of a transcription factor activation domain inhibitor

Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors

LNCaP Atlas: Gene expression associated with in vivo progression to castration-recurrent prostate cancer

<p>Abstract</p> <p>Background</p> <p>There is no cure for castration-recurrent prostate cancer (CRPC) and the mechanisms underlying this stage of the disease are unknown.</p> <p>Methods</p> <p>We analyzed the transcriptome of human LNCaP prostate cancer cells as they progress to CRPC <it>in vivo </it>using replicate LongSAGE libraries. We refer to these libraries as the LNCaP atlas and compared these gene expression profiles with current suggested models of CRPC.</p> <p>Results</p> <p>Three million tags were sequenced using <it>in vivo </it>samples at various stages of hormonal progression to reveal 96 novel genes differentially expressed in CRPC. Thirty-one genes encode proteins that are either secreted or are located at the plasma membrane, 21 genes changed levels of expression in response to androgen, and 8 genes have enriched expression in the prostate. Expression of 26, 6, 12, and 15 genes have previously been linked to prostate cancer, Gleason grade, progression, and metastasis, respectively. Expression profiles of genes in CRPC support a role for the transcriptional activity of the androgen receptor (<it>CCNH, CUEDC2, FLNA, PSMA7</it>), steroid synthesis and metabolism (<it>DHCR24, DHRS7</it>, <it>ELOVL5, HSD17B4</it>, <it>OPRK1</it>), neuroendocrine (<it>ENO2, MAOA, OPRK1, S100A10, TRPM8</it>), and proliferation (<it>GAS5</it>, <it>GNB2L1</it>, <it>MT-ND3</it>, <it>NKX3-1</it>, <it>PCGEM1</it>, <it>PTGFR</it>, <it>STEAP1</it>, <it>TMEM30A</it>), but neither supported nor discounted a role for cell survival genes.</p> <p>Conclusions</p> <p>The <it>in vivo </it>gene expression atlas for LNCaP was sequenced and support a role for the androgen receptor in CRPC.</p