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

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

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

Gene expression in prostate cancer

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 (L0ngSAGE) libraries. We identified 35 genes with novel associations to androgen signalling and validated 24 of these genes using quantitative real time-polymerase chain reaction (qRT-PCR). These genes were: ARL6IF5, BL VRB, C]9orf48, C]orfJ22, C6orf66, CAMK2NJ, CCNI, DERA, ERRFI], GLUL, GOLFH3, HMJ3, HSP9OB], MANEA, NANS, NIPSNAP3A, SLC4JA], SOD], SVIF, TAOK3, TCP], TMEM66, USP33, and VTAJ. The physiological relevance of these expression trends was evaluated in vivo using the LNCaP Hollow Fibre model. There is no cure for castration-recurrent prostate cancer (CRPC), and the mechanisms underlying the disease are not known. To address this problem, we assayed the transcriptome of LNCaP human prostate cancer cells as they progress to castration-recurrence in vivo using replicate L0ngSAGE libraries. We identified 96 novel genes consistently differentially expressed in CRPC. The expression profiles support a role for the transcriptional activity of the androgen receptor genes (CCNH, CUEDC2, FLNA, and FSMA 7), steroid synthesis and metabolism genes (DHCR24, DHRS7, ELOVL5, HSDJ 7B4, and OPRKJ), neuroendocrine cell genes (ENO2, MAOA, OPRK], SJOOA]O, and TRPM8), and proliferation genes (GAS5, GNB2L], MT-ND3, NKX3-], PCGEM], PTGFR, STEAFJ, and TMEM3OA) in castration-recurrence. Screening for prostate cancer using serum levels of prostate-specific antigen has resulted in the over-treatment of indolent disease. Novel diagnostic and prognostic markers for prostate cancer are required. To address this need, the levels of 27 transcripts were investigated with qRT-PCR. Expression of POP3 (100 kb from EST CF140309) was prostate-specific, with restricted expression ofADAM2, POP1 (50 kb from AK000023), POP4 (truncated TMEFF2), POP 10 (intron ofADAM2), ELOVL5, RAMP], and SPON2. ELO VL5, NGFRAP1, POP5 (intron of NCAM2), POP8 (intron of EFNA5), RAMP], SPON2, and TMEM66 were differentially expressed between laser microdissected tumour and normal clinical samples of prostatic tissue. These studies suggest that ADAM2, ELOVL5, POP 1, POP3, POP4, POP 10, RAMP], and SPON2 may be good candidates for biomarkers of prostate cancer.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Novel Biomarkers for Prostate Cancer Including Noncoding Transcripts

Levels of 27 transcripts were investigated as potential novel markers for prostate cancer, including genes encoding plasma membrane proteins (ADAM2, ELOVL5, MARCKSL1, RAMP1, TMEM30A, and TMEM66); secreted proteins (SPON2, TMEM30A, TMEM66, and truncated TMEFF2 (called POP4)); intracellular proteins (CAMK2N1, DHCR24, GLO1, NGFRAP1, PGK1, PSMA7, SBDS, and YWHAQ); and noncoding transcripts (POP1 (100 kb) from mRNA AK000023), POP2 (4 kb from mRNA AL832227), POP3 (50 kb from EST CFI40309), POP5 (intron of NCAM2, accession DO668384), POP6 (intron of FHIT), POP7 (intron of TNFAIP8), POP8 (intron of EFNA5), POP9 (intron of DSTN), POP10 (intron of ADAM2, accession DO668396), POP11 (87kb from EST BG194644), and POP12 (intron of EST BQ226050)). Expression of POP3 was prostate specific, whereas ADAM2, POP1, POP4, POP10, ELOVL5, RAMP1, and SPON2 had limited tissue expression. ELOVL5, MARCKSL1, NGFRAP1, PGK1, POP2, POP5, POP8, PSMA7, RAMP1, and SPON2 were significantly differentially expressed between laser microdissected malignant versus benign clinical samples of prostate tissue. PGK1, POP2, and POP12 correlated to clinical parameters. Levels of CAMK2N1, GLO1, SDBS, and TMEM30A transcripts tended to be increased in primary prostate cancer from patients who later had biochemical failure. Expression of GLO1, DHCR24, NGFRAP1, KLK3, and RAMP1 were significantly decreased in metastatic castration-recurrent disease compared with androgen-dependent primary prostate cancer. These novel potential biomarkers may therefore be useful in the diagnosis/prognosis of prostate cancer